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Cardiac adenylate metabolism : possible relationship to autoreguation of coronary blood flow Nakatsu, Kanji 1971

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CARDIAC  ADENYLATE  METABOLISM:  AUTOREGULATION  OF  POSSIBLE  CORONARY  RELATIONSHIP  BLOOD  FLOW  by  KANJI NAKATSU B.Sc, M.Sc,  U n i v e r s i t y o f A l b e r t a , 1964. U n i v e r s i t y o f A l b e r t a , 1968.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of Pharmacology  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1971.  TO  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfilment of  the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the L i b r a r y s h a l l I  f u r t h e r agree  make i t  freely available  that permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  study. thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department by h i s of  this  written  representatives. thesis  It  for financial  i s understood that gain shall  permission.  Department of  PA. f  t A#  The U n i v e r s i t y o f B r i t i s h Vancouver 8. Canada  c Columbia  y - •>/  that  or  copying o r p u b l i c a t i o n  not be allowed without my  (i)  ABSTRACT  The m e t a b o l i s m  o f 5'-AMP by 5 - n u c l e o t i d a s e , a d e n y l a t e deaminase 1  and a d e n y l a t e k i n a s e was examined i n h e a r t homogenates o f r a t , r a b b i t , dog, p i g e o n and t u r t l e .  The s t u d y was conducted  the p o s s i b i l i t y that adenosine,  i n c o n s i d e r a t i o n of  a c a t a b o l i c p r o d u c t o f 5'-AMP, may  c o n t r o l vasotone f o r the a u t o r e g u l a t i o n of coronary blood flow.  The  r e l a t i v e a c t i v i t i e s o f homogenates o f h e a r t s from v a r i o u s s p e c i e s t o form adenosine  by t h e a c t i o n o f 5 ' - n u c l e o t i d a s e g e n e r a l l y s u p p o r t e d  such a r o l e f o r t h i s n u c l e o s i d e . the l a r g e s t p o t e n t i a l requirements  Those s p e c i e s a n t i c i p a t e d t o have f o r coronary v a s o d i l a t i o n , i . e .  t h o s e whose oxygen consumption i s known t o i n c r e a s e s i g n i f i c a n t l y d u r i n g p h y s i c a l e x e r t i o n , had t h e h i g h e s t l e v e l s o f c a r d i a c tidase.  5'-nucleo-  An e x c e p t i o n t o t h i s was t h e p i g e o n w h i c h had no d e t e c t a b l e  cardiac 5'-nucleotidase;  t h e o r d e r o f l e v e l s o f t h i s enzyme i n h e a r t s  o f t h e o t h e r s p e c i e s t e s t e d was:  r a t > dog > r a b b i t > t u r t l e .  The  t u r t l e v e n t r i c l e , by v i r t u e o f i t s h i g h c o n t e n t o f a d e n y l a t e deaminase and l o w c o n t e n t o f 5 ' - n u c l e o t i d a s e appeared t o c a t a b o l i z e 5'-AMP l a r g e l y by d e a m i n a t i o n  t o IMP. Homogenates o f p i g e o n v e n t r i c l e c o n t a i n e d t h e  g r e a t e s t a c t i v i t y o f adenylate k i n a s e , i n d i c a t i n g t h a t the h e a r t o f t h i s s p e c i e s i s equipped  f o r p r e s e r v a t i o n o f ATP by r e s y n t h e s i s f r o m ADP.  Enzyme h i s t o c h e m i s t r y r e v e a l e d t h a t most 5 ' - n u c l e o t i d a s e o f mammalian h e a r t s was l o c a l i z e d i n t h e e n d o t h e l i a l c e l l s o f c a p i l l a r i e s . T h e r e f o r e , i f adenosine  i s i n v o l v e d i n r e g u l a t i o n of coronary p e r f u s i o n ,  i t s s o u r c e may be c a p i l l a r y e n d o t h e l i a l c e l l s r a t h e r than c a r d i a c m u s c l e cells. 5 ' - N u c l e o t i d a s e was p a r t i a l l y p u r i f i e d from a n a c e t o n e powder o f rat heart.  I t was a c t i v e o v e r a broad range o f pH w i t h an optimum a t  (ii)  pH 8.5. Mn  I|  T h e enzyme was s t i m u l a t e d up t o 5 - f o l d b y Mg  and N i  | [  also stimulated activity. _5  I|  in  t h e a b s e n c e o f Mg  a n d 2.3 x 10  Certain of i t s properties indicated  (K = 1.9 x 10 a  T h e K f o r 5'-AMP was 2.1 x 10 m  M); _ £^  M  M i n t h e p r e s e n c e o f 16 mM M g C ^ . that t h e production of adenosine  might  be f a v o u r e d u n d e r c o n d i t i o n s i n w h i c h c o r o n a r y v a s o d i l a t i o n w o u l d b e r e q u i r e d and v i c e - v e r s a .  F o r e x a m p l e , t h e enzyme was i n h i b i t e d b y A T P ,  whose l e v e l s a r e g r e a t e s t  i n w e l l oxygenated h e a r t s i nwhich energy  i s high.  Not a l l p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e were c o n s i s t e n t  enhanced a d e n o s i n e f o r m a t i o n a t reduced energy c h a r g e . phosphate, inhibited  charge  with  B o t h ADP a n d o r t h o -  t h e l e v e l s o f w h i c h i n c r e a s e when e n e r g y c h a r g e d e c r e a s e s , t h e enzyme;  In addition,  i n f a c t ADP was a more p o w e r f u l i n h i b i t o r  t h e enzyme was n o t s p e c i f i c  v a r i e t y o f n u c l e o s i d e 5'-monophosphates;  t h a n ATP.  f o r 5'-AMP b u t h y d r o l y z e d a a n d t h e h y d r o l y s i s o f 5'-AMP  was c o m p e t i t i v e l y i n h i b i t e d b y UMP. I |  I n t h e a b s e n c e o f Mg tive-non-competitive) type. was n o n - c o m p e t i t i v e .  , i n h i b i t i o n b y ADP was o f t h e m i x e d I n t h e p r e s e n c e o f 16 mM M g C ^ ,  (competi-  inhibition  On t h e b a s i s o f t h e s e d a t a a n d D i x o n p l o t s o f  i n h i b i t i o n a s a f u n c t i o n o f ADP c o n c e n t r a t i o n , i t i s s u g g e s t e d t h a t two c o n f o r m a t i o n s o f t h e enzyme a r e p o s s i b l e ; i n h i b i t e d b y ADP.  one w h i c h i s c o m p e t i t i v e l y  T h e s i m p l e n o n - c o m p e t i t i v e i n h i b i t i o n b y ADP, o b s e r v e d I |  i n t h e p r e s e n c e o f 16 mM M g C ^ , i s a t t r i b u t e d for the l a t t e r conformation.  t o Mg  -induced preference  (iii)  TABLE  OF  CONTENTS Page  INTRODUCTION  1  METHODS AND MATERIALS A.  General  B.  Survey  17 o f C a r d i a c Enzymes w h i c h  Utilize  5'-AMP a n d A d e n o s i n e  17  C.  P e r f u s i o n o f R a t , R a b b i t and T u r t l e H e a r t s  .21  D.  Histochemistry  E.  Assay  F.  Materials'  24  A.  Preliminary  25  B.  C a r d i a c Enzymes w h i c h  22  of Partially Purified  5'-Nucleotidase  22  RESULTS  Utilize  5'-AMP a n d  Adenosine  26  C.  E f f e c t o f Adenosine on Coronary  Flow  D.  Histochemical Localization of Cardiac  30  5'-Nucleotidase  31  E.  P a r t i a l P u r i f i c a t i o n of 5'-Nucleotidase  36  F.  Properties of 5'-Nucleotidase  39  DISCUSSION Nature  52 o f ADP I n h i b i t i o n  LITERATURE C I T E D  63  67  (iv)  LIST  OF  TABLES  Page  TABLE I .  Survey of V e n t r i c u l a r Adenosine Deaminase  29(a)  TABLE I I .  Summary of P u r i f i c a t i o n  38(a)  TABLE I I I .  E f f e c t of Ca** on 5'-Nucleotidase  A3(a)  (v)  LIST  Standard curve  OF  FIGURES  f o r assay of inorganic  L i n e a r i t y of 5'-nucleotidase  Survey o f v e n t r i c u l a r  phosphate.  a s s a y w i t h time and p r o t e i n .  5'-nucleotidase.  Survey o f v e n t r i c u l a r adenylate  deaminase.  Survey o f v e n t r i c u l a r adenylate  kinase.  E f f e c t o f adenosine on coronary  flow.  Histochemical  l o c a l i z a t i o n of 5'-nucleotidase  i n ventricle  sections. Plate  •A.  Rat  ventricle;  s u b s t r a t e 5'-AMP.  P l a t e B.  Rat  ventricle;  substrate  P l a t e C.  Dog-ventricle;  P l a t e ' D.  Human p a p i l l a r y m u s c l e ;  s u b s t r a t e 5'-AMP.  P l a t e E.  Human p a p i l l a r y m u s c l e ;  control using  P l a t e F.  Human p a p i l l a r y m u s c l e ;  hematoxylin  P l a t e G.  Guinea p i g v e n t r i c l e ;  s u b s t r a t e 5'-AMP.  P l a t e H.  Guinea p i g v e n t r i c l e ;  control.  Plate I.  Mouse v e n t r i c l e ;  Plate J.  Guinea p i g v e n t r i c l e ;  P l a t e K.  Pigeon v e n t r i c l e ;  s u b s t r a t e 5'-AMP.  P l a t e L.  Turtle ventricle;  s u b s t r a t e 5'-AMP.  5'-AMP  s u b s t r a t e 5'-AMP.  3'-AMP.  and e o s i n .  s u b s t r a t e 5'-AMP. s u b s t r a t e 5'-AMP.  Figure 8.  Summary of 5'-nucleotidase  purification.  Figure 9,  A c t i v i t y of 5'-nucleotidase as a f u n c t i o n of pH.  Figure 10.  E f f e c t of Mg, ,-Mn  Figure 11.  Substrate s p e c i f i c i t y .  Figure 12.  E f f e c t of 5'-AMP concentration.  Figure 13.  I n h i b i t i o n by ATP and ADP a t high concentrations of 5'-AMP,  Figure 14.  ADP i n the absence of Mg  Figure 15.  I n h i b i t i o n by ADP i n the,t presence of MgC^.  Figure 16.  E f f e c t of ADP concentration on i n h i b i t i o n .  Figure 17.  I n h i b i t i o n by UMP and orthophosphate i n the presence of MgC^.  Figure 18.  E f f e c t of MgC^ concentration on i n h i b i t i o n by ADP and ATP.  , and N i  on 5'-nucleotidase.  ;  (vii)  A b b r e v i a t i o n s and T r i v i a l  Names  Customary b i o l o g i c a l a b b r e v i a t i o n s and t r i v i a l throughout.the text explained  of this thesis.  names h a v e b e e n u s e d  The o f f i c i a l  names o f t h e s e a r e  below.  A d e n o s i n e D e a m i n a s e : A d e n o s i n e A m i n o h y d r o l a s e , E.C.  3.5.4.4.  A d e n y l a t e D e a m i n a s e : AMP A m i n o h y d r o l a s e , E.C. 3.5.4.6. A d e n y l a t e K i n a s e : ATP:AMP P h o s p h o t r a n s f e r a s e , E.C. 2.7.4.35 ' - N u c l e o t i d a s e : 5 ' - R i b o n u c l e o t i d e P h o s p h o h y d r o l a s e , E.C. 3 . 1 . 3 . 5 .  5'-AMP, a d e n o s i n e 5 ' - m o n o p h o s p h a t e ; ADP, a d e n o s i n e 5 ' - d i p h o s p h a t e ;  3'-AMP, a d e n o s i n e  ATP, adenosine 5 ' - t r i p h o s p h a t e ;  CMP, c y t i d i n e  5'-monophosphate;  CTP, c y t l d i n e  5 ' - t r i p h o s p h a t e ; GMP, g u a n o s i n e  GDP, g u a n o s i n e 5 ' - d i p h o s p h a t e ; P_^, o r t h o p h o s p h a t e ; UDP, u r i d i n e  A small "d" as the f i r s t indicates  Tris,  CDP, c y t l d i n e  5'-diphosphate; 5'-monophosphate;  GTP, g u a n o s i n e 5 ' - t r i p h o s p h a t e ;  UMP, u r i d i n e  5'-diphosphate;  3'-monophosphate;  5'-monophosphate;  3GP, 3 - g l y c e r o p h o s p h a t e ;  UTP, u r i d i n e 5 ' - t r i p h o s p h a t e .  letter  of an a b b r e v i a t i o n f o ra n u c l e o t i d e  t h e 2 ' - d e o x y - compound.  tr±s,(hydroxymethyl)amino-methane;  tetraacetlc  acid.  EDTA, e t h y l e n e d i a m i n e  (viii)  \  •  '  •  \  ACKNOWLEDGEMENTS  I would l i k e to express my g r a t i t u d e to Dr. George I . Drummond who, by word and example, gave guidance, encouragement and i n s t r u c t i o n during the course of t h i s work.  I also thank a l l those who contributed to my education of the past three years.  I wish to express my a p p r e c i a t i o n to the H.R. MacMillan Family and the Medical Research Council of Canada f o r f i n a n c i a l support.  For My Friends  INTRODUCTION The  d e l i v e r y o f o x y g e n t o c a r d i a c m u s c l e i s a symphony f o r w h i c h  the music i s s t i l l for  anoxic  unwritten.  function;  180  f o r c e o f c o n t r a c t i o n by  The  mammalian h e a r t  seconds of anoxia  70%  i s poorly  equipped  r e d u c e s t h e r a t e by  (Williamson, 1966).  of e n e r g y a v a i l a b l e f r o m c a r d i a c g l y c o l y s i s , an  Due  60%  and  to the l i m i t e d  amount  increase i n cardiac  energy  u t i l i z a t i o n m u s t be m a t c h e d by  processes  consumption  h e a r t , u n l i k e o t h e r o r g a n s s u c h as b r a i n ,  kidney  and  (Opie,  for  little  by  i t s arterial  supply  (Van  C i t t e r s , 1965).  o x y g e n a v a i l a b l e f o r f u r t h e r e x t r a c t i o n , any  o x y g e n must be Blood  pressure,  The  i n increased  s k e l e t a l m u s c l e , e x t r a c t s a l a r g e p o r t i o n ( o v e r 75%)  oxygen p r o v i d e d is  1968).  which r e s u l t  fulfilled  by  an  f l o w i n the coronary  v a s c u l a r r e s i s t a n c e and  flow, while coronary 4  times  f l o w i s examined under b a s a l , u n s t r e s s e d greater during d i a s t o l e than during  r a t e may  increase.  a c c o m p l i s h e d p r i m a r i l y by  The  rate  An  the p r o p o r t i o n of  t i m e s p e n t i n d i a s t o l e (Van  in  the  restricts  blood  Since  there  with respect  is little  o r no  If  1965). and  to e x e r c i s e i s Citters  and  i n a decrease i n 1965).  flow i n periods  This of  While d i a s t o l i c  to p e r f u s i o n of  change i n d i a s t o l i c  tends  increased as w e l l  pressure.may i n c r e a s e w i t h increased c a r d i a c output,  f o r m e r a r e more i m p o r t a n t  vasculature.  I t i s phasic  Citters,  ( R u s h m e r , Van  Citters,  work because f l o w i s g r e a t e s t d u r i n g d i a s t o l e . s y s t o l i c blood  perfusion  both s t r o k e volume  i n c r e a s e i n r a t e , however, r e s u l t s  reduce r a t h e r than enhance c o r o n a r y  flow.  c o n d i t i o n s , i t i s about  s y s t o l e (Van  F r a n k l i n , 1963).  to  demand  v e s s e l s and  i n c r e a s e d c a r d i a c o u t p u t due  increased heart  there  i s relatively unrestricted.  I n r e s p o n s e t o a demand f o r g r e a t e r c a r d i a c o u t p u t , heart  blood  c o n t r a c t i o n s of the h e a r t .  flow through the r e l a x e d heart  the  Since  c i r c u l a t i o n i s a f f e c t e d by  because c o n t r a c t i o n of the myocardium compresses blood  of  increased  increase i n coronary  oxygen  the  as  changes  cardiac  pressure  2.  d u r i n g , e x e r c i s e (Bevegard, Holmgren and J o n s s o n , 1 9 6 0 ) , i t i s u n l i k e l y t h a t a change i n p e r f u s i o n p r e s s u r e  can account f o r l a r g e , e x e r t i o n -  induced  f l o w such as t h a t observed  increases i n coronary  subjected  to mild  blood  tread-mill exercise  (Gregg, 1963).  that neither increased perfusion pressure  It  I t i s apparent  nor increased duration of  d i a s t o l e c a n a c c o u n t f o r enhancement o f b l o o d to t h e s t r e s s e d  f l o w and oxygen d e l i v e r y  heart.  f o l l o w s , t h e r e f o r e , that a l t e r a t i o n s i n coronary  r e s i s t a n c e must  p l a y a major r o l e i n t h e maintenance of adequate oxygenation cardium.  The a b i l i t y  of the heart  d e m o n s t r a t e d some y e a r s  pressure steady  pressure,  t o r e g u l a t e v a s c u l a r r e s i s t a n c e was ;  a r t e r i e s were c a n n u l a t e d  r e s i s t a n c e was c o n s t a n t .  was i n c r e a s e d , r e s i s t a n c e i n c r e a s e d  f l o w was m a i n t a i n e d  -  thus a  d e s p i t e p r e s s u r e .changes.  s e v e r a l hypotheses a r e discussed  M y o g e n i c , n e u r a l and m e t a b o l i c  and p e r f u s e d  However, i f t h e p e r f u s i o n  rapidly;  explanation of t h i s autoregulation of coronary sought;  o f t h e myo-  ago b y E c k e l e _ t a _ l . ( 1 9 4 9 ) i n a n e s t h e t i z e d ,  o p e n - c h e s t d o g s . ' When c o r o n a r y at constant  blood  A  relatively  satisfactory  flow i s s t i l l  c o n t r o l a r e t h e three main  Bayliss  sized  from hypoxic  hearts.  Myogenic c o n t r o l , as f i r s t  r e c e n t l y by Berne  (1964),  and/or  nora-  blood  f l o w c o u l d be m a i n t a i n e d  suggested by  i s based on t h e  that muscle c o n t r a c t s i n response to s t r e t c h .  that coronary  While  c o n t r o l t h e o r i e s s u g g e s t t h e r e l e a s e of;; v a s o a c t i v e  (1902) and r e v i e w e d  observation  explanations  resistance.  n e u r a l c o n t r o l i s thought t o i n v o l v e a c e t y l c h o l i n e , a d r e n a l i n e drenaline, metabolic  being  below.  which have been o f f e r e d f o r t h e r e g u l a t i o n o f coronary  metabolites  i n dogs  I t i s hypothe-  a t a constant  r a t e by  the c o n t r a c t i o n o f smooth m u s c l e o f r e s i s t a n c e v e s s e l s i n r e s p o n s e t o t h e stretch stimulus of increased pressure arterial  pressure  were i n c r e a s e d ,  and v i c e - v e r s a .  F o r example, i f  t h e w a l l s o f r e s i s t a n c e v e s s e l s w o u l d be  s t r e t c h e d and  w o u l d r e s p o n d by  c o n t r a c t i o n to t h e i r o r i g i n a l  w o u l d r e m o v e t h e s t i m u l u s f o r f u r t h e r c o n t r a c t i o n and  size.  could account f o r  the maintenance of r e s i s t a n c e v e s s e l diameter  at a constant  maintenance of c o n d u i t  f o r maintenance of  constant  sufficient  r a t e o f p e r f u s i o n b e c a u s e f l o w w o u l d i n c r e a s e due  increased a r t e r i a l were t e n s i o n of is  s i z e i s not  pressure.  This  value.  to  Such  a  the  However, i f t h e s t i m u l u s f o r c o n t r a c t i o n  smooth m u s c l e f i b r e s r a t h e r t h a n l e n g t h , t h i s  criticism  s a t i s f i e d because r e s i s t a n c e v e s s e l s could c o n s t r i c t u n t i l w a l l  returned  to c o n t r o l v a l u e s .  pressure  i s p r o p o r t i o n a l t o v e s s e l w a l l t e n s i o n and  to r a d i u s  (P = T / r ) . .  An  According  returned  to the o r i g i n a l v a l u e .  coronary  blood  According  range of a r t e r i a l p r e s s u r e .  A  e x p l a i n . r e g u l a t i o n of coronary the maintenance of a c o n s t a n t  flow rate.  hypothesis,  r a t e over  a wide to  Myogenic r e g u l a t i o n , as  d i a s t o l i c pressure  described,  i s unchanged  (Bevegard  1960).  i n the r e g u l a t i o n of coronary  sympathetic  For  example, i n open-chest dogs, v a g a l p e r f u s i o n d e s p i t e decreased  treatment  blood  v a s o d i l a t i o n of coronary  pressure decreased after  was  i n c r e a s e d oxygen d e l i v e r y to the h e a r t d u r i n g e x e r c i s e ,  and  coronary  tension  of t h i s myogenic theory  C e r t a i n n e u r a l . o r neurohumoral substances mediators  result  r e s i s t a n c e i s t h a t i t can o n l y account f o r  when c a r d i a c demands a r e h i g h b u t et a l . ,  might w e l l  to t h i s myogenic  at a constant  shortcoming  internal  inversely proportional  than c o n t r o l or u n t i l  f l o w m i g h t be m a i n t a i n e d  permit  relationship,  increase i n a r t e r i a l pressure  i n a decrease i n v e s s e l s i z e to l e s s  could not  to the Laplace  tensions  13%  ( F e i g l , 1969).  have been suggested  flow.  Both  as  parasympathetic  v e s s e l s have been demonstrated.  stimulation resulted i n increased  perfusion pressures;  t h e mean a o r t i c  T h i s v a s o d i l a t i o n was  with atropine, a competitive  not  observed  i n h i b i t o r of a c e t y l c h o l i n e  4.  at muscarinic receptors. F e i g l (1969) could not e x p l a i n t h i s i n terms of a l t e r a t i o n s i n oxygen tension or metabolism, because vagal s t i m u l a t i o n has a negative i n o t r o p i c e f f e c t .  Therefore, he concluded that the vaso-  d i l a t i o n observed was t r u l y a parasympathetic phenomenon. Lochner (1966) demonstrated  Bussmann and  sympathetic coronary v a s o d i l a t i o n i n  i s o l a t e d guinea p i g h e a r t s , using adrenaline and i s o p r o t e r e n o l .  In  Langendorf preparations (see Wedd, 1931), i n f u s i o n of these catecholamines increased both rate and coronary flow.  The increased rate of perfusion  could not be a t t r i b u t e d to hypoxia secondary to increased metabolic r a t e j because venous pO^ was a c t u a l l y greater than control.' I f the v a s o d i l a t i o n had been a r e s u l t of hypoxia, venous pC^ should have been reduced rather than increased. These workers concluded that a d i r e c t S-adrenergic receptor e f f e c t was involved because the enhancement of coronary flow by i s o p r o t e r e n o l and adrenaline was i n h i b i t e d by the 3-receptor b l o c k i n g agents, propranolol and pronethalol.  This i n h i b i t i o n was s p e c i f i c f o r  g-adrenergic receptors; the increase i n coronary flow induced by other v a s o d i l a t o r substances (dipyridamol, ATP, adenosine and n i t r o g l y c e r i n e ) was not blocked.  These experiments serve to i l l u s t r a t e the p o t e n t i a l of  catecholamines and a c e t y l c h o l i n e i n the c o n t r o l of blood flow through the myocardium. Although neural r e g u l a t i o n of*the coronary c i r c u l a t i o n may be a c t i v e i n normal hearts i n s i t u , t h i s i s not l i k e l y . t h e case i n denervated and i s o l a t e d hearts.  For example, i n i s o l a t e d h e a r t s , neural and neuro-  humoral substances are precluded because nerves and blood v e s s e l s which might convey vasoactive substances are severed. (Markwalder and S t a r l i n g , 1913;  In these cases  Berne, Blackmon and Gardner,  1957)  5.  metabolic  r e g u l a t i o n may b e i n v o l v e d ;  i n whole animals,  metabolic  and n e u r a l f a c t o r s c o n t r i b u t e t o r e g u l a t i o n o f  perhaps  both  coronary  perfusion. Metabolic ago  c o n t r o l of coronary  by Markwalder and S t a r l i n g  l a t i o n was i n c r e a s e d  blood  (1913).  years  Flow through the coronary  5 - f o l d by asphyxia  H i l t o n and E i c h h o l t z (1925) c o n c l u d e d  f l o w was i m p l i c a t e d many  i n t h e dog h e a r t - l u n g  circu-  preparation.  t h a t o x y g e n was t h e r e g u l a t o r o f  v a s o t o n e b e c a u s e a n inverse'< r e l a t i o n s h i p was f o u n d b e t w e e n c o r o n a r y f l o w and oxygen s a t u r a t i o n i n t h e dog h e a r t - l u n g l a c t a t e a n d CC^ w e r e e x c l u d e d flow only  slightly,  administered.  as v a s o r e g u l a t o r s  i n coronary  Both  because both a l t e r e d  e v e n when u n p h y s i o l o g i c a l l y l a r g e q u a n t i t i e s w e r e  Other m e t a b o l i t e s  w e r e e l i m i n a t e d b e c a u s e no v a s o d i l a t o r  s u b s t a n c e s c o u l d be d e m o n s t r a t e d i n b l o o d f o r one hour.  preparation.  blood  w h i c h had been  recirculated  T h i s c o n c l u s i o n was r e a c h e d b e c a u s e t h e r e was no d e c r e a s e  blood  f l o w when r e c i r c u l a t e d b l o o d  was r e p l a c e d w i t h  fresh  blood,  a s w o u l d b e e x p e c t e d i f v a s o d i l a t o r compounds h a d a c c u m u l a t e d  during  the period of r e c i r c u l a t i o n .  bility  that vasoactive  i n blood. occurred delivered  T h i s would n o t exclude  s u b s t a n c e s were r e l e a s e d b u t degraded r a p i d l y  P e r h a p s no r e l e a s e o f v a s o a c t i v e m e t a b o l i t e s  should  have  under t h e c o n d i t i o n s used, because w e l l oxygenated blood to the heart.  consistent with  those  conducted s i m i l a r  The r e s u l t s o f t h e s e  o f B e r n e , Blackmon and G a r d n e r  experiments on open-chest dogs.  demonstrated i f coronary  was  e a r l y workers a r e not (1957),  who  The l a t t e r  t h a t an i n v e r s e c o r r e l a t i o n between f l o w and oxygen c o n t e n t be  the possi-  s i n u s oxygen l e v e l s f e l l  found  could  only  b e l o w 5.5 v o l u m e s %.  They c o n c l u d e d  that coronary  f l o w was n o t d e p e n d e n t o n a r t e r i a l  oxygen content  p e r s e b u t was a f u n c t i o n o f t h e d e g r e e o f  blood'  hypoxia.  6.  In a d d i t i o n to pO^j l a c t a t e and pCO^j other metabolites have been invoked to e x p l a i n coronary v a s o d i l a t i o n i n response to hypoxia. Histamine, a known v a s o d i l a t o r , has been examined because i t appeared i n venous blood of dog gastrocnemius a f t e r c o n t r a c t i o n (Anrep and Barsoum, 1935).  I t was also reported i n cardiac venous blood of the  dog heart-lung preparation a f t e r hypoxia 1936).  (Anrep, Barsoum and Talaat,  However, since these observations could not be repeated  (Code,  Evans and Gregory, 1938) and histamine i s released from lung by (Hauge, 1968;  hypoxia  Hauge and Melmon,, 1968) ; t h i s has not been an a t t r a c t i v e ;  p o s s i b i l i t y . . Potassium causes an increase i n coronary flow at low concentrations but decreases i t at higher concentrations (Katz and Lindner, 1938).  Potassium i s given l i t t l e consideration as a mediator  of coronary autoregulation, because the maximum increases i n flow r a t e induced by t h i s c a t i o n are much smaller than those induced by asphyxia or adrenaline.  I n . a d d i t i o n , increases i n sinus potassium concentrations  do not c o r r e l a t e w e l l w i t h the increases i n coronary blood flow ( D r i s c o l and Berne, 1957). The substance which appears to claim the most experimental as mediator of coronary v a s o d i l a t i o n i s adenosine.' Drury and  support  Szent-  GyeJrgyi (1929) f i r s t demonstrated both general and coronary v a s o d i l a t o r y e f f e c t s of adenosine and adenylic a c i d . adenosine.(10 mg)  These workers showed that  increased coronary blood flow 5-fold i n both atropine  treated arid non^atropinized dog heart-lung preparations.  That t h i s  e f f e c t was due to a reduction of r e s i s t a n c e alone was assured  by  maintenance of perfusion pressure at 82 mm Hg and maintaining a constant rate by e l e c t r i c a l pacing.  Adenosine acts as a coronary v a s o d i l a t o r not  only i n i s o l a t e d preparations;  the v a s o d i l a t o r properties of adenosine  7.  and  i t s n u c l e o t i d e s were demonstrated i n dog coronary  i n s i t u b y Wedd a n d D r u r y  (1934).  a d m i n i s t r a t i o n of adenosine blood  f l o w t o 3 times  I n these  animals  ( 7 . 5 mg) i n c r e a s e d  control.  vasculature  the intravenous  the r a t e of coronary  S u c h v a s o d i l a t i o n l a s t e d 20 m i n u t e s  when e i t h e r a d e n o s i n e o r 5'-AMP was i n j e c t e d a n d 1 0 0 m i n u t e s when 3'-AMP was i n j e c t e d . The  heart  itself  was e s t a b l i s h e d a s t h e s i t e o f a c t i o n o f  a d e n o s i n e b y t h e e x p e r i m e n t s o f Wedd a d e n o s i n e was 20 t i m e s vasodilator  more p o t e n t  no due  as a  coronary In  p e r f u s i o n i s e f f e c t e d through an a o r t i c  escape r o u t e  t h e h e a r t was p e r f u s e d  pulmonary c o n n e c t i o n s ,  that  by t h e Langendorf method.  and t h e p e r f u s a t e must p a s s t h r o u g h t h e c o r o n a r y  because t h e only other Since  He d e m o n s t r a t e d  than sodium n i t r i t e  i n rabbit hearts perfused  t h i s preparation, coronary cannula  (1931).  circulation,  i s sealed by the a o r t i c  with Ringer's  valve.  s o l u t i o n and i s o l a t e d  with  t h e a c t i o n o f a d e n o s i n e c o u l d n o t have been  t o t h e r e l e a s e o f some f a c t o r f r o m t h e b l o o d  o r l u n g s , b u t must  have been a d i r e c t a c t i o n on t h e h e a r t ; The  possibility  t h a t d e r i v a t i v e s o f adenine might be i n v o l v e d  i n 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 blood (1932). blood  f l o w was r a i s e d b y R i g l e r  He s u g g e s t e d t h a t r e l e a s e o f a d e n i n e n u c l e o t i d e s c o u l d  f l o w i nmuscle t o match i t s requirements.  demonstrated that t h i s r e g u l a t i o n could occur adenine.  C l a r k e t a l . (1952) only with derivatives of  They e s t a b l i s h e d t h a t a d e n o s i n e a n d i t s 2 - s u b s t i t u t e d  ( 2 - a c e t a m i d o - , 2-amino-, 2 - m e t h y l ^ , 2-hydroxy-, and 2 - c h l o r o - ) a c t i v e as vasodepressors and  xanthosine  dilatory  i n t h e whole c a t , whereas guanosine,  were i n a c t i v e .  I n p u r s u i t of these  response of t h e coronary  regulate  concepts,  analogues were  inosine the vaso-  v a s c u l a t u r e o f o p e n - c h e s t dogs were  shown t o b e s e l e c t i v e f o r t h e a d e n i n e n u c l e o t i d e s a n d a d e n o s i n e  (Wolf  and Berne, 1956).  When ATP, ADP, AMP or adenosine was infused into the  coronary a r t e r i e s , a large increase i n coronary flow followed.  The dog  cardiac v e s s e l s , l i k e the general systemic supply of c a t s , were not responsive to non-adenine d e r i v a t i v e s such as hypoxanthine, i n o s i n e , IMP, IDP, guanine, guanosine, GDP, GTP, c y t i d i n e , CMP, CDP, CTP, u r a c i l , u r i d i n e UMP and UDP. vasodilation.  Both ITP and UTP were exceptions i n that they caused  Hence i f R i g l e r ' s suggestion i s confined to the heart, the  release of adenine d e r i v a t i v e s would be most l i k e l y to match blood flow with i t s metabolic demands. Berne, Blackmon and•Gardner (1957) demonstrated the importance of the metabolic s t a t e of the myocardium as a determinant of coronary blood flow. They perfused the coronary vasculature open-chest dogs a t high pressures so that a high coronary sinus pC^ was obtainable.  This technique combined  with c o n t r o l l e d deoxygenation of blood using N^, allowed them to i n v e s t i g a t e the e f f e c t s of blood oxygen content on coronary flow over a wide range of sinus pO^. As long as coronary sinus blood oxygen content was greater than 5.5,.ivolumes %, perfusion r a t e was not affected, by changes i n a r t e r i a l or venous blood oxygen content. When sinus blood oxygen content was l e s s than 5.5 volumes %, the perfusion r a t e was i n v e r s e l y p r o p o r t i o n a l to the oxygen content of coronary venous'blood.  Thus a l t e r a t i o n s i n blood oxygen content  alone were not s u f f i c i e n t to a l t e r vasotone; coronary r e s i s t a n c e was not a simple f u n c t i o n of blood oxygen content but was a f u n c t i o n of myocardial oxygen s u f f i c i e n c y .  The discrepancy between these data and those of  H i l t o n and E i c h h o l t z (1925) can be r e c o n c i l e d . These e a r l y workers were probably l i m i t e d to measurements i n j o n l y the oxygen dependent zone because the s p e c i a l high perfusion pressures necessary f o r demonstration of oxygen independence were not used.  D i s s o c i a t i o n of a d i r e c t l i n k  9.  between blood oxygen content and vascular tone, and establishment of the importance of the metabolic s t a t e l e d to attempts to demonstrate the release of v a s o d i l a t o r adenine d e r i v a t i v e s from the heart. I n i t i a l attempts to e x t r a c t adenosine or i t s nucleotides from perfusates of m e t a b o l i c a l l y d e b i l i t a t e d cat hearts were unsuccessful (Jacob and Berne, 1960).  However, other data of considerable importance  was obtained from these experiments.  Further evidence of a d i r e c t l i n k  between coronary blood flow and metabolic s t a t e were e l i c i t e d from experiments using Langendorf preparations of cat hearts.  When anoxia  was simulated by uncoupling o x i d a t i v e phosphorylation with d i n i t r o phenol, coronary flow increased by 25 to 50%.  Although adenine d e r i v a -  t i v e s were not found i n the perfusate, i n d i r e c t evidence f o r the release of adenosine was obtained because the quantity of degradation products of adenosine  (inosine and hypoxanthine)  increased up to 48-fold.  found i n the e f f l u e n t was  These workers also showed that adenosine, i n  the coronary vasculature, was extremely s h o r t - l i v e d and that adenosine 14 crossed c e l l membranes r e a d i l y .  When adenosine-8-C  was infused, 50%  of the r a d i o a c t i v i t y was trapped by the heart as adenine nucleotides. The other 50% was degraded to inosine and hypoxanthine.  Hence the  impaired metabolic s t a t e induced by d i n i t r o p h e n o l was thought to r e s u l t i n the formation of adenosine, which could then d i f f u s e out of the c e l l s . Recovery of adenosine per se could not be expected because of i t s rapid degradation. The suggestion that inosine and hypoxanthine, found i n coronary perfusate, o r i g i n a t e d from adenosine a f t e r i t s d i f f u s i o n from cardiac c e l l s was supported by the f i n d i n g s of Gerlach, Deuticke and Driesbach (1963).  The major metabolic pathway f o r 5'-AMP degradation i n r a t heart  10.  was demonstrated to involve dephosphorylation to adenosine followed by deamination to i n o s i n e . This i s i n contrast to s k e l e t a l muscle i n which the major route of 5'-AMP catabolism i s deamination to IMP (Imai, R i l e y and Berne, 1964). Incorporating the knowledge that adenosine: coronary v a s o d i l a t o r ;  ( i ) i s a potent  ( i i ) penetrates membranes r e a d i l y ;  produced i n preference to IMP i n heart; ;in the coronary c i r c u l a t i o n ;  (iii) is  ( i v ) i s deaminated r a p i d l y  and (v) i t s degradation products are  found i n the perfusate of d i n i t r o p h e n o l - t r e a t e d and hypoxic  dog-hearts,  an elegant theory f o r autoregulation of coronary blood flow has been proposed by Berne (1963).  He has suggested that i n response to  increased oxygen u t i l i z a t i o n or decreased oxygen d e l i v e r y , the oxygen tension of the myocardial c e l l would decrease;  .this decrease would  lead to a breakdown of adenine nucleotides to produce adenosine. Adenosine could then d i f f u s e through the i n t e r s t i t i a l f l u i d to r e s i s tance vessels to induce coronary d i l a t i o n and increase the blood and oxygen supply to the myocardium; t h i s would act as a feedback mechanism to reduce the production of adenosine as oxygen tension returned to an adequate l e v e l .  This has become known as the "adenosine hypothesis".  In support of t h i s hypothesis, the formation of adenosine has been demonstrated i n severely anoxic r a t hearts (Gerlach et al_. -, 1963). Respiring r a t hearts were placed i n a "moist" anoxic environment at 37° and analysed f o r nucleoside and nucleotide content a f t e r various incubation times.  Before i n i t i a t i o n of anoxia, n e i t h e r adenosine nor  i t s degradation products could be detected.  A f t e r 5 minutes of anoxia,  small but s i g n i f i c a n t amounts of adenosine, inosine and hypoxanthine were found.  A f t e r 60 minutes, considerable q u a n t i t i e s - o f these  substances  11.  were r e c o v e r e d w h i l e the ATP and ADP content of the h e a r t s greatly.  decreased  T h i s experiment has been repeated and i d e n t i c a l r e s u l t s were  obtained using r a b b i t hearts  (Imai, R i l e y and Berne, 1963).  these workers, e x t r a c t e d t r a c e s of hypoxanthine, from h y p o x i c  perfused hearts.  had been presented indirect  Moreover,  i n o s i n e and  To t h i s p o i n t , no d i r e c t  f o r the e x t r a c e l l u l a r presence  adenosine  evidence  o f adenosine,  evidence was p r o v i d e d by the experiments o f Klibler and  Bretschneider  (1964).  These s t u d i e s showed t h a t the coronary  d i l a t o r d i p y r i d a m o l c o u l d i n h i b i t the removal of adenosine  adenosine  i n h e a r t s , they suggested  p r e s e r v i n g adenosine.  I t would f o l l o w then t h a t the v a s o d i l a t i o n  of endogenous o r i g i n .  f i r s t d i r e c t demonstration  I f t h i s were the c a s e ,  of adenosine  of b l o o d .  (1964).  Langendorf p r e p a r a t i o n s of r a b b i t h e a r t s were t r e a t e d w i t h coumarin f o r 20 minutes to e f f e c t u n c o u p l i n g  no adenosine  was r e c o v e r e d  When bishydroxy-  of o x i d a t i o n from phosphory-  from the r e c i r c u l a t e d p e r f u s a t e .  However,  was d e t e c t e d i n the p e r f u s a t e of the same p r e p a r a t i o n a f t e r  f o r 20 minutes.  In the presence  deaminase i n h i b i t o r , adenosine uncoupled  adenosine  i n the e x t r a c e l l u l a r  f l u i d of h e a r t s was r e p o r t e d by Riehman and Wyborny  l a t i o n , 'adenosine  induced  (Kadatz, 1959) was due to  per se must have been p r e s e n t i n the e x t r a c e l l u l a r f l u i d The  administered  t h a t i t s mechanism of a c t i o n was by  by the a d m i n i s t r a t i o n of d i p y r i d a m o l a l o n e p r e s e r v i n g adenosine  vaso-  from b l o o d .  S i n c e d i p y r i d a m o l a l s o p o t e n t i a t e d the e f f e c t s of exogenously  hypoxia  although  and a n o x i c h e a r t s .  guinea p i g and c a t h e a r t s  of 8-azaguanine, an  adenosine  c o u l d be d e t e c t e d i n p e r f u s a t e s of both These o b s e r v a t i o n s were confirmed i n  ( K a t o r i and Berne, 1966).  Adenosine was  r e c o v e r e d from p e r f u s a t e s of Langendorf p r e p a r a t i o n s of these a f t e r a s i n g l e pass o f the p e r f u s a t e through  hearts  the v a s c u l a t u r e , which  12.  compares w e l l w i t h t h e r e c i r c u l a t i o n of adenosine  The  coronary  s i n u s b l o o d o f o p e n - c h e s t d o g s u n d e r much more ( R u b i o , B e r n e and  degradation of adenosine aid  of a s p e c i a l  has  o f R i c h m a n and  (1964).  conditions  presence  technique  s i n c e been d e t e c t e d  K a t o r i , 1969).  by a d e n o s i n e  sinus cannula.  "One  d e a m i n a s e , was  physiological  overcome w i t h  through  i t was  of  both d i l u t i o n and-cooling would e f f e c t i v e l y  adenosine b l o o d by  saline;  deaminase a c t i v i t y . c e n t r i f u g a t i o n and  resulting  fluid  By u s i n g t h i s  was  such  plasma p r o t e i n s were heat  concentrated  and  technique, adenosine  the a r t e r i a l preparation  was  s h o r t a s 30  calculated  t o be  (0.56  (Rubio  yM)'.  and  Berne, 1969).  because f o r adenosine  effect vasoconstriction.  asphyxia.  By  recovery  extra-  fluid  This.was g r e a t e r  As  increasing coronary  i n adenosine  from the p e r i c a r d i a l  fluid  dog-  significant  estimated  a t 1.09  determination f o r anoxic hearts  yM  (Rubio  blood  flow.  o r 55%  f o r the adenosine was  than  in.this  concentration which  f u r t h e r support  of  been e x t r a c t e d from  This i s p a r t i c u l a r l y  In normally oxygenated dog-hearts  c o n c e n t r a t i o n was  has  sinus  t o be a t r u e a u t o r e g u l a t o r , t h e r e s h o u l d be  allows f o r a decrease  adenosine  yM.  The  o x y g e n a t e d as w e l l as h y p o x i c  mechanism f o r r e d u c i n g as w e l l as finding  i n the coronary  i n the e x t r a c e l l u l a r 0.75  reduce  content.  t o 60 s e c o n d s .  In a d d i t i o n , adenosine  space of normally  volumes  denatured.  for nucleoside  detected  to  diluted  c o n c e n t r a t i o n r e q u i r e d f o r maximal v a s o d i l a t i o n  the p e r i c a r d i a l hearts  analysed  the c o n c e n t r a t i o n of adenosine  a n o x i c h e a r t s was  t o two  C e l l s were removed f r o m t h i s  b l o o d a f t e r p e r i o d s of a n o x i a as polation,  c o o l e d w i t h one  the  the cannula  the c o l l e c t i o n tube, ice-cold  and  i n the  of the g r e a t e s t problems,  As b l o o d p a s s e d  diluted  Wyborny  greater than  et a l . , 1969).  the  Values  This  could hypothesis,  i n c r e a s e d 70% by  the e x t r a c e l l u l a r  a  partial  adenosine previous obtained  by  13.  the l a t t e r procedure produce s u f f i c i e n t  might lead  adenosine  t o t h e c o n c l u s i o n t h a t normal  hearts  t o m a i n t a i n maximal v a s o d i l a t i o n a t a l l  times.  Olsson  cardium  o f dog, u s i n g a q u i c k - f r e e z e t e c h n i q u e combined w i t h a  assay which  (1970) d e t e c t e d a d e n o s i n e  exploited  rongeurs which  the s p e c i f i c i t y  had been c h i l l e d  samples o f v e n t r i c l e  i n n o r m a l l y oxygenated  of adenosine  deaminase.  i n l i q u i d n i t r o g e n were used  and f r e e z e them s i m u l t a n e o u s l y .  myosensitive Modified  to take  Extracts of these  s a m p l e s w e r e made a n d t h e a d e n o s i n e  c o n t e n t was d e t e r m i n e d .  was  enzymatically converted to u r i c  a c i d and t h e change i n absorbance  was  monitored  w i t h a dual wavelength,  When t h e c o r o n a r y adenosine  split-beam recording  supply to the v e n t r i c l e  c o n c e n t r a t i o n i n c r e a s e d about  Despite this  accepted.  hypothesis because l i d o f l a z i n e , sparing agent,  spectrophotometer.  was o c c l u d e d f o r 15  seconds,,  6-fold.  w e l l documented s u p p o r t , t h e a d e n o s i n e  not been u n i v e r s a l l y  Adenosine  F o r example, Afonso  hypothesis has  (1969) q u e s t i o n e d t h e  a c o r o n a r y v a s o d i l a t o r and a d e n o s i n e  p o t e n t i a t e d v a s o d i l a t i o n i n d u c e d by exogenous  but n o t by h y p o x i a i n dog h e a r t s i n s i t u .  He s u g g e s t e d  adenosine  that i f adenosine  were r e l e a s e d i n a p p r o p r i a t e amounts d u r i n g h y p o x i a , v a s o d i l a t i o n by  this  c o n d i t i o n should have been p o t e n t i a t e d by l i d o f l a z i n e .  induced  This  o b j e c t i o n s h o u l d be tempered b e c a u s e t h e e x a c t mechanism and s i t e o f action, of l i d o f l a z i n e  as c o r o n a r y v a s o d i l a t o r have n o t been  established.  K l l b l e r , Spieckermann and B r e t s c h n e i d e r (1970) have demonstrated the coronary v a s o d i l a t o r , d i p y r i d a m o l i n h i b i t e d of  adenosine  adenosine  by t h e c a n i n e myocardium.  were n e c e s s a r y  They s u g g e s t e d  and r e l e a s e  that i f release of  f o rr e g u l a t i o n of coronary flow, dipyridamol  should induce . v a s o c o n s t r i c t i o n by i n h i b i t i o n these cells.-  both uptake  that,  Hence t h e y c o n c l u d e d  of adenosine  r e l e a s e ,by  t h a t s u c h d a t a was n o t c o n s i s t e n t  14.  w i t h the adenosine hypothesis.  However, vasotone i s not l i k e l y a  f u n c t i o n of adenosine release per se but may be r e l a t e d to e x t r a c e l l u l a r adenosine concentration.  The quantity of adenosine i n the e x t r a c e l l u l a r  pool would be expected to be a f f e c t e d by movement of adenosine i n t o and away from i t .  Dipyridamol i n h i b i t s movement i n both d i r e c t i o n s (Kdbler  et: a l . , 1970).  I f i n h i b i t i o n of adenosine penetration into c e l l s were  i n h i b i t e d to a greater degree than flow out, an increase i n i n t e r s t i t i a l adenosine concentration would be p o s s i b l e i n the face of an i n h i b i t e d rate of release.  This p o s s i b i l i t y i s supported by the observation that  dipyridamol has only minimal v a s o d i l a t o r y properties i n hearts perfused with Tyrode's s o l u t i o n (Kadatz and Schroter, 1962) i n which one of the major sources of adenosine i n a c t i v a t i o n , the red blood c e l l s (Ktibler and Bretschneider, 1964) i s absent.  In summary, the adenosine hypothesis  has considerable data to support i t , but there are i n c o n s i s t e n c i e s w i t h i n t h i s data, and the questions posed by Afonso (1969) and Ktibler et a l . (1970) have helped to maintain channels of doubt. Although i t has not been unequivocally established whether adenosine mediates the r e g u l a t i o n of coronary flow,,the formation of adenosine i n the mammalian heart i s w e l l documented (Gerlach et_ a l . , 1963; Imai et a l . , 1964;  Richman and Wyborny, 1965;  Olsson, 1970).  Adenosine  i s produced by the dephosphorylation of 5'-AMP by 5'-nucleotidase according to the r e a c t i o n  5'-AMP  5'-nucleotidase •.—» adenosine + P  I t i s evident that a f u l l understanding of the properties of t h i s enzyme i n cardiac t i s s u e , e s p e c i a l l y with respect to i t s l o c a l i z a t i o n , k i n e t i c s and r e g u l a t i o n , could y i e l d important information regarding the p o s s i b l e  15.  r o l e of adenosine  i n coronary a u t o r e g u l a t i o n .  5 ' - N u c l e o t i d a s e i s not c o n f i n e d t o c a r d i a c t i s s u e , i t i s almost universally distributed  i n the l i v i n g w o r l d ;  5 ' - n u c l e o t i d a s e s have  been s t u d i e d from b a c t e r i a , y e a s t , p l a n t s and numerous t i s s u e s h i g h e r animals  ( f o r review see Drummond and Yamamoto, 1971).  from The  c h a r a c t e r i s t i c s of such enzymes a r e almost as v a r i e d as t h e i r s o u r c e s . For  example, t h e 5 ' - n u c l e o t i d a s e from b o v i n e p i t u i t a r y i s s o l u b l e  ( L i s o w s k i , 1966) w h i l e t h a t from E h r l i c h A s c i t e s - t u m o u r c e l l s brane bound  (Murray and F r i e d r i c h s , 1969).  h y d r o l y s i s of 5 ' - n u c l e o t i d e s are  quite different  a soluble 1967) of a K  The r e l a t i v e r a t e s of  by the enzyme from d i f f e r e n t  (Drummond and Yamamoto, 1971).  ( F r i t z s o n , 1969) and a membrane bound  these enzymes a r e s u b s t a n t i a l l y d i f f e r e n t .  optimum o f 6.2.  (Song and Bodansky, The p r o p e r t i e s  The s o l u b l e enzyme had  an a b s o l u t e requirement f o r Mg  and a pH  In c o n t r a s t , the membrane bound enzyme had a  (5'-AMP) o f 0.022 mM.and pH optima a t 7.5 and 9.3; activity  sources  In a d d i t i o n , both  5 ' - n u c l e o t i d a s e have been e x t r a c t e d from r a t l i v e r .  (5.'-AMP) o f 6.8 mM,  i s mem-  but was not e s s e n t i a l .  Mg  enhanced  From these d a t a , i t i s apparent  that  p r o p e r t i e s o f the enzyme s h o u l d not be e x t r a p o l a t e d from one source t o another and t h a t c a u t i o n should be e x e r c i s e d i n comparison pretation.  and i n t e r -  T h e r e f o r e , t h e involvement o f 5 ' - n u c l e o t i d a s e i n c o r o n a r y  a u t o r e g u l a t i o n must be d i s c u s s e d w i t h s p e c i f i c r e f e r e n c e to the enzyme from h e a r t . Fortunately, 5'-nucleotidase i s present i n cardiac t i s s u e s e v e r a l s p e c i e s i n q u a n t i t i e s q u i t e adequate Drummond and Duncan, 1966).  f o r examination  from  (Baer,  By t h e use of h i s t p c h e m i c a l and' e l e c t r o n  m i c r o s c o p i c t e c h n i q u e s , the enzyme from r a t h e a r t has been  demonstrated  16.  in and  the T-system, i n t e r c e l l u l a r Behnke, 1959).  spaces and e n d o t h e l i a l c e l l s  Baer e t a l . ,  (Rostgaard  (1966) r e p o r t e d t h a t 83% of t h e r a t  h e a r t enzyme was membrane b o u n d w h i l e 1 7 % was s o l u b l e ;  t h e membrane  b o u n d enzyme c o u l d b e s o l u b i l i z e d w i t h s o d i u m d e o x y c h o l a t e . • The (5'-AMP) o f t h e 5 ' - n u c l e o t i d a s e was 1.8 x 10 ~* M; b y ATP.  pH  f r o m membrane, a f t e r  o p t i m u m was 9.5 a n d i t was c o m p e t i t i v e l y i n h i b i t e d  Edwards and M a g u i r e  (1970) r e p o r t e d t h a t t h e K  1.65 x 10 ^ M, pH o p t i m u m 7.6 a n d ATP i n h i b i t e d non-competitive enzyme.  solubilization,  manner;  m  (5'-AMP) was  i n a mixed  competitive-  these d a t a were a l s o d e r i v e d from r a t h e a r t  I f c a r d i a c 5'-nucleotidase produces adenosine  for vasodilation,  a membrane b o u n d enzyme w o u l d a p p e a r m o s t s u i t a b l e f o r t h e e f f i c i e n t , disposal of this  function.' Formation  of adenosine  i n the v i c i n i t y of  t h e c e l l membrane w o u l d p r o v i d e f o r t h e s h o r t e s t d i s t a n c e o f d i f f u s i o n to the e f f e c t i v e  site;  i t w o u l d a l s o r e d u c e t o a minimum t h e p o s s i b i l i t y  of d e g r a d a t i o n by adenosine 1966).  Therefore,  adenosine  (Baer e t a l . ,  studies of 5'-nucleotidase, with reference to the  hypothesis, should  preference  deaminase o f t h e cytoplasm  i n v o l v e t h e membrane b o u n d enzyme i n  t o t h e s o l u b l e enzyme.  T h u s , a s t u d y o f membrane b o u n d , c a r d i a c 5 ' - n u c l e o t i d a s e may relevant  t o f u r t h e r i n g our knowledge of coronary  perfusion.  An u n d e r -  s t a n d i n g o f t h e mechanisms w h i c h ^ e f f e c t r e g u l a t i o n o f c o r o n a r y flow i s p e r t i n e n t to both  t h e d e s i g n and p r e s c r i p t i o n o f  agents used i n t r e a t i n g d i s e a s e s of coronary attempts  origin.  blood  pharmacological  This thesis  t o c o n t r i b u t e t o such a p o o l o f knowledge by e x a m i n a t i o n  levels of 5'-nucleotidase  be  describes of:  the  i n hearts of s e v e r a l species, the l o c a l i z a t i o n of  5'-nucleotidase  i n c a r d i a c t i s s u e a n d t h e p r o p e r t i e s o f membrane  5'-nucleotidase  isolated  from heart.  bound  17.  METHODS AND MATERIALS  A.  General 1.  Phosphate Assay  D e t e r m i n a t i o n s of i n o r g a n i c phosphate were made by a m o d i f i c a t i o n of the F i s k e and SubbaRow (1925) method as d e s c r i b e d below.  Acid-  molybdate reagent c o n t a i n e d 2.5% (NH^^MoO^ i n 5N H^SO^.  Fresh reducing  s o l u t i o n was made every 4 o r 5 days.  I t was prepared by d i s s o l v i n g 0.25 g  of powder c o n t a i n i n g 7.70% l - a m i n o - 2 - n a p h t h o l - 4 - s u l f o n i c a c i d , 46.15% Na2S0^ and 46.15% NaHSO^ i n 10 ml water. mixed w i t h 50 u l acid-molybdate, a f i n a l volume of 1.0 ml. minutes the absorbance photometer.  2.  An a l i q u o t to be assayed  was  20 u l r e d u c i n g s o l u t i o n and water i n  A f t e r i n c u b a t i o n a t room temperature  f o r 10  was measured a t 720 nm i n a Beckman DU s p e c t r o -  The l i g h t path was 1.0 cm.  P r o t e i n Determination  P r o t e i n c o n c e n t r a t i o n s of e x t r a c t s were e s t i m a t e d by the b i u r e t method except i n the case of the p a r t i a l l y p u r i f i e d 5 ' - n u c l e o t i d a s e , i n which the o p t i c a l assay was used  (Laynej 1957).  Ammonium i o n s were removed  from p r o t e i n p r e p a r a t i o n s by d i a l y s i s a g a i n s t d i s t i l l e d water when required.  B.  Survey of C a r d i a c Enzymes which U t i l i z e 5'-AMP and For a l l experiments  d e s c r i b e d i n t h i s s e c t i o n , enzymes were  prepared and assayed on the same day; c a r r i e d out a t 0-4°. immediately  Adenosine  p r e p a r a t i o n of enzymes was  In a l l c a s e s , h e a r t s were removed from  animals  a f t e r s a c r i f i c e , washed to remove b l o o d and used d i r e c t l y o r  s t o r e d a t -80° f o r enzyme p r e p a r a t i o n at a l a t e r  date.  1.  5'-Nucleotidase  F r e s h o r f r o z e n h e a r t s were trimmed o f n o n - v e n t r i c u l a r t i s s u e , minced and homogenized w i t h a P o t t e r - E l v e h j e m 50 mM 2-amino-2-methyl-l, 3 - p r o p a n e d i o l 2.8 mM M g C ^ and 0.15 M K C l .  homogenizer, i n 10 volumes o f b u f f e r , pH 9.0 c o n t a i n i n g  The homogenate was s t r a i n e d t h r o u g h  cheese c l o t h t o remove c o n n e c t i v e t i s s u e .  I n o r d e r t o remove endo-  genous o r t h o p h o s p h a t e , one volume o f s a t u r a t e d (NH^^SO^ s o l u t i o n was  added s l o w l y t o the- homogenate, w i t h c o n s t a n t  stirring.  The  m i x t u r e was c e n t r i f u g e d a t 30,000 x g f o r 10 m i n u t e s , t h e s u p e r n a t a n t d i s c a r d e d and the w a l l s o f t h e c e n t r i f u g e tube c a r e f u l l y r i n s e d w i t h the above b u f f e r .  The p e l l e t was d i s p e r s e d i n b u f f e r , the pH was  a d j u s t e d t o 9.0 and the volume was a d j u s t e d t o t h a t o f t h e o r i g i n a l homogenate. For t h e assay:, 20 y l o f 0.10 M 5'-AMP was added t o 230 y l o f . t h e t i s s u e suspension, 37°  and t h i s m i x t u r e was i n c u b a t e d f o r 10 minutes a t  i n a water b a t h w i t h m e c h a n i c a l s h a k i n g .  C o n t r o l s were c a r r i e d  out i n w h i c h 5'-AMP was r e p l a c e d w i t h 3'-AMP, 3 g l y c e r o p h o s p h a t e o r -  water.  A l l a s s a y s were performed i n d u p l i c a t e .  The r e a c t i o n was  stopped by a d d i t i o n o f 1.0 ml of' i c e - c o l d 3% t r i c h l o r o a c e t i c a c i d ; a f t e r c e n t r i f u g a t i o n t o remove denatured p r o t e i n , a l i q u o t s ' o f t h e supernatant expressed wet 2.  f l u i d were assayed f o r i n o r g a n i c phosphate.  Activity i s  as ymoles per minute per mg p r o t e i n o r p e r gram o f t i s s u e  weight. Adenylate  Deaminase  F r e s h o r f r o z e n h e a r t s were trimmed of. n o n - v e n t r i c u l a r t i s s u e , minced and homogenized, w i t h a P o t t e r - E l v e h j e m 0.10  homogenizer i n 10 volumes o f  M p o t a s s i u m phosphate b u f f e r pH 7.5, c o n t a i n i n g 0.15 M K C l .  The  19.  h o m o g e n a t e was c e n t r i f u g e d a t 3 0 , 0 0 0 x g f o r 20 m i n u t e s s u p e r n a t a n t was k e p t f o r a s s a y . 20  The i n c u b a t i o n m i x t u r e c o n t a i n e d  p i o f 0.10 M 5'-AMP, a n a p p r o p r i a t e amount o f enzyme s o l u t i o n a n d  homogenizing proceeded 250  buffer  i n a f i n a l v o l u m e o f 250 y l . The r e a c t i o n  a t 37° f o r 10 m i n u t e s  a n d was s t o p p e d b y t h e a d d i t i o n o f  y lo f 10% p e r c h l o r i c a c i d .  A control  t u b e was p r e p a r e d  i d e n t i c a l m a n n e r , e x c e p t t h a t t h e enzyme was a d d e d a f t e r acid.  F o l l o w i n g removal  a l i q u o t s o f each of  and t h e c l e a r  1.0 m l .  o f denatured  spectrophotometer.  perchloric  p r o t e i n by c e n t r i f u g a t i o n ,  s u p e r n a t a n t were d i l u t e d w i t h b u f f e r  The a b s o r b a n c e  i n an  20 V I  to a final  volume  a t 265 nm was m e a s u r e d i n a U n i c a m SP 8 2 5  The d e c r e a s e  i n absorbance  of the experimental  t u b e s a t t h i s wave l e n g t h c o m p a r e d w i t h t h e c o n t r o l was u s e d t o c a l c u l a t e t h e amount o f s u b s t r a t e d e a m i n a t e d the assay r e s u l t s from t h e f a c t of  t o IMP.  The v a l i d i t y o f  that themolecular e x t i n c t i o n  IMP i s 4 0 % o f t h a t o f 5'-AMP u n d e r t h e s e c o n d i t i o n s (Ae 265  3 8.1  x 10 ) .  due  t o d e p h o s p h o r y l a t i o n o f 5'-AMP b y a p h o s p h a t e  of  The p o s s i b i l i t y  disclosed  3.  of an a r t i f a c t u a l  decrease  a d e n o s i n e w a s e x c l u d e d , b e c a u s e no p h o s p h a t a s e  Chromatographic  in  coefficient  a n a l y s i s .(paper;  nm  i n absorbance  f o l l o w e d by deamination c o u l d be d e t e c t e d .  i s o p r o p a n o l : NH^OH: w a t e r ,  7:1:2)  t h a t o n l y 5'-AMP a n d i t s d e a m i n a t i o n p r o d u c t , I M P , w e r e p r e s e n t  t h e r e a c t i o n m i x t u r e a t t h e end o f i n c u b a t i o n . Adenylate  Kinase  E x t r a c t s o f v e n t r i c l e were p r e p a r e d as f o r a d e n y l a t e deaminase d e t e r minations, except pH  that  t h e homogenizing  7.0, c o n t a i n i n g 1 0 mM M g C l  2  b u f f e r was 0.1 M T r i s - H C l ,  a n d 0.10 M KC1.  The 30,000 x g s u p e r -  n a t a n t w a s a s s a y e d u s i n g ADP a s s u b s t r a t e a n d 5'-AMP f o r m e d by c o u p l i n g w i t h a n e x c e s s o f p u r i f i e d  adenylic  deaminase.  was  deaminated  20.  The r e a c t i o n s a r e : Adenylate Kinase 2 ADP  ^  *  ATP + 5'-AMP  Excess A d e n y l a t e Deaminase 5'-AMP » 5'-IMP The assay was performed  i n spectrophotometric c e l l s  ( l i g h t path = 0.5 cm)  which c o n t a i n e d 20 y l of 10 mM ADP, 20 y l o f a d e n y l i c deaminase s o l u t i o n (75 yg p r o t e i n ) , an a p p r o p r i a t e amount of 30,000 x g supernatant and homogenizing  b u f f e r i n a f i n a l volume of 1.5 ml.  added was s u f f i c i e n t these c o n d i t i o n s .  The a d e n y l i c deaminase  to deaminate 1.5 ymoles o f 5'-AMP p e r minute under  A b l a n k was prepared i n t h e same manner as t h e  e x p e r i m e n t a l , except ADP was o m i t t e d . a d d i t i o n of h e a r t e x t r a c t  The r e a c t i o n was s t a r t e d by  ( d i l u t e d w i t h b u f f e r as r e q u i r e d ) and t h e  r a t e of d e c r e a s e i n absorbance  a t 265 nm was determined  u s i n g a Unicam  SP 825 r e c o r d i n g spectrophotometer w i t h a 1.0 A a t t e n u a t o r (to a l l o w use o f h i g h s u b s t r a t e c o n c e n t r a t i o n s ) . ' Assays were conducted Activity  at 30°.  i s expressed as i n i t i a l v e l o c i t y i n ymoles ADP u t i l i z e d p e r  minute p e r mg p r o t e i n o r per gram of t i s s u e wet weight.  4.  Adenosine  Adenosine  Deaminase  deaminase assays were c a r r i e d out on e x t r a c t s prepared f o r  a d e n y l a t e k i n a s e assay. cells  (light  Reaction mixtures, i n spectrophotometric  path = 0.5 cm), c o n t a i n e d 50 y l of 10 mM adenosine, 5 o r  10 y l o f h e a r t e x t r a c t , and 0.10 M c i t r a t e 0.10  M K C l i n a f i n a l volume o f 1.5 m l .  b u f f e r pH 6.5, c o n t a i n i n g  The b l a n k c o n t a i n e d a l l  components of t h e assay except adenosine.  The r e a c t i o n r a t e was  determined  a t 265 nm as f o r a d e n y l a t e  from t h e decrease i n absorbance  21.  deaminase.  Assays were conducted  a t 30  C.  Activity  i s expressed  as i n i t i a l v e l o c i t y i n umoles per minute p e r mg p r o t e i n .  C.  P e r f u s i o n o f Rat, Rabbit and T u r t l e  Hearts  Hearts were removed as q u i c k l y as p o s s i b l e a f t e r s a c r i f i c e and placed i n perfusion f l u i d .  A f t e r extraneous  c o n n e c t i v e t i s s u e was  trimmed away, h e a r t s were c a n n u l a t e d and p e r f u s e d by the Langendorf technique.  -Rat and r a b b i t h e a r t s were c a n n u l a t e d by the a o r t a and  p e r f u s e d w i t h Krebs-Ringer c o n t a i n e d 118.5 mM NaCl,  bicarbonate s o l u t i o n a t 37°.  This  2.75 mM KC1, 2.54 mM C a C l , 118 mM 2  solution  KH P0 , 2  4  1.18 mM MgSO^, 24.9 mM NaHC0 , 1.8 g/1 g l u c o s e and was a e r a t e d w i t h 3  95% 0  2  5 % C 0 . " T u r t l e h e a r t s were c a n n u l a t e d 2  through  (Robb, 1965) and were p e r f u s e d w i t h Mine's s o l u t i o n and  B a r b o r i a k , 1965) a t room temperature.  101 mM NaCl,  3  2  2  were connected  was 50 cm water.  to a f o r c e t r a n s d u c e r  Hardman  2.0 mM C a C l , 3.0 mM  KC1, 1.0 g/1 g l u c o s e and was a e r a t e d w i t h 100% 0 . p r e s s u r e i n a l l experiments  (Meester,  Mine's s o l u t i o n c o n t a i n e d  5.0 mM H B 0 , 3.9 mM N a ^ H . ^ , 3  the r i g h t a o r t a  The p e r f u s i o n  The a p i c e s of the h e a r t s  (Grass FT 03) which was coupled t o  a s t r i p c h a r t r e c o r d e r (Grass 5D).  The d i a s t o l i c t e n s i o n was 1 g.  r a t e s were determined  t h e p e r f u s a t e f o r s e v e r a l 1 minute  by c o l l e c t i n g  Flow  periods.  Rates o f coronary p e r f u s i o n , i n the absence and presence of  adenosine,  were r e c o r d e d o n l y a f t e r an e q u i l i b r a t i o n p e r i o d ;  hearts  were c o n s i d e r e d to be e q u i l i b r a t e d when b o t h f l o w r a t e and c o n t r a c t i l e f o r c e had reached  a steady s t a t e .  C o n t r o l f l o w r a t e s were r e c o r d e d w h i l e  h e a r t s were b e i n g p e r f u s e d w i t h the s o l u t i o n s d e s c r i b e d above; mental r a t e s were r e c o r d e d w h i l e h e a r t s were b e i n g p e r f u s e d w i t h c o n t a i n i n g the d e s i r e d c o n c e n t r a t i o n o f adenosine. p e r f u s i o n a r e expressed  as p e r c e n t o f the c o n t r o l .  experisolutions  Rates o f c o r o n a r y  22.  Histochemistry The l o c a l i z a t i o n o f 5 ' - n u c l e o t i d a s e of W a c h s t e i n and M e i s e l a t t h e enzyme is  (1957) i n w h i c h l i b e r a t e d p h o s p h a t e i s t r a p p e d  s i t e by p r e c i p i t a t i o n as the lead s a l t .  subsequently  made v i s i b l e b y c o n v e r s i o n  as d a r k brown o r b l a c k d e p o s i t s Fresh  was d e t e r m i n e d b y t h e m e t h o d  deposition  t o the s u l f i d e which  appears  i nthe tissue section.  t i s s u e was m o u n t e d o n c r y o s t a t m i c r o t o m e p l a n c h e t t e s  c o m m e r c i a l m o u n t i n g medium (Ames) a n d q u i c k f r o z e n . were c u t , mounted on c o v e r r a t i o n s were i n c u b a t e d  s l i p s and a l l o w e d  with  Sections, lOy thick,  to a i r dry.  These prepa-  f o r 4 5 ^ m i n u t e s a t 37° i n a medium c o n t a i n i n g  4 m l o f 0.2 M T r i s b u f f e r a d j u s t e d  t o pH 7.2 w i t h m a l e i c  0.10 Mg S 0 , 0.6 m l o f 2% l e a d n i t r a t e , 4  a c i d , 1 ml of  0.4 m l H 0 a n d 4 m l o f 2.5 mM  5'-AMP a s t h e n e u t r a l i z e d p o t a s s i u m s a l t . treated  Lead  2  Control preparations  i n e x a c t l y t h e same m a n n e r , e x c e p t 5'-AMP was r e p l a c e d  3'-AMP ( 2 . 5 mM) o r g - g l y c e r o p h o s p h a t e  (4 mM) o r w a t e r .  were with  The r e a c t i o n  was s t o p p e d b y r e m o v i n g t h e s e c t i o n s f r o m t h e i n c u b a t i o n medium a n d placing  them i n 1 0 % f o r m b l  s a l i n e ( 4 % f o r m a l d e h y d e i n 0.9% s a l i n e ) .  A f t e r thorough washing i n w a t e r , s e c t i o n s were p l a c e d for  1 minute.  Following  i ndilute  thorough washing i nwater, t h e cover  (NH^^S  slips  with  s e c t i o n s were mounted on m i c r o s c o p e s l i d e s w i t h g l y c e r i n e j e l l y and sealed with n a i l  lacquer.  Assay o f P a r t i a l l y P u r i f i e d In order  5'-Nucleotidase  to determine the properties of 5'-nucleotidase, i t s  a c t i v i t y was m e a s u r e d u n d e r a b r o a d r a n g e o f c o n d i t i o n s w h i c h a r e described these  i ndetail  i nconjunction with results.  To a c c o m m o d a t e a l l  c o n d i t i o n s , two d i f f e r e n t a s s a y p r o c e d u r e s w e r e n e c e s s a r y .  ' At  23.  substrate was  (5'-AMP) c o n c e n t r a t i o n s o f 0.16 mM o r h i g h e r , the a c t i v i t y  determined by the e s t i m a t i o n o f l i b e r a t e d phosphate  previously.  In g e n e r a l , the assay m i x t u r e s c o n t a i n e d b u f f e r  5 ' - n u c l e o t i d a s e , s u b s t r a t e and i n some cases s t i m u l a t o r s inhibitors. minutes,  solution,  and/or  A f t e r i n c u b a t i o n w i t h mechanical shaking a t 37° f o r 10  t h e r e a c t i o n s were stopped by the a d d i t i o n o f i c e - c o l d  t r i c h l o r o a c e t i c a c i d which denatured was  as d e s c r i b e d  the enzyme;  The denatured  protein  removed by c e n t r i f u g a t i o n and an a l i q u o t o f t h e supernatant was  assayed f o r phosphate of orthophosphate  content.  Enzyme a c t i v i t y i s expressed as ymoles  l i b e r a t e d per minute.  the s e n s i t i v i t y of the phosphate  assay.  T h i s method was l i m i t e d by Hence, a t 5'-AMP c o n c e n t r a t i o n s  l e s s than 0.16 mM, 5 ' - n u c l e o t i d a s e was assayed by a m o r e . s e n s i t i v e , d i r e c t o p t i c a l method.  T h i s method depends on the d e g r a d a t i o n o f newly  formed adenosine t o i n o s i n e by excess adenosine deaminase and measurement of t h e r e s u l t i n g change i n absorbance  a t 265 nm.. I n c u b a t i o n s were  c a r r i e d out i n s p e c t r o p h o t o m e t r i c c e l l s m a i n t a i n e d a t 37° by a thermos t a t i c a l l y c o n t r o l l e d water j a c k e t .  The assay m i x t u r e s c o n t a i n e d b u f f e r  s o l u t i o n , 5 ' - n u c l e o t i d a s e , 5'-AMP, excess adenosine deaminase and i n some experiments,  s t i m u l a t o r s and/or  inhibitors.  The r e a c t i o n s a r e :  5'-nucleotidase 5'-AMP  •—}  adenosine  excess adenosine deaminase adenosine  •—••  •  ->  inosine  3 At 265 nm, the m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t of adenosine i s 8.1 x 10 / cm g r e a t e r than t h a t o f i n o s i n e .  T h e r e f o r e , the r e a c t i o n r a t e was d e t e r -  mined from the decrease i n absorbance w i t h a Unicam SP 825 spectrophotometer  a t t h i s wavelength and r e c o r d e r .  which was r e c o r d e d  T h i s assay was  24.  l i m i t e d by the low s i g n a l to noise r a t i o at high substrate concentration.  Materials Sources of materials and t i s s u e s are l i s t e d below: Calbiochem, Los Angeles:  IMP, c r e a t i n e phosphate, glucose-1-phosphate,  fructose-l-phosphate, fructose-1, 6-diphosphate, UMP, sodium deoxycholate and adenosine deaminase (calf i n t e s t i n a l mucosa). Sigma Chemical Co., St. Louis:  5'-AMP, 3'-AMP, 3-glycerophosphate,  ADP, ATP, glucose-6-phosphate, dUMP, dCMP, dGMP and adenosine. N u t r i t i o n a l Biochemical Co., Cleveland: ribose-5-phosphate, r i b u l o s e 5-phosphate, and galactose-6-phosphate. Schwarz Bipresearch Inc., Mt. Vernon: Ames Co., E l k h a r t :  CMP.  Tissue-tek mounting medium.  U n i v e r s i t y of B r i t i s h Columbia Animal S i t e :  Swiss albino mice, Wistar  r a t s , albino r a b b i t s and mongrel dogs. College B i o l o g i c a l Supplies, S e a t t l e :  turtles.  Pigeons were obtained l o c a l l y . Human p a p i l l a r y muscle was obtained as biopsy m a t e r i a l though the courtesy of Dr. Peter A l l a n , Vancouver General H o s p i t a l . Substrates were obtained as sodium or potassium s a l t s or were converted to the potassium s a l t p r i o r to use. reagent grade from various sources.  A l l other materials were  25.  RESULTS  Preliminary One method f o r d e t e r m i n a t i o n of 5 ' - n u c l e o t i d a s e a c t i v i t y depends on the e s t i m a t i o n of i n o r g a n i c phosphate l i b e r a t e d from added n u c l e o tide.  F i g . 1 shows the standard curve f o r d e t e r m i n a t i o n of o r t h o -  phosphate by a m o d i f i c a t i o n of the F i s k e and Absorbance a t 720 nm 0.30  umoles.  SubbaRow (1925) method.  i s a l i n e a r f u n c t i o n of orthophosphate  T h i s standard curve was  1.0  cm,  phosphate;  an absorbance of 0.380 r e p r e s e n t e d 0.1 t h i s v a l u e was  used  to  repeated s e v e r a l times over a  2 % year p e r i o d , and a l l results...were i d e n t i c a l . was  up  When the l i g h t  path  umole of o r t h o -  i n the c a l c u l a t i o n of r a t e s .  The v a l i d i t y of the 5 ' - n u c l e o t i d a s e assay depends on more than the r e p r o d u c i b i l i t y of i n o r g a n i c phosphate d e t e r m i n a t i o n . of orthophosphate  l i b e r a t e d i n the enzymatic  t i o n a l to time of i n c u b a t i o n and  The q u a n t i t y  r e a c t i o n must be  enzyme c o n t e n t .  This l i n e a r  s h i p i s d e p i c t e d i n F i g . 2, i n which enzyme a c t i v i t y was  proporrelation-  determined  a c c o r d i n g to c o n d i t i o n s • d e s c r i b e d f o r the survey experiments  (Methods,  s e c t i o n B. 1 ) .  experiments  S i m i l a r r e s u l t s were o b t a i n e d from analogous  w i t h the p a r t i a l l y p u r i f i e d enzyme;  i n t h i s case l i n e a r i t y was  observed  w e l l beyond the time and amount of p r o t e i n used r e g u l a r l y i n a s s a y s .  JUMOLES Figure 1.  ORTHOPHOSPHATE  Standard curve f o r assay of inorganic phosphate. Graph of absorbance at ,720 nm versus ymoles of orthophosphate  Mg PROTEIN (A) 0.8 1.2 1.6  LU  I— <  o_ to o Q_ O IE I— C£  o  cn LU  O  3  6 8 MINUTES (O) F i g u r e 2.. L i n e a r i t y o f 5 ' ^ n u c l e o t i d a s e  assay w i t h time and p r o t e i n .  The enzyme p r e p a r a t i o n used was crude homogenate of r a t v e n t r i c l e . Experiments t o show l i n e a r i t y w i t h t i m e (O) were conducted u s i n g 1.5 mg o f p r o t e i n ; t h o s e t o show l i n e a r i t y w i t h p r o t e i n ( A ) were conducted u s i n g 10 minute i n c u b a t i o n p e r i o d .  26.  B.  C a r d i a c Enzymes which U t i l i z e 5'-AMP and 1.  5'-Nucleotidase On  the b a s i s of the adenosine  5 ' - n u c l e o t i d a s e might be expected might a l s o be expected the s p e c i e s .  of animals  h y p o t h e s i s , the l e v e l s of c a r d i a c  to v a r y from s p e c i e s to s p e c i e s and  to be a f u n c t i o n of t h e ^ p h y s i c a l a c t i v i t y of  I f the adenosine  range of animals,  and  Adenosine  hypothesis  one might expect  i s a p p l i c a b l e to a wide  a c o r r e l a t i o n between the  ability  to impose l a r g e l o a d s and oxygen demands on t h e i r  the a b i l i t y of t h e i r h e a r t s to produce adenosine.  hearts  Hence, l e v e l s  of 5 ' - n u c l e o t i d a s e i n h e a r t s might be r e l a t e d to the p o t e n t i a l n e c e s s i t y f o r , and magnitude o f , coronary v a s o d i l a t i o n . was  The  t u r t l e , f o r example,  a n t i c i p a t e d to have low l e v e l s of the c a r d i a c enzyme, s i n c e i t can  function anaerobically f o r long periods. 17 hours i n t o t a l a n o x i a  ( B e l k i n , 1963)  l a r g e l o a d s oh t h e i r h e a r t s . expected  T u r t l e s s u r v i v e f o r up and  a p p a r e n t l y do not  place  On the o t h e r hand, b i r d s and mammals were  to have r e l a t i v e l y h i g h l e v e l s of c a r d i a c 5 ' - n u c l e o t i d a s e ,  which would a l l o w r a p i d p r o d u c t i o n of adenosine. then be equipped  These animals  to accommodate h i g h c a r d i a c demands imposed  increased a c t i v i t y . running  The  (Taylor, Schmidt-Nielsen  and Raab, 1970), and and  consumption of pigeons  times  (LeFebvre,  1964).  by  during f l i g h t  i s 8.5  similar  results  Shepherd, 1967). the r e s t i n g  I f energy u t i l i z a t i o n and oxygen d e l i v e r y are  mechanism f o r the f o r m a t i o n of adenosine.  h y p o t h e s i s might p r e d i c t a h i g h e r l e v e l of c a r d i a c  t i d a s e i n pigeons  than i n r a t s .  Energy  values  of the amount of blood pumped by the h e a r t , both r a t and pigeon a w e l l developed  would  r a t i n c r e a s e s oxygen consumption 3 . 5 - f o l d by  were o b t a i n e d from human s u b j e c t s (Bevegard  adenosine  to  indicative should have  In f a c t j  the  5'-nucleo-  27.  The l e v e l s o f 5 ' - n u c l e o t i d a s e  found  i n homogenates o f h e a r t s  f r o m v a r i o u s s p e c i e s a r e shown i n F i g . 3. A s s a y s w e r e c a r r i e d pH 9.0 t o i n a c t i v a t e a d e n y l a t e s u b s t r a t e , 5'^AMP ( L e e , 1 9 5 7 ) . activity  i s expressed  wet w e i g h t  deaminase w h i c h c o u l d compete f o r t h e Comparison o f values  T h e r e w e r e m a r k e d d i f f e r e n c e s i n enzyme  i n hearts of thevarious species studied;  activity  f o l l o w e d b y d o g , mouse a n d r a b b i t .  Homogenates  The 5 ' - n u c l e o t i d a s e  than 10% of r a t  o f t u r t l e v e n t r i c l e c o n t a i n e d no d e t e c t a b l e  5 ' - n u c l e o t i d a s e a c t i v i t y under these c o n d i t i o n s . a n o n - s p e c i f i c p h o s p h a t a s e was f o u n d , was  the specific  But i n t h i s activity  0.0054 u m o l e s p e r m i n u t e p e r mg p r o t e i n when e i t h e r  or B-glycerophosphate the assay,  pigeon  activity  r a t hearts demonstrated t h e  o f r a b b i t v e n t r i c l e h o m o g e n a t e s was l e s s  ventricle.  i s s i m i l a r whether  a s u m o l e s p e r m i n u t e p e r mg p r o t e i n o r p e r g r a m  of tissue.  highest a c t i v i t y  out a t  was u s e d a s s u b s t r a t e .  tissue,  of which  3'-AMP, 5'-AMP  Under t h e c o n d i t i o n s o f  v e n t r i c l e h o m o g e n a t e s c o n t a i n e d no d e t e c t a b l e  5'-nucleotidase o r n o n - s p e c i f i c phosphatase  activity.  <  \  o  0.02 -  O  o  0.01 -  \ \ \ \ \ \  \ \ \ \ \ \  \ \ \  s Figure 3.  2.0  CO CO  o o  Q  H CO ZD  \ \ \ \ \  LJJ  I  o  o  \ \ \ \  1.0 g '  BIT  0.03  CO <  o UJ  O  \  Survey of v e n t r i c u l a r 5"-nucleotidase. Bars depict 5'-nucleotidase a c t i v i t y of crude homogenates of hearts of various species. Enzyme a c t i v i t y i s represented on the basis of p r o t e i n (open bars) and t i s s u e wet weight (hatched b a r s ) . Lines represent standard e r r o r of the mean. Values f o r mouse v e n t r i c l e were obtained from 12 pooled hearts.  2.  A d e n y l a t e Deaminase and A d e n y l a t e  Kinase  The  adenosine  s u b s t r a t e , 5'-AMP, f r o m w h i c h  a c t i o n o f 5 ' - n u c l e o t i d a s e , can a l s o be u t i l i z e d  i s formed by t h e  by o t h e r enzymes.  A d e n y l a t e d e a m i n a s e c a t a l y z e s t h e c o n v e r s i o n o f 5'-AMP t o IMP. ate kinase c a t a l y z e s a r e v e r s i b l e r e a c t i o n which o f 5'-AMP a n d o n e m o l e c u l e  relative activities  c o n v e r t s one m o l e c u l e  o f ATP t o two m o l e c u l e s  t h e n t h a t t h e f a t e o f 5'-AMP i n c e l l s  Adenyl-  o f ADP.  c o u l d be d e t e r m i n e d  I t follows  by t h e  o f t h e s e two enzymes a s w e l l a s 5 ' - n u c l e o t i d a s e .  For t h i s r e a s o n , b o t h a d e n y l a t e deaminase and a d e n y l a t e k i n a s e were estimated  i n h e a r t s o f t h e above s p e c i e s .  from h e a r t a r e s o l u b l e assays were performed  (Lee, 1957a;  detected.  Adenylate  supernatants of various species a r e  I n a l l c a s e s , e x c e p t d o g h e a r t s , enzyme a c t i v i t y w a s  Turtle v e n t r i c l e contained a t least  f i v e t i m e s a s much  a d e n y l a t e deaminase as t h a t from r a t , r a b b i t o r pigeon. this  enzyme i n t h i s  s k e l e t a l muscle,  which  i s shown i n F i g . 4 f o r c o m p a r a t i v e  s p e c i e s i s g i v e n i n F i g . 5.  i nventricular  kinase  highest  (Fig. 5).  activity  W h i l e t u r t l e v e n t r i c l e was  t o s k e l e t a l muscle w i t h respect t o adenylate  ( F i g . 4 ) , i t was l e a s t  purposes.  supernatants of various  Pigeon v e n t r i c l e possessed  f o l l o w e d by r a b b i t , d o g , r a t and t u r t l e .  activity  Activity of  t i s s u e was c o n s i d e r a b l y l e s s t h a n t h a t o f r a b b i t  Adenylate kinase a c t i v i t y  most s i m i l a r  enzymes  C o l o w i c k and K a l c k a r , 1943),  u s i n g 30,000 x g s u p e r n a t a n t s .  deaminase l e v e l s o f v e n t r i c u l a r shown i n F i g . 4.  Since both these  deaminase  similar with respect to adenylate  Figure 4.  Survey of v e n t r i c u l a r adenylate deaminase. Adenylate deaminase was estimated i n the 30,000 x g supernatant of heart homogenates. Enzyme a c t i v i t y i s represented on the basis of p r o t e i n (open bars) and t i s s u e wet weight (hatched b a r s ) .  Figure 5.  Survey of v e n t r i c u l a r adenylate kinase. Adenylate kinase of the 30,000 x g supernatant of v e n t r i c u l a r homogenates. Enzyme a c t i v i t y i s represented on the basis of p r o t e i n (open bars) and t i s s u e wet weight (hatched b a r s ) . Lines represent standard error of the mean.  29.  3.  Adenosine Deaminase I f adenosine  mediates coronary  a u t o r e g u l a t i o n , v a s o t o n e must  a f u n c t i o n of e x t r a c e l l u l a r adenosine  c o n c e n t r a t i o n i n the h e a r t .  c o n c e n t r a t i o n m u s t be a f u n c t i o n o f a d e n o s i n e d e a m i n a s e as w e l l as f o r m a t i o n by e x t r a c e l l u l a r adenosine values  the content adenosine  5 -nucleotidase.  of adenosine  The  s i g n i f i c a n t adenosine (5'-nucleotidase  H e n c e i t was  the  high  t h a t the r a t e of  d e s i r a b l e to  estimate  deaminase i n h e a r t s of v a r i o u s s p e c i e s .  deaminase a c t i v i t i e s  between s p e c i e s .  adenosine,  slow.  This  adenosine  For example,  1  c o n c e n t r a t i o n of h e a r t s could a t t a i n  sufficiently  turtle hearts i s l i s t e d  ments  d e g r a d a t i o n by  i n s p i t e of low r a t e s of f o r m a t i o n p r o v i d e d  d e g r a d a t i o n was  detected  i n Table  1.  be  i n r a t , r a b b i t , dog,  The  pigeon  Only minor d i f f e r e n c e s were  and  found  m o s t n o t a b l e o b s e r v a t i o n was  the d e t e c t i o n of  deaminase i n pigeon  In previous e x p e r i -  survey), pigeon  hearts.  h e a r t s appeared unable  i . e . they lacked 5'-nucleotidase.  t h a t they s h o u l d have a w e l l developed  I t was  to  form  surprising therefore,  means t o d e g r a d e t h i s  nucleoside.  29(a)  Adenosine Deaminase from Hearts of Various Species  ymoles/min/mg P r o t e i n 3 XIO  s.e.m. 3 XIO  number  Rat  9.00  0.27  10  Rabbit  3.38  0.95  3  Dog  8.28  Pigeon  4.03  0.31  6  Turtle  2.92  0.41  4  2  TABLE I . Survey of v e n t r i c u l a r adenosine deaminase. Adenosine deaminase of the 30,000 x g supernatants of homogenates of v e n t r i c l e s from various species i s represented on the basis o f . p r o t e i n . Assays were conducted by the d i r e c t o p t i c a l method. Standard error of the mean was c a l c u l a t e d i f more than two hearts were sampled.  30.  C.  E f f e c t o f A d e n o s i n e on Coronary S i n c e homogenates o f p i g e o n  Flow  and t u r t l e v e n t r i c l e c o n t a i n e d  d e t e c t a b l e 5 ' - n u c l e o t i d a s e , i t seemed p o s s i b l e t h a t t h e s e were i n c a p a b l e of adenosine  formation.  determine  could mediate coronary  hearts.  whether adenosine  Some p r e l i m i n a r y e x p e r i m e n t s  e f f e c t s of adenosine turtle,  on c o r o n a r y  r a b b i t and r a t s .  Pigeon  I t was o f i n t e r e s t  successfully  from s a c r i f i c e to  using Langendorf i n F i g . 6.  significant.  preparations  Adenosine  f l o w and i n c r e a s e d r a b b i t  The e f f e c t o f a d e n o s i n e  r a t e was n o t s t a t i s t i c a l l y  d i l a t i o n i n these  h e a r t s c o u l d n o t be  o f r a t and t u r t l e h e a r t s a r e r e p r e s e n t e d  f l o w 5 4 % a t 3 yM.  then to  flow i n i s o l a t e d perfused hearts of  The r e s u l t s o f e x p e r i m e n t s  a l a r g e increase i n r a t coronary  tissues  were performed to examine t h e  perfused because they d i d not s u r v i v e the i n t e r v a l cannulation.  no  induced  coronary  on t u r t l e c o r o n a r y  perfusion  40  RAT  A  ~  -  30 <  20  —  10  O <  o  0  s y \  *  b  o  •  TURTLE  V\  \  -10  i  i  2 F i g u r e 6.  E f f e c t of adenosine  •  1  1  2 6 8 ADENOSINE (juM) on coronary  1  10  flow.  Experiments were conducted u s i n g Langendorf p r e p a r a t i o n s of r a t ( A ) and t u r t l e (O) h e a r t s . Changes i n p e r f u s i o n r a t e a r e expressed as percent of c o n t r o l r a t e determined immediately b e f p r e e x p e r i m e n t a l . Verticle lines depict standard e r r o r of the mean where 4 o r more v a l u e s were obtained.  31.  Histochemical.Localization of Cardiac 5'-Nucleotidase According to the adenosine hypothesis o f coronary adenosine  i s formed  interstitial  fluid  i n c a r d i a c muscle (Rubio and Berne,  c e l l s and then d i f f u s e s 1969).  d i f f u s e to coronary resistance vessels, (Provenza and S c h e r l i s , 1959), oxygen d e l i v e r y machinery cells.  into the  The n u c l e o s i d e w o u l d  such as p r e c a p i l l a r y  then  sphincters  i n d u c e v a s o d i l a t i o n and t h u s i n c r e a s e  t o t h e myocardium.  f o radenosine  autoregulation,  I f this  i s s o , the enzymatic  p r o d u c t i o n s h o u l d be found  i n heart  Most o f t h e c a r d i a c 5 ' - n u c l e o t i d a s e i s a s s o c i a t e c i  l a t e f r a c t i o n s o f h e a r t homogenates  (Baer e t a l . ,  muscle with  particu-  1966), hence i t w o u l d /  b e r e a s o n a b l e t o e x p e c t t h i s enzyme t o b e l o c a t e d o n o r i n membranes o f heart muscle of adenosine. would  result  cells.  T h i s l o c a t i o n would  Formation of adenosine  (Baer e t a l . ,  phosphate  a t s i t e s elsewhere  i n these  efflux cells  i n slower e f f l u x because of g r e a t e r d i f f u s i o n d i s t a n c e s ;  a d d i t i o n adenosine might plasm  be most s u i t e d f o r r a p i d  be degraded  1966).  by adenosine  deaminase o f t h e c y t o -  Some h i s t o c h e m i c a l e v i d e n c e o f i n o r g a n i c  l i b e r a t i o n f r o m 5'-AMP b y c a r d i a c m u s c l e  been r e p o r t e d (Rostgaard and Behnke, 1959; R p s t g a a r d and Behnke  i n  (1959) found  T-system' and e n d o t h e l i a l c e l l s .  c e l l s have a l r e a d y  Sommer a n d S p a c h ,  this activity These workers  1964).  i n intercellular  spaces,  c a r r i e d out examinations of 1  t i s s u e s e c t i o n s by e l e c t r o n m i c r o s c o p y and u s i n g p r e f i x e d  r a thearts. , In  t h e p r e s e n t s t u d i e s , the; s i t e o f 5 ' - n u c l e o t i d a s e i n h e a r t s was d e t e r m i n e d by t h e method o f W a c h s t e i n a r e employed.  and M e i s e l (1957) i n w h i c h u n f i x e d s e c t i o n s  These u n f i x e d , f r o z e n s e c t i o n s were chosen  generally accepted  because i ti s  t h a t s u c h p r e p a r a t i o n s p r o v i d e t h e b e s t enzyme p r e s e r -  v a t i o n and s h o u l d be most s u i t a b l e f o r such s t u d i e s  (Pearse, 1960).  In  t h i s method, t h e l o c a l e o f 5 ' - n u c l e o t i d a s e i s c h a r a c t e r i z e d by b l a c k o r d a r k brown d e p o s i t s o f l e a d  sulfide.  32.  U s i n g t h i s method, s e c t i o n s o f a l l mammalian h e a r t s especially  those o f r a t , contained  incubation with H, a n d J ) . rather  lead deposition  after  5'-AMP a s t h e s u b s t r a t e • ( F i g . 7, p l a t e s A, B, C, D, G,  That  t h i s d e p o s i t i o n was d u e t o s p e c i f i c  than n o n - s p e c i f i c phosphatases  :  sections  incubated  which represents  5'-nucleotidase  i s a p p a r e n t upon c o m p a r i s o n o f  i n t h e p r e s e n c e o f 5'-AMP t o t h o s e i n c u b a t e d  3'-AMP o r 3 - g l y c e r o p h o s p h a t e .  with  considerable  examined,  F o r example,  with  c o m p a r e F i g . 7 p l a t e D,  a s e c t i o n o f human p a p i l l a r y m u s c l e a f t e r  5'-AMP, t o p l a t e E f o r w h i c h 3'-AMP was u s e d .  Since  incubation the only  d i f f e r e n c e i n t h e s e e x p e r i m e n t s was t h e p o s i t i o n o f t h e p h o s p h a t e moiety of adenylatej represent  t h e d a r k brown and b l a c k  theaction of specific  presence of s p e c i f i c which represents  a r e a s o f p l a t e D must  5'-nucleotidase.  5'-nucleotidase  i s s e e n o n c o m p a r i s o n o f p l a t e G,  a section of guinea p i g heart  t o p l a t e H, w h i c h i s r e p r e s e n t a t i v e 3-glycerophosphate, In contrast  (1957).  w i t h 5'-AMP, incubated  ester.  a f t e r t r e a t m e n t by t h e method o f  T h e r e was a c o m p l e t e a b s e n c e  d e p o s i t i o n when e i t h e r 5'-AMP, 3'-AMP, 3 - g l y c e r o p h o s p h a t e e s t e r was p r e s e n t i n t h e i n c u b a t i o n medium ( p l a t e K ) . in thehistochemical whether  t e s t showed f a i n t l e a d  the presence of non-specific  f r o m d i r e c t .enzyme a s s a y  5'-nucleotidase  pattern  o r no p h o s p h a t e  Turtle ventricle  ester  phosphatase  occurred  (plate L ) ,  as had been  ascertained  (Fig. 3 ) . In fact, the i n t e n s i t y of lead  s i t i o n i nv e n t r i c u l a r sections the  of lead  deposition but this  5'-AMP, o r 3'-AMP was t h e a d d e d p h o s p h a t e  indicating  with  sections, pigeon v e n t r i c l e sections  c o n t a i n e d , no l e a d d e p o s i t i o n w h a t s o e v e r W a c h s t e i n and M e i s e l  incubated  of s i m i l a r sections  3'-AMP o r no p h o s p h a t e  t o mammalian h e a r t  S i m i l a r l y , the  from t h e v a r i o u s  species  appeared  a c t i v i t y determined by t e s t tube assay  of lead deposition  i n mammalian h e a r t  sections  depo-  to parallel  ( F i g . 3 ) . The  revealed  t h a t 5'-  33.  nucleotidase the  was d i s c r e t e l y l o c a l i z e d  entire ventricle.  trated  i n s p e c i f i c regions  The u n i f o r m i t y o f t h i s d i s t r i b u t i o n i s i l l u s -  i n F i g . 7, p l a t e A w h i c h i s a l o w m a g n i f i c a t i o n  of r a t v e n t r i c l e s e c t i o n incubated was d e p o s i t e d restricted  with  5'-AMP.  t h r o u g h o u t t h e s e c t i o n and t h a t  to d i s t i n c t  areas.  no e v i d e n c e o f a c t i v i t y c o u l d  heart  endothelial cells.  this deposition  eosin  was  magnification,  (plate B).  Virtually a l l  s e c t i o n s was f o u n d i n s t r u c t u r e s  s u c h s t r u c t u r e s a p p e a r e d t o be  Evidence that  stained  (plate F).  nuclei containing  the regions  identical  of lead deposition i n  f o r n u c l e i w i t h h e m a t o x y l i n and  regions  of routinely stained  t h i s method, most o f t h e c a r d i a c  endothelial cells  counterstained  sections, which  of c a p i l l a r i e s .  represented  Thus i t a p p e a r s  This  p l a t e s C, D, G a n d I , r e s p e c t i v e l y . c l e a r l y v i s i b l e , b u t no d e p o s i t s  d i s t r i b u t i o n was o b s e r v e d i n d o g ,  confined  I n each case, heart  o f l e a d c a n be d e t e c t e d  i n  muscle c e l l s a r e within  them;  were found i n d i s c r e t e areas between muscle c e l l s .  contrast, lead deposition  that,  5 ' - n u c l e o t i d a s e was l o c a l i z e d i n  human, g u i n e a p i g a n d mouse v e n t r i c l e s e c t i o n s w h i c h a r e r e p r e s e n t e d  lead deposits  from  A r e a s h a v i n g 5 ' - n u c l e o t i d a s e were i d e n t i c a l t o  endothelial c e l l s of the coronary m i c r o c i r c u l a t i o n . by  lead  s e c t i o n s w e r e i n d e e d o c c u p i e d b y e n d o t h e l i a l c e l l s was p r o v i d e d  adjacent sections with  I t i s clear that  be o b s e r v e d i n c a r d i a c m u s c l e c e l l s o r i n  5'-nucleotidase a c t i v i t y of r a t heart which surround muscle c e l l s ;  photomicrograph  Upon e x a m i n a t i o n a t g r e a t e r  t h e p l a s m a membrane o f c a r d i a c m u s c l e c e l l s  with  throughout  In  i n ' t u r t l e v e n t r i c l e , a l t h o u g h f a i n t , was n o t  t o e n d o t h e l i a l c e l l s b u t appeared t o be g e n e r a l l y d i s t r i b u t e d  in a l l cells.  The p a t t e r n o f l e a d d e p o s i t i o n d e m o n s t r a t e d i n t h e s e  p h o t o g r a p h s was h i g h l y r e p r o d u c i b l e ; i n d u p l i c a t e from each species  three  always w i t h  to s i x hearts  were  identical results. .  sampled  34.  Although by  no  5'-nucleotidase  t h e h i s t o c h e m i c a l method u s e d , o t h e r  t h e enzyme was  detectable  Behnke  who  (1959),  Sommer a n d  Spach  the  and  triads  This  ADP,  intercallated  involved;  indicative  AMP,  not  be  By  similar  t h e W a c h s t e i n and  Hardpnk  (1968)  (1957) t e c h n i q u e  was  used.  was  not  observation  I t i s possible that this an a r t i f a c t  When T r i s - H C l and  of  used.  This  near the  T r i s - H C l was  o f l e a d on  superficial  p r e s e n c e o f T r i s - H C l , made i t i m p o s s i b l e of  activity surface  5'-nucleotidase Hardonk  b u f f e r s were compared i n  far superior.  random a g g r e g a t i o n  s e c t i o n s when i t was  sections;  t h e T r i s - H C l b u f f e r u s e d by  Tris-maleate  t h e l a t t e r was  reported  Whether  of  the  the  inferior  surfaces of t i s s u e  lead precipitation,  to r e l i a b l y  ATP,  which i s  i n muscle c e l l s of r a t heart  or l o c a l i z e d  b e c a u s e t h e r e was  hypothesis  ^-glycerophosphate  extended throughout the muscle c e l l s  study,  hearts.  demonstrated i n the presence of  p y r o p h o s p h a t e and  Meisel  i n m u s c l e c e l l s was  at  Behnke (1959) b e c a u s e a n o n - s p e c i f i c phospha-  was  present  techniques,  as p e r t i n e n t to t h e a d e n o s i n e  a c t i v i t y was  thiamine  and  e l e c t r o n microscopy,  d i s c s i n s e c t i o n s o f p r e f i x e d dog  s m a l l amounts of 5 ' - n u c l e o t i d a s e  (1968).  that  p r e v i o u s l y mentioned, Rostgaard  of a n o n - s p e c i f i c phosphatase.  clear.  cells  (1964) d e m o n s t r a t e d t h e p r e s e n c e o f a p h o s p h a t a s e  t h a t o f R o s t g a a r d and  IDP,  As  w i t h i n muscle  l a b o r a t o r i e s have r e p o r t e d  i n the T-system of r a t h e a r t .  i n f o r m a t i o n may  t a s e was  there.  detected  u s e d enzyme h i s t o c h e m i s t r y and  observed a c t i v i t y  as  c o u l d be  identify  the  in  the  site  5'-nucleotidase. Sections  contained  from guinea  pig ventricle  and  human p a p i l l a r y  muscle  i n t e n s e l e a d d e p o s i t s w i t h i n the w a l l s of l a r g e r blood  i n a d d i t i o n to those  deposits  i n endothelial cells.  F i g . 7, p l a t e J i s  a low m a g n i f i c a t i o n photomicrograph of a s e c t i o n from guinea showing s u b s t a n t i a l 5 ' - n u c l e o t i d a s e  activity  vessels,  pig  i n the c e n t r e of the  heart field.  35.  Examination of such vessels at higher magnification revealed that lead deposition was l a r g e l y confined to the intima and media; had only s l i g h t a c t i v i t y .  the adventia  Figure  7.  Histochemical sections.  l o c a l i z a t i n of 5 -nucleotidase 1  i n ventricle  The enzyme i s l o c a t e d i n t h e d a r k b r o w n o r b l a c k lead deposition.  P l a t e A.  areas of  Rat v e n t r i c l e ;  substrate  5'-AMP  M a g n i f i c a t i o n 90X, showing g e n e r a l i z e d h i g h a c t i v i t y between muscle c e l l s .  P l a t e B.  Rat ventricle; 5'-AMP.  substrate  M a g n i f i c a t i o n 900X, s h o w i n g l o c a l i z a t i o n of 5'-nucleotidase i n c a p i l l a r y endothelium.  P l a t e C.  Dog v e n t r i c l e ; s u b s t r a t e  5'-AMP  M a g n i f i c a t i o n 900X, showing r e a c t i o n product l i m i t e d to c a p i l l a r y endothelium between muscle c e l l s .  P l a t e D.  Human p a p i l l a r y m u s c l e ; s u b s t r a t e 5'-AMP.  L o n g i t u d i n a l l y sectioned endothelial c e l l s e x h i b i t high 5'-nucleotidase activity. M a g n i f i c a t i o n 900X.  P l a t e E.  Human p a p i l l a r y muscle; c o n t r o l u s i n g 3'-AMP.  M a g n i f i c a t i o n 360X. I d e n t i c a l r e s u l t s were o b t a i n e d w i t h 3-glycerophosphate o r w a t e r .  P l a t e F.  Human p a p i l l a r y m u s c l e ; hemat o x y l i n and e o s i n .  M a g n i f i c a t i o n 360X. Note t h e v a s c u l a r i z a t i o n adjacent to myocardial c e l l s i n v o l v i n g s t r u c t u r e s which c o n t a i n clearly v i s i b l e n u c l e i , providing evidence that the s t r u c t u r e s c o n t a i n i n g lead deposits are e n d o t h e l i a l c e l l s .  P l a t e G.  Guinea p i g v e n t r i c l e ; substrate 5'-AMP.  M a g n i f i c a t i o n 900X.  P l a t e H.  Guinea p i g v e n t r i c l e ; control.  Water replaced 5'-AMP i n the incubation medium. Analogous r e s u l t s were obtained with human, r a t , dog and mouse v e n t r i c l e sections when water, g-glycerophosphate or 3'-AMP replaced 5'-AMP i n the incubation medium. M a g n i f i c a t i o n 360X.  P l a t e I . Mouse v e n t r i c l e ; 5'-AMP.  substrate  5'-Nucleotidase a c t i v i t y l o c a l i z e d i n areas between myocardial c e l l s . Magnif i c a t i o n 360X.  Plate J .  Guinea p i g v e n t r i c l e ; 5'-AMP.  substrate  M a g n i f i c a t i o n 90X, showing 5'-nucleot i d a s e located i n l a r g e blood v e s s e l s i n a d d i t i o n to c a p i l l a r i e s .  P l a t e K.  Pigeon v e n t r i c l e ; 5'-AMP.  substrate  M a g n i f i c a t i o n 360X. Note absence of lead d e p o s i t i o n ; r e s u l t s c o r r e l a t e with l a c k of 5'-nucleotidase measured by biochemical assay.  P l a t e L. T u r t l e v e n t r i c l e ; substrate 5'-AMP. M a g n i f i c a t i o n 90X. D i f f u s e lead d e p o s i t i o n obtained with 3'-AMP as w e l l as 5'-AMP i n d i c a t i n g n o n - s p e c i f i c phosphatase a c t i v i t y .  36.  P a r t i a l P u r i f i c a t i o n of Considerable has  interest  5 -Nucleotidase 1  i n the p r o p e r t i e s of cardiac  a r i s e n r e c e n t l y because of the p o s s i b l e involvement  r e g u l a t i o n of coronary cardiac  blood  t i s s u e was u n d e r t a k e n  flow.  Astudy  5'-nucleotidase of adenosine  of 5'-nucleotidase  i n t h i s l a b o r a t o r y i n an attempt  more a b o u t t h e f o r m a t i o n o f a d e n o s i n e  i n heart.  p r o g r e s s , p a p e r s by Edwards and M a g u i r e  While  in  from to learn  t h i s w o r k was i n  (1970) and S u l l i v a n and A l p e r s  (1971) were p u b l i s h e d i n w h i c h s e v e r a l c h a r a c t e r i s t i c s o f c a r d i a c 5 ' - n u c l e o t i d a s e were d e s c r i b e d . 5'-Nucleotidase  h a s b e e n r e p o r t e d i n t h e s o l u b l e a n d membrane-  containing, particulate 1966).  I n order  r o l e of adenosine  f r a c t i o n s o f r a t h e a r t homogenates  t h a t t h e s e s t u d i e s m i g h t be r e l e v a n t t o t h e p o s s i b l e i n coronary  a u t o r e g u l a t i o n , examination  b o u n d 5 ' - n u c l e o t i d a s e was deemed more p e r t i n e n t t h a n s o l u b l e enzyme. they contained majority  (Baer e t a l . ,  examination  Rat h e a r t s were chosen as t h e source the highest l e v e l of 5'-nucleotidase  o f t h e membrane  o f enzyme,  Some p r o p e r t i e s o f t h e enzyme f r o m t h i s  been e l u c i d a t e d (Baer e t a l . ,  1966).  Before  because  ( F i g . 3) a n d t h e  ( 8 3 % ) o f enzyme f r o m t h e s e h e a r t s was membrane b o u n d  a l . , 1,966) .  of the  source  ( B a e r eit  have a l r e a d y  a n e x t e n s i v e s t u d y o f 5'-  n u c l e o t i d a s e c o u l d be c a r r i e d o u t , i t s p u r i f i c a t i o n was n e c e s s a r y . particular,  i t was e s s e n t i a l t o r e m o v e a l l enzymes w h i c h c o u l d  w i t h the b a s i c assays of these  In  interfere  f o r 5 ' - n u c l e o t i d a s e and t h e s u b s e q u e n t m o d i f i c a t i o n s  assays.  I n a t y p i c a l p r e p a r a t i o n , 10-15 f r o z e n , washed r a t h e a r t s were trimmed o f n o n - v e n t r i c u l a r t i s s u e and minced.  Minced v e n t r i c l e s  h o m o g e n i z e d i n 10 v o l u m e s o f a c e t o n e a t - 1 5 ° .  The h o m o g e n a t e  c e n t r i f u g e d a t 3 0 , 0 0 0 x g f o r 10 m i n u t e s a t - 1 5 ° ;  the  were  was  supernatant  37.  acetone was  discarded.  volume of acetone.  The p e l l e t was  A f t e r the t h i r d  e x t r a c t e d twice w i t h the same  e x t r a c t i o n , the p e l l e t was  dried  under vacuum, p r o v i d e d by water aspirator., f o r 1 hour, broken up a powder and d r i e d under o i l pump vacuum f o r 3 hours. was  s t o r e d a t -80° u n t i l Procedures  f o r e x t r a c t i o n of 5 ' - n u c l e o t i d a s e from the Acetone powder (1.5 g) was  T r i s - H C l b u f f e r , pH 7.5  a smooth p a s t e was  obtained.  added and  The p a s t e was  The p e l l e t was  T r i s - H C l b u f f e r pH 7.5  T r i s - H C l b u f f e r pH The  until  and c e n t r i f u g e d a t 30,000 x g f o r 10  w i t h 50 ml of 50 mM  T r i s - H C l b u f f e r , pH 7.5,  was  was  e x t r a c t e d w i t h a f u r t h e r 50 ml of 50  d i s c a r d e d . - The p e l l e t was  7.5  suspension  the supernatant  The supernatant was  c e n t r i f u g a t i o n was  mixed w i t h  t r a n s f e r r e d to a g l a s s  the mixture homogenized t h o r o u g h l y .  c e n t r i f u g e d a t 30,000 x g f o r 10 minutes and discarded.  acetone  i n a c h i l l e d g l a s s mortar  P o t t e r - E l v e h j e m homogenizer, 40 ml of 50 mM was  T h i s powder  used.  powder were c a r r i e d out a t 4 ° . 10 ml of 50 mM  into  mM  minutes.  then e x t r a c t e d 9 times  c o n t a i n i n g 2 M KBr.  The  a t 30,000. x g f o r 10 minutes and a l l s u p e r n a t a n t s  were d i s c a r d e d . . The KBr remaining i n the p e l l e t was  removed by  extracting  the p e l l e t  as above.  The  was  twice w i t h 50 ml of 50 mM  then homogenized i n 20 ml of-50 mM  deoxycholate. for  10 minutes.  The p e l l e t was at  T r i s - H C l pH 7.5  T h i s was  T r i s - H C l , pH 7.5  pellet  c o n t a i n i n g 1%  Na  s t i r r e d f o r 3 hours and c e n t r i f u g e d a t 30,000 x g  The supernatant which c o n t a i n e d the enzyme was e x t r a c t e d w i t h 3 ml of 50 mM  30,000 x g f o r 10 minutes and  saved.  T r i s - H C l pH 7.5, c e n t r i f u g e d  t h i s r e s u l t i n g p e l l e t was d i s c a r d e d .  The s u p e r n a t a n t s were pooled f o r immediate use or c o u l d be s t o r e d a t -20° without l o s s of a c t i v i t y .  38.  The respect was  deoxycholate  e x t r a c t was b r o u g h t t o 1 5 % s a t u r a t i o n w i t h  t o (NH)^SO^ b y a d d i t i o n o f a s a t u r a t e d  solution.  The  mixture  c e n t r i f u g e d a t 30,000 x g f o r 10 m i n u t e s a n d t h e p r e c i p i t a t e was  discarded.  The r e s u l t i n g  supernatant  was d i a l y z e d a g a i n s t  30 v o l u m e s  o f 50 mM T r i s - H C l pH 7.2 f o r 3 h o u r s w i t h  2 changes o f d i a l y s i s  For  t o remove  e x p e r i m e n t s i n w h i c h i t was n e c e s s a r y  cation, to  the supernatant  buffer.  endogenous. divalent ;  was b r o u g h t t o 10 mM EDTA f o r 10 m i n u t e s  prior  dialysis. T h i s p u r i f i c a t i o n scheme i s s u m m a r i z e d i n F i g . 8 a n d t h e y i e l d s  from a t y p i c a l fied  preparation are l i s t e d  i n T a b l e 2.  1 6 - f o l d f r o m t h e a c e t o n e powder w i t h a y i e l d  y i e l d was p r o b a b l y  o f 107%.  This  purihigh  due t o s o l u b i l i z a t i o n w i t h spdium d e o x y c h o l a t e ,  may h a v e e n h a n c e d t h e a v a i l a b i l i t y final  The enzyme was  of substrate to the active site.  enzyme p r e p a r a t i o n was o b t a i n e d  a s s a y p r o c e d u r e s and p e r m i t t e d  which  i n s o l u b l e form, which  The  facilitated  the use of d i r e c t o p t i c a l assays.  This  f i n a l p r e p a r a t i o n was u s e d f o r enzyme c h a r a c t e r i z a t i o n s t u d i e s .  I t was  sufficiently  deaminase,  adenylate  f r e e of ATPase, n o n - s p e c i f i c phosphatase, adenylate  k i n a s e and a d e n o s i n e deaminase t h a t i n t e r f e r e n c e w i t h  t i d a s e assays were Further  5'-nucleo-  obviated.  efforts  to purify  t h e enzyme, i . e . t o o b t a i n a h i g h e r  v i t y p e r mg o f p r o t e i n ( s p e c i f i c a c t i v i t y ) , w e r e u n s u c c e s s f u l . and  v a r i o u s forms o f c e l l u l o s e  and  S e p h a d e x (DEAE-, QAE-) w e r e u s e d i n a t t e m p t s t o p u r i f y  acti-  Dowex 1  (DEAE-, CM-, p h o s p h o - , P A B - , E C T E 0 L A - )  t i d a s e on t h e b a s i s o f m o l e c u l a r  charge.  5'-nucleo-  S e p h a r o s e 6-B a n d u l t r a -  f i l t r a t i o n w e r e u s e d i n a t t e m p t s t o p u r i f y t h e enzyme o n t h e b a s i s o f molecular  size.  precipitation. specific activity.  Other techniques A l l these  t r i e d were e t h a n o l  and i s o e l e c t r i c  methods were abandoned because t h e i n c r e a s e o f  a c t i v i t y was n o t s i g n i f i c a n t o r t h e r e was e x c e s s i v e  loss of  38(a)  Specific Activity ymole/min/mg P r o t e i n  1.  2.  Acetone Powder  0.0093  Sodium Deoxycholate Extract  0.085  1 5 % Ammonium Sulfate Supernatant  0.149  T a b l e 2.  Total  Protein mg  Total Activity ymole/min  1,190  11.1  200  17.1  79.8  11.9  Summary o f P u r i f i c a t i o n . Y i e l d and i n c r e a s e o f s p e c i f i c a c t i v i t y o b t a i n e d during a t y p i c a l preparation of 5'-nucleotidase s t a r t i n g f r o m t h e a c e t o n e p o w d e r . The a s s a y s were conducted by d e t e r m i n a t i o n o f o r t h o p h o s p h a t e liberated. The i n c u b a t i o n m i x t u r e s c o n t a i n e d e n z y m e , 5 mM 5'-AMP, 16 mM M g C l 2 a n d 50 mM T r i s H C l b u f f e r , pH 7.2 i n a f i n a l v o l u m e o f 1.0 m l . F u r t h e r p u r i f i c a t i o n by c h r o m a t o g r a p h i c methods was u n s u c c e s s f u l .  Summary of 5'-nucleotidase  Figure 8.  purification  Ventricles a.  Acetone E x t r a c t i o n Acetone Soluble m a t e r i a l discarded Powder  b.  Aqueous E x t r a c t i o n -> Supernatant discarded Pellet  c.  2 M KBr E x t r a c t i o n Supernatant discarded N1/  Pellet d.  Sodium deoxycholate extraction •> P e l l e t discarded Supernatant  e.  15% ( N H ) S 0 4  2  4  -> P e l l e t discarded  Supernatant, f i n a l enzyme preparation  39.  P r o p e r t i e s of C a r d i a c If  adenosine  i n response  mediates v a s o d i l a t i o n of the coronary  to h y p o x i a ,  this condition.  5'-Nucleotidase  One  5 ' - n u c l e o t i d a s e may  i t s r a t e o f f o r m a t i o n m u s t be  might expect, be  i n h i b i t e d by  oxygenated h e a r t s but  s t i m u l a t e d by  t h e c o n c e n t r a t i o n o f ATP  substances  i s a potent  inhibitor  tissue.  (Gerlach et a l . ,  of 5'-nucleotidase  1963;  the f o r m a t i o n of adenosine.  5 -nucleotidase  c o u l d be  1  r e g u l a t e d by  c h a n g e when t h e o x y g e n s u p p l y  1964;  p h o s p h a t e may  Williamson,  (Baer e t a l . ,  o t h e r m e t a b o l i t e s whose  be  of the p r o p e r t i e s of  For (Imai, by  were designed  e m p h a s i s on p o t e n t i a l p h y s i o l o g i c a l  D e p e n d e n c e on 5'-Nucleotidase  5'-nucleotidase  r e g u l a t e d c o u l d c o n t r i b u t e to  to i n v e s t i g a t e these  10.  levels  so s t i m u l a t i o n o f t h e enzyme  f o l l o w i n g experiments  5 t o pH  may  increases during hypoxia  i n coronary  pH  1966).  1966),  hearts  e v a l u a t i o n of the r o l e of adenosine  1.  oxygenated  c  t h e w a y s i n w h i c h i t may  particular  example,  a p p r o p r i a t e f o r the c o r r e c t i o n of t h i s c o n d i t i o n .  Obviously, a precise understanding and  For  adequately  I t i s also quite possible that  content  W i l l i a m s o n , 1 9 6 6 ) , and be  in  t o t h e h e a r t becomes i n a d e q u a t e .  example, c a r d i a c orthophosphate et a l . ,  accu-  5'-nucleo-  which are present  therefore a reduction i n i t s concentration i n hypoxic facilitate  which  Conversely,  i n hypoxic  of  I t follows  i s substantially greater i n well  hearts than i n anoxic hearts ATP  substances  are d e f i c i e n t  under  t h e r e f o r e , that the a c t i v i t y  mmulate i n oxygen d e f i c i e n t c a r d i a c t i s s u e . t i d a s e m i g h t be  enhanced  enhanced d u r i n g oxygen d e f i c i t .  t h a t 5 ' - n u c l e o t i d a s e m i g h t be  vasculature  the  autoregulation.  The  features with  significance.  pH d e p e n d e n c e o n pH was  determined  A c o m b i n e d T r i s - m a l e a t e b u f f e r was  over  the range of  u s e d so t h a t  the  40.  v a r i o u s pH v a l u e s c o u l d be a c c o m m o d a t e d w i t h a minimum o f d i f f e r e n c e f r o m one o f HC1  o r KOH  mixture per no  assay  to the next.  Hence t h e o n l y a l t e r a t i o n s w e r e a d d i t i o n s  w h i c h were used to a d j u s t a l l a d d i t i o n s to the i n c u b a t i o n  t o t h e a p p r o p r i a t e pH v a l u e s .  s e . a p p e a r e d t o have any  Neither K  e f f e c t on  the assay;  e f f e c t on 5 ' - n u c l e o t i d a s e a c t i v i t y .  u m o l e s p e r m i n u t e p e r mg  protein after  The  the experimental, except  up  t o 0.15  activity  ions M KC1  had  i s expressed  as  s u b t r a c t i o n of n o n - s p e c i f i c  phosphatase c o n t r o l v a l u e s which were o b t a i n e d to  i o n s nor CI  +  i n a manner  that B-glycerophosphate  identical  (10 mM)  replaced  5'-AMP.. The is  pH p r o f i l e  shown i n F i g . 9.  p o s s e s s i n g 85% T h i s was found  The  enzyme was  range of  7 and  t o t h e r e s u l t s o f Edwards and M a g u i r e  6 t o 8.8.  o r m o r e o f maximum a c t i v i t y  10.  (1970),  who  5 0 % o r more o f m a x i m a l  Maguire  f r o m pH  6 to 10.5.  pH,  pH  activity  B a e r e t a l . ( 1 9 6 6 ) r e p o r t e d t h a t t h e enzyme  o p t i m u m i s shown t o be 8.5,  et  5'-nucleotidase  o r m o r e o f m a x i m a l a c t i v i t y b e t w e e n pH  similar  by Edwards and  rat heart  a c t i v e over a broad  t h a t r a t h e a r t enzyme m a i n t a i n e d  f r o m pH 50%  of p a r t i a l l y p u r i f i e d  possessed  In F i g . 9 the  w h i c h i s h i g h e r t h a n t h e v a l u e , 7.6,  ( 1 9 7 0 ) and  lower  t h a n t h a t o f 9.5  pH reported  r e p o r t e d by  Baer  i s quite different  from  a l . (1966) The. pH. p r o f i l e o f r a t heart.; 5 ' - n u c l e o t i d a s e  t h a t o f t h e enzyme i s o l a t e d M i t s u i and maximally over  Tsushima  from s e v e r a l other  (1967) found  a c t i v e a t pH  6.5  and  intestinal  smooth m u s c l e  one  and  a t 5.5  another  (Burger  a t 9.3.  For  example, I t o h ,  t h a t t h e enzyme f r o m c h i c k e n l i v e r  possessed  a n a r r o w r a n g e o f o n l y 1.5  sources.  pH u n i t s . and  50% o r more of m a x i m a l 5'-Nucleotidase  Lowenstein,  Similarly  1970)  had  was  activity  from p i g two  t h e membrane b o u n d  pH  optima,  5'-nucleo-  41.  t i d a s e from r a t l i v e r  (Song and Bodansky,  1967) had pH optima a t 7.5  and 9.3 The d i s c r e p a n c y between t h e d a t a r e p r e s e n t e d i n F i g . 9 and t h a t of Edwards and Maguire  (1970) and Baer et^ al_.  (1966) may be due to  d i f f e r e n c e s i n i s o l a t i o n procedure o r assay c o n d i t i o n s . I |  m i x t u r e s of Edwards and Maguire p r e s e n t assay employed  The assay  (1970) c o n t a i n e d no Mg , w h i l e the I j | | 10 mM Mg and Baer e t a l . (1966) used 8 mM Mg  The h i g h a c t i v i t y r e p o r t e d by Baer ejt al_. a t pH v a l u e s above 8.5 may have been c o n t r i b u t e d to by n o n - s p e c i f i c phosphatases.  Examination o f  F i g . 9 r e v e a l s t h a t t h e pH p r o f i l e would have been c o n s i d e r a b l y if  different  t h e r a t e o f phosphate l i b e r a t i o n from g-glycerophosphate had not been  s u b t r a c t e d from t h a t from 5'-AMP. A l t h o u g h the pH optimum f o r 5 ' - n u c l e o t i d a s e was found to be 8.5, t h i s pH was n o t used i n the remaining s t u d i e s .  A l l o t h e r experiments  were conducted a t pH 7.2 which decreased t h e p o s s i b i l i t y o f e r r o r due to n o n - s p e c i f i c phosphatase a c t i v i t y physiological.  ( F i g . 9) and was c o n s i d e r e d to be more  I f the enzyme were to f u n c t i o n i n t r a c e l l u l a r l y , pH 7.0  would have been a more a p p r o p r i a t e c h o i c e  (Woodbury, 1965).  there i s some e v i d e n c e t h a t i t may f u n c t i o n e x t r a c e l l u l a r l y .  However, Baer and  Drummond (1968) found t h a t phosphate was removed from 5'-AMP a f t e r  exposure  of o n l y a few seconds to the v a s c u l a t u r e o f r a t h e a r t s p e r f u s e d by t h e :  Langendorf Method.' When r a d i o a c t i v e 5'-AMP was i n f u s e d i n t o t h e c o r o n a r y c i r c u l a t i o n , most of t h e l a b e l was r e c o v e r e d i n t h e p e r f u s a t e as adenosine or i n o s i n e , which a r e breakdown p r o d u c t s o f 5'-AMP.  Since i t i s generally  a c c e p t e d t h a t n u c l e o s i d e phosphates do n o t r e a d i l y e n t e r c e l l s , these workers suggested t h a t t h e a c t i v e s i t e of 5 ' - n u c l e o t i d a s e was a c c e s s i b l e to e x t r a c e l l u l a r s u b s t r a t e .  I f t h i s enzyme f u n c t i o n e d e x t r a c e l l u l a r l y , the  Figure 9. A c t i v i t y of 5'-nucleotidase as-a function-of pH. Rate of phosphate l i b e r a t e d from 5'-AMP by s p e c i f i c 5'-nucleotidase (O) d from 3 glycerophosphate by non-specific phosphatase (^) p l o t t e d against pH. Points represent values obtained i n d u p l i c a t e . F i n a l concentrations i n the incubation were Tris-maleate, 40 mM; MgCl2, 10 mM; 5'-AMP, 10 mM; enzyme, 0.62 mg/ml i n a f i n a l volume of 0.25 ml. a n  _  42.  pH of i t s n a t u r a l m i l i e u could be 7.4 (Spector, 1956).  Since the exact  d i s p o s i t i o n of 5'-nucleotidase was not known, the pH value chosen f o r f u r t h e r c h a r a c t e r i z a t i o n studies was a compromise of 7.2. F.  2.  E f f e c t s of Divalent Cations In determining the e f f e c t s of d i v a l e n t cations on 5'-nucleotidase, I|  one source of a r t i f a c t could be endogenous metal ions such as Mg  . In  order to reduce t h i s problem to a minimum, the enzyme was treated w i t h 10 mM EDTA, a c h e l a t i n g agent which binds d i v a l e n t c a t i o n s . The EDTAc a t i o n chelate and excess EDTA were removed from enzyme by d i a l y s i s . Enzyme a c t i v i t y was determined by estimation of phosphate l i b e r a t e d . A l l values of F i g . 10 are represented as the percent of maximal a c t i v i t y i n the presence of UgCl^ a f t e r s u b t r a c t i o n of. c o n t r o l values obtained by s u b s t i t u t i o n of 3-glycerophosphate  f o r 5'-AMP i n the incubation  mixture. A d d i t i o n of d i v a l e n t c a t i o n was not e s s e n t i a l to the demonstration of 5'-nucleotidase a c t i v i t y ;  t h i s enzyme from r a t heart was a c t i v e even  a f t e r EDTA treatment to remove d i v a l e n t metal ions ( F i g . 10).  This obser-  v a t i o n was s i m i l a r to those of Edwards and Maguire (1970) and S u l l i v a n and Alpers (1971), who also found that t h i s enzyme was a c t i v e i n the absence of added Mg  .  5'-Nucleotidase from r a t heart was s i m i l a r to  that from r a t cerebellum (Bosmann and P i k e , 1971) which was a l s o a c t i v e i n the absence of d i v a l e n t metal i o n .  In c o n t r a s t , the soluble 5'-nucleo-  tidases of r a t ( F r i t z s o n , 1969) and chicken l i v e r (Itoh, et a l . , 1967) I |  had absolute requirements f o r Mg observed unless Mg  was added.  ; no 5'-nucleotidase a c t i v i t y could be Although the r e s u l t s of the present study  and those of Edwards and Maguire (1970) and S u l l i v a n and Alpers (1971) a l l showed that 5'-nucleotidase was a c t i v e i n the absence of d i v a l e n t  43.  c a t i o n s , t h e r e were s u b s t a n t i a l d i f f e r e n c e s between r e s u l t s in  the presence  of such  ions.  In this  study  16 mM M g C ^  obtained  was a b l e t o  s t i m u l a t e t h e enzyme up t o 5 - f o l d ( F i g . 1 0 ) w h i l e E d w a r d s a n d M a g u i r e ( 1 9 7 0 ) r e p o r t e d i n h i b i t i o n b y b o t h Mg  a n d Ca  .  S u l l i v a n and A l p e r s  ( 1 9 7 1 ) r e p o r t e d no s t i m u l a t i o n b y d i v a l e n t c a t i o n b u t c l o s e r of t h e i r data r a i s e s reported  the p o s s i b i l i t y  t h a t a f t e r EDTA t r e a t m e n t ,  t h e enzyme a c t i v i t y .  of another  treatment;  c a t i o n c o u l d c a u s e enzyme s t i m u l a t i o n . t h a t o f S u l l i v a n and A l p e r s all  activity  interpretation.  In both  the present  i n the assay.  i o n s more t i g h t l y  I t would  minutes) d i d not cause r e d u c t i o n of a c t i v i t y  appear  for divalent cation  t h a n d o e s EDTA.  a l t e r n a t i v e seems m o r e l i k e l y b e c a u s e p r o l o n g e d  s t u d y and  d i d not eliminate  t h a t t h e enzyme d o e s n o t h a v e a n a b s o l u t e r e q u i r e m e n t these  some d i v a l e n t  hence a d d i t i o n o f d i v a l e n t  ( 1 9 7 1 ) , EDTA t r e a t m e n t  e v e n i f EDTA w e r e p r e s e n t  or that i t binds  They  I| j | || | | Ca , Mn , Co a n d Mg increased  The p r e p a r a t i o n may h a v e c o n t a i n e d  c a t i o n w h i c h was r e m o v e d b y t h i s  examination  The  first  e x p o s u r e t o EDTA ( 6 0  g r e a t e r t h a n 10 m i n u t e s  exposure. The s t i m u l a t i o n o f 5 ' - n u c l e o t i d a s e b y Mg depicted o f Mg  i n F i g . 10.  -H-  -H++ , Ni a n d Mn i s  Enzyme a c t i v i t y was m a x i m a l a t 16 mM  w i t h no d e c l i n e i n a c t i v i t y  a t 80 mM.  i n the case  This i s d i f f e r e n t  t h e s o l u b l e 5 ' - n u c l e o t i d a s e o f r a t l i v e r w h i c h was m a x i m a l l y  from  active at  j | 0.1 M Mg  1969). (Fig.  and d e c l i n e d t o l e s s  t h a n h a l f m a x i m a l a t 0.5 M  (Fritzson, Ij | |  The s t i m u l a t i o n o f r a t h e a r t 5 ' - n u c l e o t i d a s e b y Mn 1 0 ) was m a x i m a l a t much l o w e r  respectively.  The K  f o r Mg  and N i  c o n c e n t r a t i o n s , 2 mM a n d 1  , obtained  from a Hofstee  mM  p l o t of the  ct  | [  d a t a r e p r e s e n t e d i n F i g . 10 . ( v e l o c i t y v e r s u s v e l o c i t y / M g concentration) I| | [, was 1.9 mM. Mn was a m o r e e f f e c t i v e s t i m u l a t o r o f t h e enzyme t h a n Mg ;  43(a)  MgCl  (mM)  2  16  CaCl  (mM)  2  Activity  -  100  -  15  10  28  16  5  94  4  -  73  4  5-  70  TABLE 3.  E f f e c t o f Ca  on  (%)  (control)  5'-nucleotidase.  E f f e c t s o f C a C l 2 o n EDTA t r e a t e d 5 ' - n u c l e o t i d a s e w i t h and w i t h o u t s t i m u l a t i o n by MgCl2-  >•  140  -  120  ••  > I—  o <  X  o  100  80  -  60  r  40  20  CATION F i g u r e 10.  E f f e c t o f Mg  , Mn  , and N i  (mM) on 5 ' - n u c l e o t i d a s e .  S t i m u l a t i o n o f EDTA p r e - t r e a t e d 5 ' - n u c l e o t i d a s e was e f f e c t e d b y a d d i n g M g C l 2 (O). M n C l 2 (A), a n d N i C l ( • ) . Values, obtained i n d u p l i c a t e a r e represented as the percent of maximum o b t a i n e d i n t h e p r e s e n c e o f M g C ^ . The f i n a l c o n c e n t r a t i o n s i n t h e r e a c t i o n m i x t u r e were T r i s - H C l , 50 mM, pH 7.2; 5'-AMP, 10 mM; enzyme 0.21 mg/ml a n d m e t a l c h l o r i d e i n 0.25 m l . 2  44.  maximal a c t i v i t y the  p r e s e n c e o f Mg  tidase activity at  .  Ca  I|  , 5'-nucleo-  ( 1 0 mM) h a d o n l y a s l i g h t  i n t h e p r e s e n c e o f Mg  These r e s u l t s a r e s t r i k i n g l y  and M a g u i r e  and N i  r e a c h e d an optimum a t l o w c o n c e n t r a t i o n s and d e c r e a s e d  (Table 3);  a l l .  was 1 5 4 % o f t h a t o b t a i n e d i n  I n t h e c a s e s o f b o t h Mn  higher concentrations.  effect at  i n t h e p r e s e n c e o f Mn  (1970);  I|  stimulatory  i t did not alter  activity  d i f f e r e n t f r o m t h o s e o f Edwards  t h e y f o u n d t h a t 1 0 mM Mg  I |  reduced a c t i v i t y to  I [  30% o f t h a t i n t h e absence  o f Mg  .  T h e r e d o e s n o t seem t o b e an..  apparent reason f o r t h i s  discrepancy.  are  from t h o s e o b t a i n e d from s t u d i e s o f sheep  also quite different  The d a t a r e p r e s e n t e d i n F i g . 10  I|  b r a i n 5 ' - n u c l e o t i d a s e ( I p a t a , 1968).  While N i  enhanced r a t h e a r t  5 ' - n u c l e o t i d a s e a c t i v i t y by about 4 - f o l d , i t i n h i b i t e d  t h e enzyme f r o m  sheep b r a i n by 58%. 3.  Substrate  Specificity  Substrate s p e c i f i c i t y enzyme.  Activity  s t u d i e s were performed w i t h  was e s t i m a t e d b y m e a s u r i n g  from t h e v a r i o u s phosphate  esters.  EDTA-treated  orthophosphate  The r a t e o f p h o s p h a t e  liberated  release  e a c h e s t e r was d e t e r m i n e d i n t h e p r e s e n c e o f 16 mM M g C ^ ;  i n addition,  r a t e s f o r most o f t h e s e e s t e r s were d e t e r m i n e d i n t h e absence Since s e v e r a l phosphate  esters  of MgC^.  t e s t e d were a c i d l a b i l e , c o n t r o l v a l u e s  to account f o r a c i d h y d r o l y s i s d u r i n g c o l o u r development from experimental values.  from  Activity  were  subtracted  i s represented as percent o f a c t i v i t y  i n t h e p r e s e n c e o f 16 mM M g C ^ w i t h 5'-AMP a s s u b s t r a t e . The  enzyme p o s s e s s e d a b r o a d s u b s t r a t e s p e c i f i c i t y  5'-monophosphates;  this  i stypical  s e e Drummond a n d Yamamoto, 1 9 7 1 ) .  for nucleoside  o f most 5 ' - n u c l e o t i d a s e s ( f o r r e v i e w , P h o s p h a t e was l i b e r a t e d  a v a r i e t y o f n u c l e o s i d e 5'-monophosphates t e s t e d  ( F i g . 11).  from each o f 5'-AMP was  UMP Open Bars Hatched Bars:  +Mg -Mg  120 >- 100 o <  X  IMP  80  <  60  £  40 20  CMP  5'AMP  dUMP  dCMP  s 1  Figure 11.  Substrate S p e c i f i c i t y .  dGMP  0 El  3'AMP  /3GP JZO.  Open bars represent orthophosphate l i b e r a t e d i n the presence of 16 mM MgCl2 and hatched bars i n the absence of MgCl2The f o l l o w i n g were not substrates: p-nitrophenylphosphate, pyrophosphate, ribose-5-phosphate, glucose-6-phosphate, fructose-1, 6-diphosphate, fructose-l-phosphate, r i b u l o s e 5-phosphate and galactose-6-phosphate: The f i n a l concent r a t i o n s i n the incubation mixtures were T r i s - H C l , 50 mM, pH 7.2;. MgCl2» 16 mM; enzyme, 0.49 or 0.77 mg/ml and test compound, 10 mM i n a f i n a l volume of 1.0 ml.  45.  not  the best substrate;  UMP was h y d r o l y z e d  more r a p i d l y .  The 2'-deoxy-  r i b o n u c l e o s i d e 5'-monophosphates were u t i l i z e d a t about h a l f t h e r a t e of t h e c o r r e s p o n d i n g  r i b o n u c l e o s i d e 5'-monophosphates.  Although  a c t i v i t y was observed when a number of s u b s t r a t e s c o n t a i n i n g  different  bases were t e s t e d , no enzyme a c t i v i t y was found i f t h e base was absent. For example the f o l l o w i n g sugar phosphates d i d n o t a c t as s u b s t r a t e s , r i b o s e - 5 - p h o s p h a t e , glucose-6-phosphate, f r u c t o s e - l - p h o s p h a t e , 6-diphosphate, r i b u l o s e - 5 - p h o s p h a t e  fructose-1,  and g a l a c t o s e - 6 - p h o s p h a t e .  In addition  t h i s enzyme d i d n o t r e l e a s e orthophosphate f r o m e i t h e r p - n i t r o p h e n y l :  phosphate o r pyrophosphate.  The broad s u b s t r a t e s p e c i f i c i t y does not  appear t o be due to the presence of two enzymes; and  one independent of Mg  I |  .  one r e q u i r i n g Mg  I |  The r e l a t i v e r a t e s o f h y d r o l y s i s of s u b s t r a t e s I |  were s i m i l a r i n both t h e presence and absence of Mg  .  Further  evidence  f o r the e x i s t e n c e of a s i n g l e enzyme can be i n f e r r e d from t h e f a c t the p y r i m i d i n e n u c l e o t i d e , UMP, c o m p e t i t i v e l y l a t i o n of 5'-AMP ( t h i s w i l l be d e s c r i b e d F.  4.  E f f e c t of 5'-AMP The  inhibited  the dephosphory-  later).  Concentration  enzyme^preparation used was p r e t r e a t e d w i t h  the c h e l a t i n g agent  EDTA, f o l l o w e d by d i a l y s i s to remove endogenous d i v a l e n t c a t i o n . such a p r e p a r a t i o n ,  that  the stimulatory  c o u l d be determined over t h e f u l l  e f f e c t of Mg  extent  on  With  5'-nucleotidase  of substrate concentration.  Two  methods o f assay were r e q u i r e d because t h e broad range o f 5'-AMP concent r a t i o n tested assay  (0.0033 mM t o 10 mM) c o u l d n o t be accommodated by e i t h e r  alone. For c o n c e n t r a t i o n s  of 5'-AMP from 0.16 mM to 10 mM,  5'-nucleotidase  a c t i v i t y was determined by e s t i m a t i o n o f phosphate l i b e r a t e d . The incubation mixture contained.Tris-HCl  b u f f e r , 50 mM, pH 7.2;  MgCl„, 16 mM;  46.  e n z y m e , 0.17 mg/ml a n d 5'-AMP i n a f i n a l v o l u m e o f 1.0 m l . i s represented  a s u m o l e s p e r m i n u t e p e r mg p r o t e i n .  Activity  I n t h e range o f  5'-AMP c o n c e n t r a t i o n f r o m 0.0033 mM t o 0.16 mM, t h e d i r e c t o p t i c a l was u s e d . 16 mM;  The r e a c t i o n m i x t u r e  enzyme 0.17 mg;  v o l u m e o f 3.0 m l . for  the f u l l  contained  T r i s - H C l , 50 mM, pH 7.2; 1  A corresponding  s e r i e s without  r a n g e o f 5'-AMP c o n c e n t r a t i o n .  The a c t i v i t y o f 5 ' - n u c l e o t i d a s e seen, i n F i g . 1 2 .  Rate values  are comparable t o those  MgCl , 2  e x c e s s a d e n o s i n e deaminase and 5 -AMP i n a f i n a l MgCl  obtained  2  1  was c a r r i e d o u t  Lack o f n o n - s p e c i f i c phospha-  t a s e w a s a s c e r t a i n e d b y r e p l a c i n g 5'-AMP w i t h 3'-AMP.  is  assay  -  a s a f u n c t i o n o f 5'-AMP  obtained by t e s t  t i d a s e d i s p l a y e d t y p i c a l Michaelis^-Mentbn  concentration  by d i r e c t o p t i c a l assay tube assay  (panel B ) .  (panel A)  5'-Nucleo-  dependence on s u b s t r a t e  concen-  I | t r a t i o n i n t h e p r e s e n c e a n d a b s e n c e o f Mg 0.16 mM 5'-AMP.  I n t h i s r a n g e , t h e a c t i v i t y was s t i m u l a t e d l e s s t h a n  2 - f o l d b y 16 mM M g C l 2  and  The K  i n the presence of MgCl  stimulated  2  i n t h e a b s e n c e o f M g C l was 2.1 x 1 0 ~ M —5 | | was 2.3 x 1 0 M w h i c h i n d i c a t e d t h a t Mg 5  m  2  t h e enzyme b y i n c r e a s i n g maximum v e l o c i t y r a t h e r t h a n b y  changing a f f i n i t y  f o r substrate  comparable t o those  obtained  ( F i g . 14 a n d ' 1 5 ) .  by other workers.  (1970) and S u l l i v a n and A l p e r s and  o v e r t h e r a n g e 0.0033 mM t o  These K  values are  Edwards and M a g u i r e  (1971) r e p o r t e d v a l u e s  o f 1.45 x 1 0 ~* M  2.3 x 1 0 ^ M, r e s p e c t i v e l y when e x p e r i m e n t s w e r e c o n d u c t e d i n t h e I |  a b s e n c e o f Mg 5'-Nucleotidase  a c t i v i t y d i d not increase at concentrations of I |  5'-AMP g r e a t e r mixture.  t h a n 0.16 mM i f Mg  I n t h e presence of MgCl  4 mM 5'-AMP.  was n o t p r e s e n t 2  i n the incubation  enzyme a c t i v i t y was m a x i m a l a t  A t t h i s c o n c e n t r a t i o n t h e a c t i v i t y was  0.08 Figure 12.  0.16  2 4 AMP (mM)  8  E f f e c t of 5'-AMP. concentration. 5'-Nucleotidase a c t i v i t y was determined i n the" absence (A,V) and presence (P,0) of -MgCl.2 (16 mM) . Panel A represents enzyme a c t i v i t y a t low concent r a t i o n s of 5'-AMP; assays were performed by the d i r e c t o p t i c a l method. Panel B shows a c t i v i t y a t high 5'-AMP concentrations; a c t i v i t y was estimated by determination of phosphate l i b e r a t i o n .  stimulated 4.4-fold by 16 mM MgC^, i n contrast to l e s s than 2-fold at lower 5'-AMP concentrations.  This high a c t i v i t y i n the presence  of 16 mM MgCl appeared to involve-a second  estimated to be about  2  -4 10  M.  This second apparent K may i n d i c a t e more than one enzyme or m  i t may represent one enzyme w i t h two a f f i n i t i e s f o r 5'-AMP. S i m i l a r observations of m u l t i p l e a f f i n i t i e s have been made f o r nucleoside-3' , 5 ' - c y c l i c phosphodiesterase  from bovine heart (Beavo, Hardman and  Sutherland, 1970) and r a t b r a i n (Thompson and Appleman, 1971).  Beavo  et a l . (1970) suggested that t h i s enzyme could have m u l t i p l e binding s i t e s f o r substrate and that binding at one s i t e might a f f e c t binding at another. IR i v  t r o  Regulation of 5'-Nucleotidase  As described p r e v i o u s l y , i t was expected that the a c t i v i t y of cardiac 5'-nucleotidase might be regulated by various metabolites and thus by the metabolic state of the heart.  Baer et a l . (1966) have  already shown that t h i s enzyme was markedly i n h i b i t e d by ATP and suggested that the decrease i n ATP l e v e l s during hypoxia could r e s u l t i n greater 5'-nucleotidase a c t i v i t y and increased adenosine formation. I f metabolic state regulates t h i s enzyme, a c t i v i t y could be a f u n c t i o n of more.than cardiac ATP,.concentration  alone.  More appropriately i t  could be a f u n c t i o n of energy charge.  Atkinson and Walton (1967)  defined energy charge of the adenylate system as " h a l f the average number of anhydride-bound phosphate groups per adenosine moiety". This may also be w r i t t e n (ATP energy charge =  +  ^ADP)  : (ATP + ADP + AMP)  48.  R e c e n t l y Burger and Lowenstein (1970) showed t h a t 5 - n u c l e o t i d a s e o f 1  p i g i n t e s t i n a l smooth muscle was n o t o n l y i n h i b i t e d by ATP but was more p o w e r f u l l y i n h i b i t e d by ADP. seemed important  On t h e b a s i s o f t h e a b o v e . i n f o r m a t i o n , i t  to examine t h e e f f e c t s o f these and o t h e r m e t a b o l i t e s  on r a t h e a r t 5 ' - n u c l e o t i d a s e .  The p a r t i c u l a r substances  s t u d i e d were  chosen because t h e i r c o n c e n t r a t i o n s i n c a r d i a c t i s s u e a r e s u b j e c t to c o n s i d e r a b l e change d u r i n g hypoxia  (Imai e t a l . , 1964; W i l l i a m s o n ,  1966).  Thus t h e e f f e c t s of ATP, ADP, c r e a t i n e phosphate and i n o r g a n i c phosphate on t h e enzyme were examined under a v a r i e t y o f c o n d i t i o n s . I [  F.  5.  a.  E f f e c t s o f ADP and ATP i n t h e Presence  o f Mg  a t High  C o n c e n t r a t i o n s o f 5'-AMP.  0.15  In t h e presence of 16 mM M g C ^ and a t c o n c e n t r a t i o n s of 5'-AMP mM o r g r e a t e r , 5 ' - n u c l e o t i d a s e was i n h i b i t e d by both ADP and ATP  ( F i g . 13).  I t i s u n l i k e l y t h a t t h i s i n h i b i t i o n was due to c h e l a t i o n  I [ of Mg  because the c o n c e n t r a t i o n of M g C l  than t h a t o f i n h i b i t o r .  was 16 o r 32 times  greater  ADP was c o n s i d e r a b l y more e f f e c t i v e as an  i n h i b i t o r of 5 ' - n u c l e o t i d a s e . ADP  2  A t 2 mM 5'-AMP, t h e i n h i b i t i o n by 0.5 mM  was 41% w h i l e i n h i b i t i o n by; 1.0 mM ATP was o n l y 12%.  No attempts  were made to determine t h e type of i n h i b i t i o n o r i n h i b i t o r y u s i n g data o b t a i n e d a t h i g h 5'-AMP c o n c e n t r a t i o n s  constants  (0.16 mM o r g r e a t e r ;  F i g . 13). b.  E f f e c t s of ADP, ATP and C r e a t i n e Phosphate i n the Absence [ | of Mg  The  i n h i b i t i o n o f 5 ' - n u c l e o t i d a s e by ADP i n t h e absence of Mg  i s shown i n F i g . 14, 16 and 18.  Examination  of t h e H o f s t e e  p l o t i n F i g . 14 r e v e a l s t h a t t h e enzyme was i n h i b i t e d  (1952)  i n a mixed  I |  I  Figure  13.  2 AMP (mM)  I n h i b i t i o n b y ATP a n d ADP a t h i g h  3  concentrations  4  o f 5'-AMP.  A s s a y s w e r e p e r f o r m e d a s f o r t h e e f f e c t o f 5'-AMP c o n c e n t r a t i o n ( r a n g e 0.16 mM t o 10 mM 5'-AMP), e x c e p t t h a t t h e enzyme w a s n o t t r e a t e d w i t h EDTA. T h e f i n a l i n c u b a t i o n m i x t u r e c o n t a i n e d T r i s - H C l , 5 0 mM, pH 7.2; M g C l 2 , 16 mM; e n z y m e , 0.019 mg; 5'-AMP "and A T P , 1.0 mM ( A ) o r ADP, .0.5 mM ( Q ) . Points represent values obtained i n duplicate and a f t e r s u b t r a c t i o n o f c o n t r o l s t o a c c o u n t f o r p o s s i b l e h y d r o l y s i s o f i n h i b i t o r during c o l o u r development.  The i n h i b i t i o n of 5'--nucleotidase by ADP i s represented i n t h i s Hofstee p l o t . Assays were conducted using an EDTA treated preparation i n the absence (O) and presence (Q) of ADP. The incubation mixture contained T r i s - H C l , 50 mM, pH 7.2; ADP, 0.0033 mM when required; . enzyme, 0.179 mg, excess adenosine deaminase and 5'-AMP i n a f i n a l volume of 3.0 ml. Reaction rates were determined by the d i r e c t o p t i c a l assay.  (competitive-non-competitive) decreased by 25% w h i l e to  5.0 x 10 ^ M..  extent by  manner.  the apparent  The e f f e c t  T h e maximum v e l o c i t y w a s was i n c r e a s e d  f r o m 2.1 x 10 ^ M  o f i n c r e a s i n g c o n c e n t r a t i o n o f ADP o n t h e  o f i n h i b i t i o n i s shown i n F i g . 1 6 .  t h e method o f D i x o n  :  (1953),  I f these  two s t r a i g h t  data  arereplotted  lines of different  slope  are obtained;  t h i s i s i n t e r p r e t e d t o mean t h a t ADP i n h i b i t e d  by b i n d i n g  a t two s i t e s .  The o b s e r v a t i o n o f m i x e d i n h i b i t i o n i s s i m i l a r  to the  mixed i n h i b i t i o n by n u c l e o s i d e Maguire  (1970).  inhibited  triphosphates  reported  T h e s e w o r k e r s f o u n d t h a t A T P , UTP, GTP a n d I T P a l l  by a l t e r a t i o n  o f b o t h maximum v e l o c i t y  J  Sullivan  and A l p e r s  (1971) r e p o r t e d  enzyme f r o m t h e same s o u r c e , Fig. by  not  r a t heart,  In contrast,  i n o n l y a c o m p e t i t i v e manner.  18 shows t h a t t h i s enzyme was v i r t u a l l y  completely  i snot present  inhibited  i n the reaction  C r e a t i n e p h o s p h a t e was f o u n d t o i n h i b i t 1 8 % a t 0.067 mM a n d  a t a l l a t 0.0067 mM when t h e c o n c e n t r a t i o n o f 5'-AMP was 0.0083 mM.  The i n h i b i t i o n b y d i - a n d t r i p h o s p h a t e limited  to 5'-nucleotidase  non-competitive cerebellum  Pike,  esters of nucleosides  from c a r d i a c t i s s u e .  f r o m s h e e p b r a i n was i n h i b i t e d  rat  and K . m  t h a t b o t h ADP a n d ATP i n h i b i t e d t h e  0.033 mM ATP o r 0.033 mM ADP when Mg  mixture.  by Edwards and  was i n h i b i t e d  F o r e x a m p l e , t h e enzyme  b y A T P , UTP a n d CTP i n a m i x e d  way ( I p a t a , 1 9 6 8 ) .  i s not  competitive  I n a d d i t i o n , 5'-nucleotidase  from  b y A T P , UTP, I T P , C T P , a n d GTP (Bosmann a n d  1971). c.  E f f e c t s o f ADP, UMP a n d O r t h o p h o s p h a t e i n t h e P r e s e n c e o f Mg  [ |  I|  I n h i b i t i o n b y ADP, UMP a n d o r t h o p h o s p h a t e i n t h e p r e s e n c e o f Mg was e x a m i n e d u s i n g bation mixture 0.185 mg;  enzyme w h i c h h a d n o t b e e n t r e a t e d w i t h EDTA.  contained  T r i s - H C l , 50 mM, pH 7.2;  excess adenosine deaminase;  MgC^j  16 mM;  5'-AMP a n d ADP, 0.033 mM;  The i n c u enzyme, UMP,  50.  0.067 mM o r o r t h o p h o s p h a t e , AO mM  i n a f i n a l v o l u m e o f 3.0 m l .  s e r i e s was a l s o c a r r i e d o u t i n t h e a b s e n c e were determined by t h e d i r e c t o p t i c a l The MgC^  Reaction rates  i n t h e p r e s e n c e o f 16 mM  1  F i g . 16 d e m o n s t r a t e s  i s a f u n c t i o n o f ADP c o n c e n t r a t i o n .  That  that  inhibition  t h e i n h i b i t i o n b y ADP was  i n a n o n - c o m p e t i t i v e manner i s s e e n i n F i g . 1 5 , i n w h i c h t h e  d a t a have been p l o t t e d by t h e method o f H o f s t e e  (1952).  T h e maximum  v e l o c i t y was d e c r e a s e d 4 5 % b y 0.033 mM ADP w h i l e t h e a f f i n i t y d i d n o t appear  t o be a f f e c t e d .  The K  2;-16 x 10 ^ M w h i l e t h e a p p a r e n t 10~  5  M.  control  method.  i n h i b i t i o n o f 5 - n u c l e o t i d a s e b y ADP  i s shown i n F i g . 15 a n d 1 6 ;  effected  of i n h i b i t o r s .  A  of the uninhibited  f o r 5'-AMP  enzyme was  i n t h e p r e s e n c e o f ADP was 2.58 x  The c a l c u l a t e d v a l u e o f K. f o r ADP was 4 x 1 0 ~  5  M.  These  I  r e s u l t s a r e q u i t e d i f f e r e n t f r o m t h o s e o f S u l l i v a n and A l p e r s ( 1 9 7 1 ) . These workers  f o u n d no i n h i b i t i o n b y ADP i n t h e p r e s e n c e o f 8 mM  h o w e v e r o n l y 0.0017 mM ADP was u s e d c o m p a r e d t o 0.033 mM u s e d t h e d a t a o f F i g . 15.. When t h e y u s e d  t w i c e a s much ADP  MgC^,  to o b t a i n  (0.0034  mM),  I|  slight  i n h i b i t i o n was o b s e r v e d i n t h e p r e s e n c e o f Mg ATP i n t h e p r e s e n c e o f 16 mM M g C l  e x t e n t a s ADP. ( 0 . 0 3 3 mM)  2  d i d not i n h i b i t  t o t h e same  When t h e c o n c e n t r a t i o n o f 5'-AMP was 0.033 mM,  only inhibited  1 1 % w h i l e ADP  These d a t a a r e n o t c o n s i s t e n t w i t h  (0.033. mM)  inhibited  ATP  35% ( F i g . 1 8 ) .  t h e f i n d i n g s o f S u l l i v a n and A l p e r s  who r e p o r t e d no i n h i b i t i o n o f 5 ' - n u c l e o t i d a s e when t h e " c o n c e n t r a t i o n o f ATP was 0.02 mM a n d t h a t o f MgSO^ was 10 mM. crepancy  The r e a s o n f o r t h i s  dis-  i s not apparent.  UMP i n h i b i t e d t h e enzyme i n a c o m p e t i t i v e m a n n e r ( F i g . 1 7 ) . The a p p a r e n t K was 5.2 x 10 ^ M i n t h e p r e s e n c e o f UMP c o m p a r e d t o t h e m c o n t r o l v a l u e o f 2.4 x 10 ^ M; maximum v e l o c i t y was u n c h a n g e d . These  i  i  I  I  :  I  I  2  3 4 5 SPECIFIC ACTIVITY (S) mM  i  _L  6  7  Figure 15. I n h i b i t i o n by ADP i n the presence of MgC^. This Hofstee p l o t shows the i n h i b i t i o n of 5'-nucleotidase by ADP. Assays f o r the u n i n h i b i t e d (O) and ADP i n h i b i t e d (Q) reactions were conducted i n the presence of 16 mM M g C l by the d i r e c t o p t i c a l method. 2>  I  A L  0.01  I  0.02  I  0.03 ADP  Figure 16.  I  0.04  I  0.05  I  0.06  (mM)  E f f e c t of ADP concentration on i n h i b i t i o n . I n h i b i t i o n by ADP was ascertained i n the absence (A) and presence (O) °f 16 mM MgCl2- Values are depicted as percent of u n i n h i b i t e d c o n t r o l . 5'-AMP concentration was always 0.033 mM.  by the d i r e c t o p t i c a l method.  51.  r e s u l t s should substrate cates  not be unexpected, s i n c e UMP was a l s o found to be a  ( F i g . 11).  The o b s e r v a t i o n  t h a t one enzyme was r e s p o n s i b l e  of c o m p e t i t i v e  inhibition  indi-  f o r h y d r o l y s i s o f 5'-AMP and UMP.  I f t h e r e had been two enzymes, one s p e c i f i c f o r p u r i n e n u c l e o t i d e s and another s p e c i f i c f o r p y r i m i d i n e  nucleotides,  t i v e i n h i b i t i o n would have been observed. i n h i b i t e d 5'-nucleotidase manner (Fig.- 17).  The K  i t i s u n l i k e l y that  Orthophosphate, u n l i k e UMP,  i n a n o n - c o m p e t i t i v e r a t h e r than m  competi-  competitive  determined i n t h e presence of 40 mM  ortho-  phosphate was 2.3 x 10 ^ M which was unchanged from 2.4 x 10 ^ M i n i t s absence.  The v a l u e  o f K. was c a l c u l a t e d t o be 73 mM.  The e f f e c t s of  I  orthophosphate on 5 ' - n u c l e o t i d a s e on enzyme from sheep b r a i n .  was q u i t e d i f f e r e n t from i t s e f f e c t  Ipata  (1968) found t h a t the ATP-induced  i n h i b i t i o n of sheep b r a i n 5 ' - n u c l e o t i d a s e  was r e v e r s e d  by t h e a d d i t i o n  of orthophosphate. d.  E f f e c t o f Mg""" on I n h i b i t i o n by ADP and ATP. 1 1  Both ADP and ATP v i r t u a l l y at a c o n c e n t r a t i o n  eliminated  o f 0.033 mM i n the absence of MgC^.  MgCl2 t o 16 mM r e s t o r e d  enzyme t o 89% o f c o n t r o l  activity  A d d i t i o n of  the a c t i v i t y of t h e ADP i n h i b i t e d enzyme to 63%  of u n i n h i b i t e d c o n t r o l and r e s t o r e d  (1971) r e p o r t e d  a l l 5'-nucleotidase  ( F i g . 18).  t h e a c t i v i t y of t h e ATP i n h i b i t e d I n comparison, S u l l i v a n and A l p e r s  that the a d d i t i o n o f MgSO^ c o m p l e t e l y r e v e r s e d the  i n h i b i t i o n due to ATP.  Examination o f F i g . 18 r e v e a l s t h a t the a c t i v i t y  of the ADP i n h i b i t e d enzyme was maximally enhanced by 4 mM MgCl^; MgCl The  2  K  was r e q u i r e d  11 mM  f o r maximum s t i m u l a t i o n of t h e ATP i n h i b i t e d enzyme.  f o r s t i m u l a t i o n o f ADP and ATP i n h i b i t e d enzyme was about  -3 1 x 10 M MgCl  9  -3 compared t o 1.9 x 10 M f o r t h e u n i n h i b i t e d enzyme.  100 ATP (0.033 mM)  80 ADP P o  60  (0.033 mM)  o —  o  o  < o  on  40  20  12 MgCI F i g u r e 18.  2  14  (mM)  E f f e c t o f MgCl2 c o n c e n t r a t i o n on i n h i b i t i o n by ADP and ATP. A c t i v i t y o f ADP (O) and ATP ( • ) i n h i b i t e d 5 ' - n u c l e o t i d a s e as a f u n c t i o n o f MgCl2 c o n c e n t r a t i o n i s r e p r e s e n t e d as percent of u n i n h i b i t e d c o n t r o l i n the presence o f 16 mM MgCl2« Assays were performed by the d i r e c t o p t i c a l method and enzyme which had been t r e a t e d w i t h EDTA was used. The f i n a l i n c u b a t i o n m i x t u r e c o n t a i n e d T r i s - H C l , . 50 mM, pH 7.2; 5'-AMP, 0.033 mM; enzyme, 0.21 mg; excess adenosine deaminase; ADP o r ATP, 0.033 mM and MgCl2 i n a f i n a l volume o f 3.0 ml.  16  DISCUSSION  The  o b s e r v a t i o n s made i n t h e c o u r s e  measure o f support coronary warrant  of t h i s  f o r the r o l e o f adenosine  autoregulation.  The r e s u l t s o f c e r t a i n e x p e r i m e n t s ,  of ventricular  p e r f u s i o n experiments  enzymes i n v o l v i n g  suggest  r e g u l a t i o n of coronary  blood  that adenosine  some  i n the mediation of  some s k e p t i c i s m o f t h i s p o s s i b i l i t y .  the survey  study provide  however,  Data accumulated  from  5'-AMP a n d f r o m c a r d i a c may be i n v o l v e d i n a u t o - .  f l o w o f mammals b u t t h a t i t m i g h t n o t  operate  i n t h i s capacity i n other animals.  cardiac  5 ' - n u c l e o t i d a s e was p r e s e n t  The o b s e r v a t i o n t h a t m o s t  i n e n d o t h e l i a l c e l l s of the coronary  c i r c u l a t i o n a n d n o t i n h e a r t m u s c l e , c e l l s may s u g g e s t c a t i o n o f t h e model o f Rubio and Berne  (1969).  a slight modifi-  Adenosine  formed  d u r i n g h y p o x i a may n o t b e d e r i v e d f r o m m u s c l e c e l l s b u t may come f r o m endothelial  cells.  Certain properties of r a tventricle  5'-nucleotidase,  I|  such  a s i n h i b i t i o n b y ATP a n d s t i m u l a t i o n b y Mg  p r o d u c t i o n of adenosine  b y t h i s enzyme may b e e n h a n c e d d u r i n g  C e r t a i n o t h e r p r o p e r t i e s , however, i n d i c a t e j u s t The  r e s u l t s of a survey  amounts o f 5 ' - n u c l e o t i d a s e  found  i n harmony w i t h t h o s e e x p e c t e d  the adenosine  the opposite.  hypothesis.  on t h e b a s i s o f t h i s h y p o t h e s i s .  and t h u s  a w e l l developed  Hearts  means f o r t h e  substantial levels of 5'-nucleotidase.  T h i s p r e d i c t i o n was made b e c a u s e mammals a r e c a p a b l e  forced  The  i n mammalian and t u r t l e h e a r t s a r e  o f mammals w e r e p r e d i c t e d t o p o s s e s s  l a r g e l o a d s on t h e i r h e a r t s .  hypoxia.  of 5'-nucleotidase l e v e l s of hearts of  various species generally supports  production of adenosine  , indicate that the  of quickly p l a c i n g  F o r e x a m p l e , when r a t s a n d d o g s w e r e  t o r u n , oxygen c o n s u m p t i o n i n c r e a s e d 3 . 5 - f o l d and 2 . 5 - f o l d ,  respectively  (Taylor et a l . ,  1 9 7 0 ) ; t h e i n c r e a s e was s i m i l a r  i n humans  53.  (Rushmer et^ a l _ . , 1 9 6 3 ) . consequently demand  coronary  malian hearts  anoxia  ( B e l k i n , 1963)  i s probably  example, t u r t l e s and  necessary  isolated  fulfilled can  n e e d f o r o x y g e n and  i n t u r t l e h e a r t s as  a d e n o s i n e and  The  hypothesis.  this  c o n c l u s i o n c o u l d be  total  n o t be  l a c k of  i n t u r t l e v e n t r i c l e s w o u l d seem t o s u p p o r t  e f f e c t s of adenosine are equal  by  (personal observation).  v a s o d i l a t i o n may  i n mammalian h e a r t s .  p o i n t , i t has  energy  q u i t e adequately  5'-nucleotidase  preceding  expected  t u r t l e h e a r t s were found to f u n c t i o n  The  thus  Cardiac  s u r v i v e 17 h o u r s o f  s e v e r a l hours i n unoxygenated Mines s o l u t i o n  To  for  f o u n d i n mam-  unnecessary.  for  critical  oxygen  vaso-  T u r t l e v e n t r i c l e s were  t o a n o x i a a p p e a r t o be For  was  and  means f o r t h e p r o d u c t i o n o f a d e n o s i n e b e c a u s e  v a s o d i l a t i o n i n these hearts  means.  w o u l d be  That c o n s i d e r a b l e 5 ' - n u c l e o t i d a s e  i s a p p a r e n t f r o m F i g . 3.  d e f i c i e n c i e s due  a greater  I f adenosine were r e s p o n s i b l e f o r t h i s  have a p o o r l y developed  anaerobic  to f u l f i l l  s u b s t a n t i a l amounts of 5 ' - n u c l e o t i d a s e  its, production.  to  i n i n c r e a s e d h e a r t work  v a s o d i l a t i o n i n order  (Gregg, 1963).  dilation,  T h i s would r e s u l t  the  as  detectable adenosine  been assumed t h a t t h e v a s o d i l a t o r y  i n t u r t l e and repudiated  mammalian h e a r t s .  i f the coronary  The  circulation  t u r t l e w e r e much more s e n s i t i v e t o a d e n o s i n e t h a n . t h a t o f mammals. t h i s were the case^  undetectable  a b l e to produce s u f f i c i e n t c a n be fused  amounts o f 5 ' - n u c l e o t i d a s e  adenosine f o r v a s o d i l a t i o n .  e l i m i n a t e d because adenosine d i d not t u r t l e h e a r t s , i n amounts s u f f i c i e n t  dilation  i n h e a r t s o f r a b b i t and  although  not  to induce  circulation,  If be  possibility per-  s u b s t a n t i a l vaso-  These p e r f u s i o n s t u d i e s ,  e x t e n s i v e , s u g g e s t t h a t w h i l e a d e n o s i n e may  turtle hearts.  This  i n c r e a s e f l o w through  rats (Fig. 6).  r e g u l a t i o n o f t h e mammalian c o r o n a r y  might  of  mediate  i t does not  I f adenosine were to mediate coronary  do  autoso  in  autoregulation i n  54.  b i r d s , the mechanism f o r i t s f o r m a t i o n might be expected developed.  t o be w e l l  F o r example, pigeons use 8.5 times as much oxygen d u r i n g  f l i g h t as a t r e s t  (LeFebvre, 1964).  The r a t e o f energy  output o f  g u l l s and b u d g e r i g a r s w h i l e f l y i n g may be as h i g h as 20 times the r e s t i n g values  (Tucker, 1969).  I t seems r e a s o n a b l e to expect  that  h e a r t s o f these b i r d s would r e q u i r e s i m i l a r i n c r e a s e s i n oxygen s u p p l y . I f 5 ' - n u c l e o t i d a s e produced  adenosine  t o induce v a s o d i l a t i o n and thus  f a c i l i t a t e oxygen d e l i v e r y to these h e a r t s , s u b s t a n t i a l l e v e l s of t h i s enzyme might be expected.  T h i s was n o t the case;  i n f a c t , no 5'-  n u c l e o t i d a s e was d e t e c t e d i n p i g e o n h e a r t s under t h e c o n d i t i o n s of t h e d i r e c t assay o r by h i s t o c h e m i c a l examination.  T h i s o b s e r v a t i o n may  i n d i c a t e t h a t pigeons use some means, o t h e r than adenosine,  to regulate  coronary flow.• Since the l e v e l s of cardiac 5'-nucleotidase exhibited  large  v a r i a t i o n s amongst s p e c i e s , i t seemed t h a t the o v e r a l l metabolism of 5'-AMP by h e a r t s o f these s p e c i e s p r o b a b l y d i f f e r e d was  significantly.  This  supported by r e s u l t s of a survey of a d e n y l a t e k i n a s e and a d e n y l a t e  deaminase.  The c a t a b o l i s m o f 5'-AMP i n h e a r t s which are p o o r l y equipped  f o r adenosine p r o d u c t i o n c o u l d be channeled deaminase.  through IMP by a d e n y l a t e  C o n v e r s e l y , a n a b o l i c p r o c e s s e s c o u l d be i n v o l v e d and 5'-AMP  c o u l d be c o n v e r t e d to ADP by the a c t i o n o f a d e n y l a t e k i n a s e . d i f f e r e n c e i n t h e c a r d i a c l e v e l s o f these enzymes may r e f l e c t  The certain  s p e c i f i c f u n c t i o n s o f n u c l e o s i d e s and n u c l e o t i d e s and the manner i n which these h e a r t s respond  to p a r t i c u l a r demands.  F o r example,  v e n t r i c l e i s w e l l equipped  for. deamination o f 5'-AMP.  turtle  Hence, i t may  m e t a b o l i z e 5'-AMP l a r g e l y by c o n v e r s i o n to IMP and produce o n l y s m a l l amounts of adenosine, a s i t u a t i o n s i m i l a r to t h a t i n s k e l e t a l muscle  55.  (Imai et al_. , 1964).  Adenosine might not  capacity i n turtle hearts. adenylate  In c o n t r a s t , the r e l a t i v e l y lower  deaminase i n mammalian h e a r t s w o u l d tend  f i c a n t c a t a b o l i s m of a d e n y l a t e w o u l d be  function i n a regulatory  through  to allow a  adenosine r a t h e r than  p a r t i c u l a r l y a p p r o p r i a t e i f these  animals  levels  use  of  signiIMP.  This  adenosine  to  mediate r e g u l a t i o n of coronary  blood  flow because c o m p e t i t i o n f o r  the  source  t o be  restricted.  of.  of adenosine would tend  adenylate  kinase  w h e r e b y ATP  i n pigeon  c a n be  equipped to use  v e n t r i c l e provides  r e s y n t h e s i z e d f r o m ADP.  The  a w e l l developed  Thus, t h i s  b o t h h i g h e n e r g y b o n d s o f ATP  high levels  by  t i s s u e seems w e l l  its ability  adenine n u c l e o t i d e s i n a form s u i t a b l e f o r energy r e l e a s e . the pigeon work.  At  to  t h e o p p o s i t e end  ( S p e c t o r , 1956)  of  In t h i s  t h e s p e c t r u m , t u r t l e v e n t r i c l e may  by  m i g h t be  k i n a s e , s i n c e t h e i r lower  s a t i s f i e d by  anaerobic  p e r i o d s of a n o x i a of adenylate a r a p i d onset  t e s t e d i s not  need  e n e r g y demands  metabolism.  This  ( B e l k i n , 1963).  that -turtles survive  Mammals w i t h t h e i r  is  prolonged  intermediate  levels  k i n a s e appear l e s s w e l l equipped t h a n p i g e o n s to cope w i t h of h e a r t  work.  A comparison of the l e v e l s of adenosine deaminase i n the particularly  ventricle  r e v e a l i n g , but  i s important.  the presence of t h i s  the p o s s i b i l i t y  that pigeon  a c t i v i t y w h i c h was  not  hearts enzyme  A mechanism f o r the c a t a b o l i s m  a d e n o s i n e i m p l i e s a means f o r t h e f o r m a t i o n o f a d e n o s i n e .  T h i s was  way,  the o b s e r v a t i o n that t u r t l e hearts f u n c t i o n f o r s e v e r a l  h o u r s i n u n o x y g e n a t e d M i n e ' s s o l u t i o n and  i n pigeon  maintain  w o u l d be w e l l s u i t e d t o c o p e w i t h r a p i d i n c r e a s e s i n c a r d i a c  o n l y s m a l l amounts o f a d e n y l a t e  supported  mechanism  v e n t r i c l e d i d possess  of  This r a i s e d  5'-nucleotidase  d e t e c t a b l e under the c o n d i t i o n s d e s c r i b e d  the main s t i m u l u s f o r the subsequent s u c c e s s f u l search  herein. for  56.  5'-nucleotidase  i n pigeon hearts  T h i s enzyme was  found  5'-nucleotidase  of r a t h e a r t .  t o be  ( G i b s o n and  q u i t e d i f f e r e n t f r o m t h e membrane b o u n d Pigeon  s o l u b l e r a t h e r than p a r t i c u l a t e ; in  had  v e n t r i c l e 5'-nucleotidase  virtually  t h e 100,000 x g s u p e r n a t a n t .  5'-AMP and  Drummond, i n p r e p a r a t i o n ) .  a l l t h e enzyme was  I t showed a h i g h s p e c i f i c i t y  an a b s o l u t e r e q u i r e m e n t  f o r Mg  .  The  K  was  was found  for high,  3 a b o u t 10  times  g r e a t e r than  of 5'-nucleotidase the adenosine not  i n pigeon  hypothesis  the K  m  o f r a t h e a r t enzyme.  h e a r t c a n be  although  i n t e r p r e t e d as  i t s presence  discovery  support  for  a s a s o l u b l e enzyme i s  entirely consistent. The  g r e a t e s t p r o p o r t i o n o f c a r d i a c 5 ' - n u c l e o t i d a s e has  at s i t e s other than tissue,  t h e s u r f a c e membrane o f m u s c l e c e l l s .  5 ' - n u c l e o t i d a s e has  system, e n d o t h e l i a l c e l l s (Hardonk; 1968). the apparent  The  (Rostgaard  and  absence of a c t i v i t y  f o r m a t i o n at each l o c a t i o n . suggested  of coronary  f l o w may  Hence,  than a d i r e c t hypoxia  i n muscle c e l l s  "Hypoxic"  ( F i g . 7) p r o v i d e s  the b a s i s of these  be  the source  muscle  the p o t e n t i a l f o r  i n a steeper  cause a r e d u c t i o n i n p 0 e n d o t h e l i a l c e l l s may  p l a y an release.  of the c a p i l l a r y  r e l e a s e adenosine  This n u c l e o s i d e could then d i f f u s e  and an  might  f o r the r e g u l a t i o n  indirect Muscle  not  muscle  rather cell.  oxygen g r a d i e n t from c a p i l l a r i e s 2  ceils  adenosine  hypothesis  e n d o t h e l i a l c e l l s o f c a p i l l a r i e s and  r o l e as s t i m u l u s f o r a d e n o s i n e  T-  findings i t i s  of adenosine  o f h e a r t m u s c l e c e l l s may  could r e s u l t  m u s c l e and  fluid.  Specifically,  heart  i n endothelial cells  t h a t a minor m o d i f i c a t i o n of the adenosine  appropriate.  cells.  On  found  spaces,  B e h n k e , 1 9 5 9 ) , and  predominance of a c t i v i t y  been  In  been r e p o r t e d i n i n t e r c e l l u l a r  i n d i c a t i o n o f t h e p r o p o r t i o n o f enzyme and  be  The  to  endothelium.  to the  interstitial  i n t o the r e g i o n of  precapillary  57.  r e s i s t a n c e vessels and induce v a s o d i l a t i o n .  This would increase the  blood and oxygen supply to the myocardium and c o r r e c t the hypoxia as previously described (Berne, 1963).  In a d d i t i o n to i t s p o t e n t i a l  s i g n i f i c a n c e with respect to the r o l e of adenosine i n coronary autor e g u l a t i o n , t h i s d i s t r i b u t i o n of 5'-nucleotidase c a r r i e s i m p l i c a t i o n s pertinent to other areas of research such as membrane biochemistry and physiology. 5'-Nucleotidase has o f t e n been used as a membrane marker but i t s value as such should be questioned f o r two reasons.  F i r s t l y , the presence  of 5'-nucleotidase i n a given sample does not assure the presence of membrane.  For example, i f a f r a c t i o n of r a t l i v e r homogenate were found to  contain t h i s enzyme, i t might be concluded that membranes were also present.  Since r a t l i v e r contains a soluble 5'-nucleotidase ( F r i t z s o n ,  1969) contamination by t h i s s o l u b l e enzyme could give f a l s e p o s i t i v e results.  Secondly, the absence of '5'-nucleotidase does not n e c e s s a r i l y  i n d i c a t e the absence of membranes.  An i l l u s t r a t i o n of t h i s point may  be seen i n F i g . 7, p l a t e B which shows cardiac muscle c e l l membranes without 5'-nucleotidase a c t i v i t y .  I t i s p o s s i b l e then that heart muscle  c e l l membranes could be studied by the examination of a preparation i n which no 5'-nucleotidase could be detected.  While demonstration of the  presence or absence of membranes i s not the theme of t h i s t h e s i s , an . examination of the properties of membrane 5'-nucleotidase i s relevant. Analysis o f , c e r t a i n properties of r a t heart 5'-nucleotidase i n d i cates that enzyme a c t i v i t y and adenosine production may be enhanced during cardiac hypoxia and diminished during adequate oxygenation; r e g u l a t i o n by the metabolic s t a t e of the heart supports the hypothesis.  such  adenosine  On the basis of these p r o p e r t i e s , 5'-nucleotidase appears  58.  well  suited  of coronary support Mg  for,t h e appropriate formation of adenosine blood  flow.  f o r autoregulation  One c h a r a c t e r i s t i c w h i c h may b e i n t e r p r e t e d a s  f o r the adenosine  hypothesis  i s the p o s i t i v e c o r r e l a t i o n  c o n c e n t r a t i o n a n d enzyme a c t i v i t y .  S i n c e ATP i s known t o b e a n  I| e x c e l l e n t c h e l a t o r o f Mg  between  ... (Walaas, 1958),*the c e l l u l a r  concentration  ++ o f ATP c o u l d r e g u l a t e t h e amount o f Mg t h e enzyme.  Thus, a t h i g h e n e r g y c h a r g e  available f o r s t i m u l a t i o n of (Atkinson, 1964), a high  level  I | o f c e l l u l a r ATP w o u l d l i m i t  t h e amount o f u n c o m p l e x e d Mg  and r e s u l t  I j in  a l o w enzyme a c t i v i t y . . A t l o w e n e r g y c h a r g e ,  little  Mg  w o u l d be  n u c l e o t i d e b o u n d a n d a g r e a t e r q u a n t i t y w o u l d be a v a i l a b l e t o e n h a n c e 5'-nucleotidase  activity.  T h u s , s t i m u l a t i o n o f 5 ' - n u c l e o t i d a s e b y Mg  could f a c i l i t a t e p r o d u c t i o n of adenosine  during periods of hypoxia  when  energy charge i s reduced. Although  Mg  i s an e x c e l l e n t s t i m u l a t o r of 5 ' - n u c l e o t i d a s e , i t I |  may n o t n e c e s s a r i l y b e t h e c a t i o n i n v o l v e d i n v i v o . more p o t e n t  i n t h e heart  as a c a n d i d a t e  ( S p e c t o r , 1 9 5 6 ) , i t s h o u l d a l s o be  f o r the i n v i v o c o f a c t o r .  e f f e c t o n t h e enzyme a n d p r o b a b l y that N i  ficance,  is  a n d more e f f e c t i v e a s a s t i m u l a t o r o f t h i s e n z y m e , a n d i s  also present  finding  S i n c e Mn  I n comparison,  Ca  considered has  i s not involved i n i t s action.  s t i m u l a t e s t h e enzyme h a s some- m e t h o d o l o g i c a l  Specific  5 ' - n u c l e o t i d a s e a c t i v i t y has been determined  little The  signiusing  I | Ni as a s p e c i f i c i n h i b i t o r ; the value f o r 5'-nucleotidase catalyzed p h o s p h a t e r e l e a s e was c a l c u l a t e d b y s u b t r a c t i o n o f p h o s p h a t a s e a c t i v i t y  I | in  the. p r e s e n c e  nickel  of N i  from t h a t . i n i t s absence  s t i m u l a t i o n of t h i s  enzyme a n d t h e n i c k e l  (Campbell, induced  1962).  The  increase of  l e a d d e p o s i t i o n i n t i s s u e s e c t i o n s i n d i c a t e the dangers inherent i n the  I | above method.  Indeed, i n cases  i n which N i  i s without  inhibitory  59.  effect or i s stimulatory, false negative t h i s method were used t o e s t i m a t e The  r e s u l t s would be o b t a i n e d  i f  5'-nucleotidase.  regulation of 5'-nucleotidase  by energy charge i s probably  p e r f o r m e d more e f f e c t i v e l y b y a d i r e c t a c t i o n t h a n b y c h e l a t i o n o f divalent cation.  ATP i n h i b i t s  t h e enzyme;  i n h i b i t s b u t n o t a s w e l l as ATP.  c r e a t i n e phosphate a l s o  When t h e e n e r g y c h a r g e o f c a r d i a c  t i s s u e i s h i g h , t h e 5'-AMP c o n t e n t  i sdecreased  under i n v i t r o  conditions, 5'-nucleotidase  was  diminished.  s i m u l a t i o n of these During  (Williamson,  a n o x i a when e n e r g y c h a r g e i s l o w ,  1966); activity  i t i s expected  t h a t enzyme a c t i v i t y w o u l d b e e n h a n c e d b y r e m o v a l o f ATP a n d c r e a t i n e phosphate i n h i b i t i o n and by i n c r e a s e d adenosine formation of 5 ' - n u c l e o t i d a s e 5'-AMP s u p p o r t production and  speculated  The r e g u l a t i o n  by ATP, c r e a t i n e p h o s p h a t e , d i v a l e n t c a t i o n and  the adenosine hypothesis  This  because t h i s r e g u l a t i o n allows when v a s o d i l a t i o n i s r e q u i r e d  i s i n a g r e e m e n t w i t h B a e r e t a l . ' ( 1 9 6 6 ) who  i n an i n h i b i t e d  s t a t e a n d t h a t t h i s i n h i b i t i o n was removed'-!!  hypoxia. Some p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e  s t i m u l a t i o n a t low energy charge. this  Hence,  t h a t , i n w e l l o x y g e n a t e d t i s s u e , ATP c o u l d m a i n t a i n 5'-  nucleotidase during  c o u l d be enhanced d u r i n g h y p o x i a .  of adenosine during hypoxia  vice-versa.  substrate concentration.  arenot consistent with i t s  Such c h a r a c t e r i s t i c s  enzyme may n o t b e s u i t a b l e f o r a d e n o s i n e p r o d u c t i o n  when e n e r g y c h a r g e i s r e d u c e d .  Indeed, these  indicate that during  hypoxia  p r o p e r t i e s may e v e n  a b a s i s f o r some s k e p t i c i s m o f t h e a d e n o s i n e h y p o t h e s i s .  provide  F o r example,  b o t h ADP a n d o r t h o p h o s p h a t e a r e e f f e c t i v e i n h i b i t o r s o f r a t h e a r t 5'n u c l e o t i d a s e and t h e c o n c e n t r a t i o n s (Williamson, 1966).  of both increase during  hypoxia  T h u s i n c r e a s e d ADP a n d o r t h o p h o s p h a t e w o u l d  tend  60.  to  reduce adenosine  p r o d u c t i o n i n hypoxic  energy charge i s decreased. has been found ATP,  c a r d i a c t i s s u e i n which  I t must be emphasized t h a t because ADP  to be a more potent  i n h i b i t o r of 5'-nucleotidase  t h e proposed r e g u l a t o r y f u n c t i o n o f t h e l a t t e r n u c l e o t i d e  e t a l . , 1966) Two  than (Baer  must be c o n s i d e r e d d o u b t f u l .  o t h e r c h a r a c t e r i s t i c s o f 5 ' - n u c l e o t i d a s e which f o s t e r  q u e s t i o n i n g o f i t s proposed r o l e i n t h e f o r m a t i o n o f adenosine c o r o n a r y a u t p r e g u l a t i o n a r e i t s pH dependence and i t s broad specificity.  A t pH v a l u e s l e s s than optimum (8.5),  activity  decreased  with increasing a c i d i t y .  activity  could a c t u a l l y decrease  for  substrate  5'-nucleotidase  T h i s i m p l i e s t h a t enzyme  during the a c i d o s i s of anoxia.  If  the f u n c t i o n o f c a r d i a c 5 ' - n u c l e o t i d a s e i s t o produce adenosine  for  v a s o d i l a t i o n when t h e r e i s an oxygen d e f i c i t , an a c i d i c pH optimum would appear more advantageous because i t would f a v o u r i n c r e a s e d ade- • nosine formation during hypoxia.  T h i s i n t e r p r e t a t i o n should be tempered  w i t h the o b s e r v a t i o n t h a t the enzyme was a c t i v e over a broad thus pH c o n t r o l may not be an important The  substrate s p e c i f i c i t y  pH range;  r e g u l a t o r y mechanism.  o f r a t h e a r t 5 ' - n u c l e o t i d a s e was such  t h a t a l l n u c l e o s i d e 5'-monophosphates t e s t e d were h y d r o l y z e d ;  some were  b e t t e r s u b s t r a t e s than 5'-AMP.  I f t h i s enzyme i s t o c a t a l y z e the con-  v e r s i o n o f 5 '-AMP to adenosine  during hypoxia, a greater s p e c i f i c i t y f o r  '5'-AMP might have been expected. would be a h i n d r a n c e  t o adenosine  .Such a broad  substrate  specificity  f o r m a t i o n d u r i n g p e r i o d s o f low energy  charge i f s u b s t r a t e s o t h e r than 5'-AMP were b e i n g h y d r o l y z e d d u r i n g time.  Under these c o n d i t i o n s a d e n y l a t e d e p h o s p h o r y l a t i o n  tively  inhibited  ( F i g . 16) and adenosine  this  c o u l d be competi-  production consequently  diminished.  To  r e c a p i t u l a t e , the  c h a r a c t e r i s t i c s of 5'-nucleotidase  seem i n c o n s i s t e n t w i t h a r o l e f o r a d e n o s i n e i n c o r o n a r y are  i t s i n h i b i t i o n b y ADP  substrate nosine and  specificity.  hypothesis  are  and  o r t h o p h o s p h a t e , pH  which  autoregulation  profile  and  broad  Those c h a r a c t e r i s t i c s w h i c h support the the  i n h i b i t i o n b y ATP  and  ade-  c r e a t i n e phosphate  t h e p o s i t i v e r e l a t i o n s h i p b e t w e e n r e a c t i o n r a t e and  concentration  I|  o f b o t h s u b s t r a t e and The  foregoing  t i d a s e and based on  Mg  d i s c u s s i o n of  i t s p o s s i b l e r o l e i n the  two  identical with.the  the  accessible  I t was  could  i t should  by  with respect  t o ATP  and  ADP be  inhibition.  in a  -  was  be of  in vivo, is  A possibility  for vascular  still  regulation  might only  seems p a r t i c u l a r l y To  5'-  different  be  pertinent  further investigate  p r o f i t a b l e to c h a r a c t e r i z e the  the  enzyme f r o m  source.  other  d e s i r a b l e to examine c a r d i a c  T h e r e may  be  two  5'-nucleotidases  w h i c h does not  in limited  5'-nucleotidase  than r a t because c o n s i d e r a t i o n of  have been i n a p p r o p r i a t e f o r the  supply  studied.  This  the  was  enzyme i n t e r m s  i n t h e m y o c a r d i u m and  specific metabolites.  I t w o u l d be species  the  considered  i s that adenosine formation  a s s u m p t i o n , i t may  another  be  flow  vasodilation  a l s o assumed t h a t 5 ' - n u c l e o t i d a s e ,  occur at a d i s c r e t e s i t e  regulated  first  and  blood  I f t h i s proves to  to d i s c u s s  to the v a r i o u s m e t a b o l i t e s  to-be considered  may  inappropriate  5'-nucleo-  assumed t h a t  :  enzyme s t u d i e d h e r e i n .  adenosine hypothesis  cardiac  r e g u l a t i o n coronary  form adenosine f o r coronary  i t w o u l d be  framework.  p r o p e r t i e s of  S p e c i f i c a l l y , i „ i t was  assumptions.  n u c l e o t i d a s e w h i c h may  incorrect,  the  supply  study  the  rat heart  of- c o r o n a r y  in rat ventricle;  play a r o l e i n coronary  from  a  enzyme  autoregulation. one  i n abundant  a u t o r e g u l a t i o n and  which produces adenosine at a s i t e appropriate  another for  62.  vasodilation.  The  i s o l a t i o n procedures  used  t o d a t e may  i n l o s s o r d e s t r u c t i o n o f t h e l a t t e r o r i t may g r e a t e r a c t i v i t y of the former. l e v e l s a m o n g s t s p e c i e s may vasodilation. has  little  The  reflect  have  h a v e b e e n masked by  l a r g e v a r i a t i o n of  l e v e l s o f enzyme n o t  t o t a l 5 ' - n u c l e o t i d a s e , may  be a n e x c e l l e n t  involved primarily  involved i n  source  i n coronary a u t o r e g u l a t i o n .  i n c e l l s w i t h d i m i n i s h e d energy  production.  Although  to adenosine,  is  their potential for  that adenosine  I t c a n a l s o c o n v e r t IMP  i n the d e t e r m i n a t i o n of product  specific adenosine  i s formed wherever  to i n o s i n e .  formed by  of a d e n y l a t e deaminase because t h i s  the n a t u r e of s u b s t r a t e presented ventricular  the study of  5 ' - n u c l e o t i d a s e c a t a l y z e s t h e c o n v e r s i o n o f 5'-AMP  t h i s does not g u a r a n t e e  the a c t i v i t y  or  charge.  to f u r t h e r determine  t h i s enzyme i s f o u n d . importance  source  formation i n hypoxic tissue  a r e a f o r f u t u r e e n d e a v o u r s h o u l d be  t y p e s of. h e a r t c e l l s  this  It  t h e r a t h e a r t enzyme.• I t s p r o p e r t i e s  m i g h t be more a p p r o p r i a t e f o r a d e n o s i n e  Another  which because  i s p o s s i b l e t h a t the c h a r a c t e r i s t i c s of 5'-nucleotidase from q u i t e d i f f e r e n t from  a  5'-nucleotidase  T h e r e f o r e , h e a r t s of a s p e c i e s such as r a b b i t ,  t h i s enzyme m i g h t be  c o u l d be  resulted  :  Of  prime  5'-nucleotidase enzyme c a n  to 5'-nucleotidase.  dictate  For i n s t a n c e , i f  e n d o t h e l i a l c e l l s a r e r i c h i n a d e n y l a t e deaminase they  may  p r o d u c e l a r g e amounts o f i n o s i n e b u t o n l y s m a l l amounts o f  adenosine.  C o n v e r s e l y , e n d o t h e l i a l c e l l s may  adenylate  d e a m i n a s e and a d e n o s i n e If  the l a t t e r  may  i s observed,  be  o n l y have a low l e v e l of  the primary product of  e n d o t h e l i a l c e l l s m u s t be g i v e n s e r i o u s  c o n s i d e r a t i o n as t h e s o u r c e of a d e n o s i n e deaminase c o u l d determine  5'-nucleotidase.  i n heart.  whether adenosine  q u a n t i t i e s by c a r d i a c m u s c l e c e l l s .  Similarly,  adenylate  i s formed i n s i g n i f i c a n t  Therefore, the content of  adenylate  63.  deaminase of h e a r t mined.  m u s c l e c e l l s and  endothelial cells  A p r e r e q u i s i t e to these studies of  to o b t a i n  q u a n t i t i e s of pure h e a r t  required  endothelial cells.  enzymes a s of  described  t h e s e and  standard  t h e n a t u r e and certainty  by  Berry,  and  s i t e of adenosine formation  A n a l y s i s of  the  obtained  from the  crude p r e p a r a t i o n  may  from those of a p r e p a r a t i o n  and  sodium deoxycholate.  and  ATP  c o u l d be  exploited.  a d d i t i o n , analogues incapable the  general  enzyme w o u l d be  might c o n t r i b u t e  In order  With the  able  use  to e s t a b l i s h  tissue with  more  warranted;  a b o v e s o u r c e s may  w h i c h has  to use  properties  those of  Thus t h e  crude  acetone ADP  preparations,  p h o s p h a t a s e s w o u l d be  highly purified  extensive  5'-nucleotidase  5'-nucleotidase  by ADP.  pertinent.  been exposed to  very  required.  In  in  5'-nucleo-  e x a m i n a t i o n of  an  to a b e t t e r u n d e r s t a n d i n g of  T h e s e s t u d i e s and  preparations  be  reveal regulatory  Hopefully,  N a t u r e o f ADP  that cardiac  highly purified  of c a t i o n c h e l a t i o n would.be advantageous.  w e r e s i m i l a r , an  p h y s i o l o g i c a l f u n c t i o n of  powerfully  m i g h t be  i n cardiac  c h a r a c t e r i s t i c s o f c r u d e and  tidase preparations fied  isolation  I n these experiments:,analogues of b o t h  a n a l o g u e s r e s i s t a n t t o h y d r o l y s i s by  If  pure  than!has been p o s s i b l e .  5'-nucleotidase  different  potential for  a  employed  muscle c e l l s or  S c h e u e r ;.(1970).  m e t h o d s , one  C h a r a c t e r i z a t i o n o f b o t h c r u d e and of  of  tissue disintegration with proteolytic  Friend  biochemical  deter-  T h u s t i s s u e c u l t u r e m i g h t be  A n o t h e r t e c h n i q u e w h i c h has  of p a r t i c u l a r cell'.otypes i n v o l v e s  be  adequate supply  homogeneous p o p u l a t i o n the  cells.  i s an  should  the  a n a l y s i s such as the  puri-  this  i n vivo properties  and  heart.  Inhibition  S u l l i v a n and  i s i n h i b i t e d by r o l e o f ATP  as  Alpers ATP  but  ( 1 9 7 1 ) make i t c l e a r i s i n h i b i t e d more  a regulator  of  adenosine  64.  f o r m a t i o n d u r i n g h y p o x i a must be r e c o n s i d e r e d .  I n a d d i t i o n some c o n f u s i o n  e x i s t s r e g a r d i n g t h e k i n e t i c s o f ADP a n d ATP i n h i b i t i o n .  The a b i l i t y o f  I | ADP t o i n h i b i t reason  t h i s enzyme was r e d u c e d  f o rthis  i s not clear although  S u l l i v a n and A l p e r s ATP  o f Mg  there are a t least  (1971) have suggested  induced  o r impotent.  of MgC^  S p e c i f i c a l l y , Mg  pretation i s possible.  Mg  t o r e v e r s e ADP a n d  ...  was t h o u g h t  t i d e s so t h a t t h e y were e f f e c t i v e l y removed. I|  are either  5 ' - n u c l e o t i d a s e was a s c r i b e d t o I |  complex f o r m a t i o n .  the  t h a t t h e f r e e f o r m s o f ADP a n d  The a b i l i t y  i n h i b i t i o n of r a t heart  ;  two p o s s i b i l i t i e s .  a r e s t r o n g l y i n h i b i t o r y w h i l e t h e i r c o m p l e x e s w i t h Mg  much l e s s p o t e n t ATP  by t h e p r e s e n c e  to bind these  However, a n o t h e r  nucleo-  inter-  - a n t a g o n i s m o f ADP a n d ATP i n h i b i t i o n  may  I|  h a v e i n v o l v e d t h e enzyme r a t h e r t h a n t h e n u c l e o t i d e s . S i n c e Mg s t i m u l a t e d 5 ' - n u c l e o t i d a s e ( F i g . 9 ) , t h e i n c r e a s e i n a c t i v i t y due t o I|  a d d i t i o n of MgCl^  ( F i g . 1 8 ) m i g h t s i m p l y b e c a u s e d b y Mg  stimulation  o f t h e i n h i b i t e d enzyme. T h i s a l t e r n a t i v e e x p l a n a t i o n i s s u p p o r t e d by a n a l y s i s o f t h e d a t a o f F i g . 1 8 . The K f o r M g C l s t i m u l a t i o n o f ADP 0  o r ATP i n h i b i t e d  5 ' - n u c l e o t i d a s e was a b o u t , 1 0  M, v e r y  similar  to that  _3 for  t h e u n i n h i b i t e d enzyme ( 1 . 9 x 10  t h e Mg-ATP c o m p l e x , i . e . (Mg-ATP)/(Mg for  t h e Mg-ADP c o m p l e x  due  t o complex f o r m a t i o n , t h e K  M). I|  (Walaas, 1958).  r  a  The f o r m a t i o n c o n s t a n t f o r  )(ATP), i s about 8 times I f t h e MgCl^ e f f e c t were  f o r s t i m u l a t i o n o f t h e ATP  p r e p a r a t i o n should have been s u b s t a n t i a l l y lower ADP i n h i b i t e d p r e p a r a t i o n .  that  than  the  C o n t r o l o f 5 ' - n u c l e o t i d a s e b y Mg  simply  inhibited f o r the . I|  regulation  o f a d e n i n e n u c l e o t i d e i n h i b i t i o n a s d e s c r i b e d by S u l l i v a n and A l p e r s  (1971)  -Hseems cumbersome b e c a u s e a m e c h a n i s m f o r t h e b i n d i n g a n d r e l e a s e o f Mg must be h y p o t h e s i z e d . tide modulation  o f Mg  B u t i f t h e enzyme w e r e r e g u l a t e d b y a d e n i n e I|  s t i m u l a t i o n , no s u c h m e c h a n i s m n e e d b e  niicleo-  hypothesized.  65.  A means f o r t h e s y n t h e s i s and i s w e l l known. i n ADP  and  d e g r a d a t i o n o f t h e s e Mg  -complexing  I t i s quite possible that 5'-nucleotidase  ATP  c o u l d be  inhibited  d i r e c t l y by  agents  in a cell  these substances  rich  and  I j i n d i r e c t l y by cell  c h e l a t i o n o f Mg  poor i n these  ;  t h e r e v e r s e m i g h t be  expected  in a  substances.  Since t h e r e i s the p o s s i b i l i t y  t h a t two  separate,  synergistic  I | a c t i o n s o f Mg  w o r k t o i n c r e a s e t h e a c t i v i t y o f ADP  enzyme, t h e e f f e c t o f e a c h s e p a r a t e In  order  logues  a c t i o n w o u l d be  o r ATP difficult  t o r e s o l v e t h i s p r o b l e m i t m i g h t be p r o f i t a b l e  of these  inhibitors.  inhibited to  assess.  to employ ana-  Those analogues w h i c h would not  complex  I | Mg  but  inhibit  t h e enzyme w o u l d a l l o w d e t e r m i n a t i o n o f t h e s t i m u l a t o r y  I | e f f e c t o f Mg  on  inhibited  5'-nucleotidase without  i n t e r f e r e n c e by  chelation. T h e r e a p p e a r s t o be  considerable disagreement concerning  the  m a n n e r i n w h i c h r a t h e a r t 5 ' - n u c l e o t i d a s e . i s i n h i b i t e d b y ATP  and  I n h i b i t i o n by  these  competitive  (Baer e t a l . ;  1966;  non-competitive from these Mg  substances  has  S u l l i v a n and  ( E d w a r d s and  :  1970).  l a b o r a t o r i e s a r e c o m p l i c a t e d by  mixed c o m p e t i t i v e -  In a d d i t i o n the  the presence  In the present  s t u d y ADP  s u b j e c t e d to f u r t h e r a n a l y s i s i n an a t t e m p t  discrepancies. inhibited  A l p e r s , 1971) a n d  Maguire,  i n the r e a c t i o n m i x t u r e s .  b i t i o n was  been v a r i o u s l y r e p o r t e d as  S u l l i v a n and  5'-nucleotidase  Alpers  results  or absence of induced  inhi-  to e x p l a i n these  ( 1 9 7 1 ) r e p o r t e d t h a t 0.0017 mM  in a strictly  ADP.  ADP  c o m p e t i t i v e manner i n t h e  l [ a b s e n c e o f Mg  .  In comparison,  F i g . 14  shows t h a t , i n t h e a b s e n c e o f  I | Mg  , 0.0033 mM  .manner. two  ADP  inhibited  i n a mixed  competitive-non-competitive  I f t h e d a t a o f F i g . 1 6 . a r e p l o t t e d by  b i n d i n g s i t e s f o r ADP  are indicated.  the method of D i x o n  I f one  site  (1953),  of b i n d i n g were  the  66.  a c t i v e s i t e o f t h e enzyme t h i s w o u l d a c c o u n t f o r t h e c o m p e t i t i v e c o m p o n e n t of  inhibition.  component.  Binding a t another could account f o r the  When 16 mM M g C ^  was p r e s e n t  r e s u l t s were q u i t e d i f f e r e n t ;  i n the reaction mixtures  only non-competitive  inhibition  o b s e r v e d a n d o n l y o n e ADP b i n d i n g s i t e was i n d i c a t e d . these in  observations,  two f o r m s ;  non-competitive  the f o l l o w i n g model i s proposed.  was  On t h e b a s i s o f The enzyme  exists  o n e i n w h i c h ADP i n h i b i t s c o m p e t i t i v e l y and h a s a l o w  f o r ADP a n d a n o t h e r i n w h i c h ADP i n h i b i t s n o n - c o m p e t i t i v e l y a higher  the  f o r ADP.  Mg  I |  that only non-competitive  stabilizes  inhibition  t h e enzyme i n t h e l a t t e r i s observable  and has f o r m so  when t h e enzyme i s  j | s a t u r a t e d w i t h Mg  . I n addition to favouring a particular  conformation,  I| . Mg  a l s o s t i m u l a t e s t h e enzyme b y i n c r e a s i n g t u r n o v e r  r a t e which would  I | e x p l a i n t h e Mg i n d u c e d i n c r e a s e i n maximum v e l o c i t y w i t h no c h a n g e i n K . I f such conformations e x i s t i n v i v o , t h e i r s i g n i f i c a n c e t o the m ° p h y s i o l o g i c a l r o l e of 5'-nucleotidase o n l y be s p e c u l a t e d  at this  time.  or coronary  autoregulation  could  67.  LITERATURE CITED  AFONSO, S. • 1969 C o r o n a r y induced 216:  v a s o d i l a t o r responses  t o hypoxia and  t a c h y c a r d i a b e f o r e and a f t e r l i d o f l a z i n e .  Am. J . P h y s i o l .  297-300.  ANREP, G.V. a n d BARSOUM, G.S.  1935 A p p e a r a n c e o f h i s t a m i n e i n t h e  venous b l o o d d u r i n g muscular  contraction.  J .Physiol.  85_: 4 0 9 -  420. ANREP, G.V., BARSOUM, G.S. a n d TALAAT, M. mine by t h e h e a r t muscle. 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