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Xanthine oxidase in the lung Wilson, Wendy Lee 1987

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XANTHINE OXIDASE IN THE LUNG  by  Wendy Lee W i l s o n B.A., U n i v e r s i t y o f T o r o n t o , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y )  We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA vToAg. 1987 t  ©Wendy  L. W i l s o n , 1987  In presenting degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  thesis for scholarly by  this  his  or  her  Department of The University of British Columbia 1956 Main Mall ' Vancouver, Canada  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  DE-6(3/81)  the  for  an  advanced  Library shall make it  that permission  for extensive  granted  head  is  by the  understood be  that  allowed without  of  my  copying  or  my written  -i -  ABSTRACT The  generation  xanthine  oxidase  reperfusion  to  free  free  lung  injury  research  the  rat  (0^')  i n the  homogenates. by  lung  XO  hydrogen has  has  not  other  by  analysis  of  xanthine  raised  SDS-polyacrylamide  Western  blotting.  detected XO  Rat  kidney  was  by  After  immunologically and  liver  i n porcine  Thus, and  of  e n  in XO  in  Therefore, i s present  in porcine,  bovine,  oxidase  i n the  milk  tissues  XO.  Proteins  g e l e l e c t r o p h o r e s i s of  tissue  P r o t e i n s were t r a n s f e r e d from the g e l s to n i t r o c e l l u l o s e  antibody.  porcine  yB  demonstrated  to bovine  incubating  i t s r e a c t i o n with  kidney.  0 x  tissues.  filter  detectable  H  studied.  of  superoxide  t o determine whether XO  antibody  lung,  of  ^ 2^2'  to i n v e s t i g a t e i t s c h a r a c t e r i s t i c s  or  conversion  involvement  y e t been  enzyme,  post-ischemic  production  been  possible  a n t i s e r a c o n t a i n i n g the was  in  peroxide  also  designed  the c y t o s o l i c  c a t a l y z e s the  The  p o l y c l o n a l antibody  separated  filters  implicated  concomitant  and  disease.  and human l u n g and  on  the  injury  p r o j e c t was  lung and  employed  been  with  Immunochemical  were  has  radical-mediated  oxidative  in  urate  radical  inflammatory  this  (XO),  damage i n s e v e r a l organs.  hypoxanthine anion  of oxygen f r e e r a d i c a l s by  the  r a t XO.  but  antibody  to  XO  filters  t o the p u r i f i e d bovine  not  p r o t e i n was  XO  i n bovine  not  XO  XO  lung  reactivity.  detectable  bovine  XO.  with  was  XO  i n porcine  a  on  an enzyme-conjugated  detectable  a l l showed  liver  the  the  second  and  milk.  was lung  or  c r o s s - r e a c t i v e with  immunologically  detectable  in  -ii-  human  lung  p o s s i b l y because  the bovine  t h e antibody was n o t c r o s s r e a c t i v e w i t h  antibody.  In  vivo,  xanthine  oxidase  dehydrogenase  rather  dehydrogenase  (XDH) t h e enxyme  H^O^-  The a c t i v i t y  tissues pterin  using which  (IXP).  does  In t h i s  form  not produce  as  as  converted  0~" o r  i n several  although  substrate,  t o the f l u o r e s c e n t  i n porcine  liver  XO a c t i v i t y  a  xanthine  either  XDH and XO was measured  XO a c t i v i t y  protein/min  lung  protein/min,  o f both  is catalytically  _3 10 ug IXP/mg porcine  an o x i d a s e .  predominantly  a f l u o r o m e t r i c assay which uses an a r t i f i c a l  isoxanthopterin  in  than  exists  product  was o f 1.1 x  was not d e t e c t a b l e  _2 i n r a t l u n g o f 1.7 x 10 pg IXP/mg _2 o f 1.5 x 10 pg IXP/mg p r o t e i n / m i n , and  and k i d n e y ,  r a t kidney _2  rat  liver  biopsy for  o f 2.2 x 10  ug IXP/mg  samples were o b t a i n e d  viability  assayed activity  Three  indicating  tissue  result  of  limited  oxidation  of c r i t i c a l  the  conversion  o f 5.35 x 1 0  human  reversible  - 6  activity  showed  lung  by  a  t h i o l groups.  o f XDH t o XO were  oxidase  one o f t h e s e  three  yg IXP/mg p r o t e i n / m i n .  Ca^ /calmodulin  conversion  and then  NADH  o f XDH t o XO i s thought  proteolysis  whereas  samples  v i a b i l i t y , but only  conversion  protease,  o f NADH o x i d a s e  o f these  showed measurable XO a c t i v i t y Irreversible  Seven  a f t e r l u n g r e s e c t i o n and i n i t i a l l y t e s t e d  by d e t e r m i n a t i o n  f o r XO-XDH.  protein/min.  +  t o be t h e activated  o f t h e enzyme  occurs  by  S t u d i e s on the r a t e and n a t u r e o f conducted  i n r a t lung  using the  -iii-  fluorescence forms.  assay  incubation  of  conversion  activities  o f 90% o f the XDH  o f both  effects  t o XO.  of free C a  2 +  groups w i t h  caused  In c o n t r a s t , conversion of  T h i s c o n v e r s i o n was r e v e r s i b l e  80% by r e d u c t i o n o f t h i o l  enzyme  f o r 60 min  o f homogenates a t 15°C f o r 10 hours caused  o f t h e XDH t o XO.  using  catalytic  I n c u b a t i o n o f l u n g homogenates w i t h t r y p s i n  irreverisble  100%  to detect  to the extent  dithiothreitol  (DTT).  The  on t h e c o n v e r s i o n o f XDH t o X0 was examined by  EDTA, a c h e l a t o r o f Ca  2+  and o t h e r d i v a l e n t  c a t i o n s ; and EGTA,  2+ a more  specific  c h e l a t o r o f Ca  agents  d u r i n g homogenization  tissue  d i d not i n h i b i t  The presence  of e i t h e r  reversible  a c t i v i t y was r e v e r s i b l e by DTT. prepared  .  of these  chelating  normoxic o r i s c h e m i c r a t l u n g  enzyme c o n v e r s i o n .  Increased  XO  In t h e normoxic r a t l u n g , homogenates  w i t h EDTA and EGTA showed a s i m i l a r  t o XO which was r e v e r s i b l e t o 70% w i t h DTT.  c o n v e r s i o n o f 95% o f XDH In the i s c h e m i c r a t l u n g ,  samples prepared w i t h EDTA and EGTA showed a ' c o n v e r s i o n o f 80% and 95% XDH  t o XO  which  reversibility perfusion cannula  was  t o XDH  similar was  o f r a t lungs  to control  75% w i t h  with  EDTA  DTT  samples.  The e x t e n t o f  incubation.  In  addition,  and DTT v i a a pulmonary  p r i o r t o 60 min o f i s c h e m i a and homogenization  artery  d i d not a f f e c t  the e x t e n t o f XDH t o XO c o n v e r s i o n . These proteolytic in  results conversion  t h e r a t lung  indicate  that  irreversible  Ca^ -mediated +  o f XDH t o XO does not occur t o a g r e a t  during  either  normoxia  or  ischemia.  extent  However,  -iv-  reversible  conversion of XDH to XO does occur,  suggesting that  reversible thiol dependent conversion may play a role in the lung under both physiological and pathophysiological states.  -V-  TABLE OF CONTENTS  Page ABSTRACT  i  TABLE OF CONTENTS  iv  ABBREVIATIONS  v i i  LIST OF TABLES  ix  LIST OF FIGURES  X  ACKNOWLEDGEMENTS  ,  x i i  DEDICATION  xiii  INTRODUCTION Xanthine  1 oxidase i n the lung  9  Oxygen f r e e r a d i c a l damage a s s o c i a t e d w i t h reperfusion  ischemia-  i n j u r y i n other t i s s u e s  12  B i o c h e m i c a l aspects o f x a n t h i n e o x i d a s e  16  Purpose o f these s t u d i e s  23  METHODS Methods f o r d e t e c t i o n of x a n t h i n e  24 dehydrogenase-  oxidase a c t i v i t y  24  Experiments u s i n g p u r i f i e d x a n t h i n e o x i d a s e  29  Experiments i n p o r c i n e t i s s u e  36  Experiments i n r a t t i s s u e  43  Experiments i n human l u n g t i s s u e  49  -vi-  Page STATISTICAL ANALYSIS  50  RESULTS  51  Experiments w i t h p u r i f i e d XO  51  Experiments  i n porcine tissue  61  Experiments  in rat tissue  71  Experiments  i n human l u n g t i s s u e  83  DISCUSSION  86  Use o f p u r i f i e d b o v i n e m i l k XO i n t h e s e s t u d i e s  86  Use o f IXP assay i n t h e s e experiments  87  Comparison o f r e s u l t s from these s t u d i e s w i t h those listed  i n the l i t e r a t u r e  C o n v e r s i o n o f XDH t o XO i n the r a t l u n g  91 94  Consquences o f c o n v e r s i o n o f XDH t o XO f o r the organism.... 97 REFERENCES  101  -vii-  ABBREVIATIONS  ARDS  Adult respiratory  ATP  Adenosine t r i p h o s p h a t e  BSA  Bovine  Ca  Divalent  2 +  serum  albumin  calcium  DNA  Deoxyribonucleic  DTT  Dithiothreitol  E  M(295)  d i s t r e s s syndrome  cation acid  Molar e x t i n c t i o n  c o - e f f i c i e n t a t 295 nm i n  gm-moles/1 w i t h a 1cm l i g h t  path  EDTA  Ethylenediamine t e t r a a c e t i c  acid  EGTA  Ethylene g l y c o l t e t r a a c e t i c  acid  ELISA  Enzyme-linked-immunosorbant  assay  FAD  F l a v i n adenine  2 2  dinucleotide  Hydrogen p e r o x i d e  HBSS  Hanks b a l a n c e d s a l t  HMW  High m o l e c u l a r weight  HRP  Horseradish peroxidase  IXP  Isoxanthopterin  KDa  Kilodaltons  KPi  P o t a s s i u i m phosphate  M199  Gibco Medium 199  MW  Molecular  weight  solution standards  buffer  -viii-  NAD  N i c o t i n a m i d e adenine  dinucleotide  NADH  N i c o t i n a m i d e adenine  d i n u c l e o t i d e i n t h e reduced  form "  Superoxide  anion f r e e  radical  OH  Hydroxyl f r e e  PBS  Phosphate b u f f e r e d s a l i n e  PMN  Polymorphonuclear  PMSF  radical  leukocyte  Phenylmethylsulfonylfluoride  SE  Standard  SDS-PAGE  Sodium d o d e c y l / s u l p h a t e ( d e n a t u r i n g ) p o l y a c r y l a m i d e gel  e r r o r o f t h e mean  electrophoresis  XO  Xanthine  oxidase  XDH  Xanthine  dehydrogenase  XDH-XO  Xanthine  o x i d a s e - x a n t h i n e dehydrogenase  enzyme  activity  total  - i xLIST OF TABLES Page  I  D i s e a s e s f o r which Xanthine Oxidase M e d i a t i o n has been suggested  II  V a l u e s f o r XDH + XO and XO i n Normoxic Rat Lung a f t e r treatment w i t h C a l c i u m C h e l a t i n g Agents  III  76  V a l u e s f o r XDH + XO and XO i n Ischemic Rat Lung a f t e r treatment w i t h C a l c i u m C h e l a t i n g Agents  IV  2  80  E f f e c t o f P e r f u s i o n w i t h EDTA and DTT on t h e C o n v e r s i o n of XDH t o XO  85  -X-  LIST OF FIGURES  Page 1.  Reactions  of R e a c t i v e Oxygen S p e c i e s  .  2.  E f f e c t s of Oxygen Free R a d i c a l s on C e l l Components  3.  A c t i v a t i o n of N e u t r o p h i l s by Oxygen Free  7  Radical  D e r i v e d Chemotractants of XO  4  17  4.  Reactions  5.  SDS-PAGE of P u r i f i e d Bovine XO  52  6.  Western B l o t of P u r i f i e d XO  54  7.  Western B l o t of Bovine T i s s u e s  55  8.  Western B l o t of Rat T i s s u e s  56  9.  Western B l o t of P o r c i n e T i s s u e s  58  10.  Western B l o t of Human Lung T i s s u e  59  11.  Lineweaver-Burke P l o t of XO  60  12.  XO A c t i v i t y  i n Porcine L i v e r  62  13.  XO A c t i v i t y  i n P o r c i n e Lung  63  14.  XO A c t i v i t y  i n P o r c i n e Kidney  64  15.  I n h i b i t i o n of P u r i f i e d XO  16.  Heat S t a b i l i t y of A c t i v e F a c t o r s i n P i g T i s s u e  67  17.  E f f e c t of P i g T i s s u e E x t r a c t on R e l a t i v e F l u o r e s c e n c e  68  18.  M i c r o v a s c u l a r E n d o t h e l i a l C e l l s I s o l a t e d by C e n t r i f u g a l Elutriation  i n Ischemia and R e p e r f u s i o n  by P i g T i s s u e  22  65  70  -xi-  Page 19.  XO  i n Rat Kidney  73  20.  XO  i n Rat Lung  74  21.  Effect  22.  E f f e c t of O v e r n i g h t I n c u b a t i o n at 15°C on  of T r y p s i n Treatment  on  (XDH  + XO)  XO R a t i o (XDH  +  XO):  XO R a t i o 23.  78  E f f e c t o f C a l c i u m on R e v e r s i b i l i t y  of XDH  to  XO C o n v e r s i o n i n Normoxic Rat Lung 24.  Effect  77  of C a l c i u m on R e v e r s i b i l i t y o f XDH  79 t o XO  i n Ischemic Rat Lung  81  25.  XO A c t i v i t y i n Human Lung  26.  C o n v e r s i o n o f Rat L i v e r  27.  Proposed  Role of XO  Capillary  Conversion  from XDH  84 t o XO  92  i n Defence Mechanism o f  Endothelial Cells  98  -xiiACKNOWLEDGEMENTS Many p e o p l e have had important c o n t r i b u t i o n s t o t h i s p r o j e c t w i t h o u t which none o f t h i s would be p o s s i b l e . There a r e those who c o n t r i b u t e d t h e i r l e a r n i n g and t e c h n i c a l e x p e r t i s e as w e l l as those whose i n s i g h t support and i n s p i r a t i o n were i n v a l u a b l e . I would l i k e t o thank my s u p e r v i s o r , Dr. Anne A u t o r f o r h e r t e a c h i n g , Dr. Margo Moore f o r t e a and sympathy and e x p e r t g u i d a n c e , Dr. P e t e r Dodek f o r h i s u s e f u l commentary and c e l l i s o l a t i o n s u p e r v i s i o n , M i c h a e l B r e e d v e l d f o r the i s o l a t i o n o f XO and comic r e l i e f , Dr. Rob. T h i e s and Dr. J i n n y Hobson f o r t h e i r p e r s p e c t i v e , M a r y e t t e Mar f o r h e r r e s o u r c e f u l n e s s , c e l l c u l t u r e and t o l e r a n c e , Dr. M i c h a e l Weaver f o r the ELISA t e c h n i q u e , Joe Comeau and B a r r y Wiggs f o r computer h e l p , M i c h e l l e M u l l e n who taught me t h e a r t i n s c i e n c e , Dr. Marlene R a b i n o v i t c h my f i r s t mentor, t h e o t h e r Wendy W i l s o n , C a r l a Westerbeek-Marres, L o r i e Keenan, Wendy B a r r e t t , I n g r i d Pacey and B r i d g e t Milsom f o r t h e i r i n s i g h t f u l d i s c u s s i o n . E x t r a s p e c i a l thanks t o Jochen Wolfgang Boehm f o r bunny r a b b i t care and l i b a t u m . Many thanks t o D e n i s e J a c k s o n and Lee Kowk f o r e x p e r t t y p i n g a s s i s t a n c e .  DEDICATION  I  would  like  to  dedicate  this  to  all  those who were  instrumental in my leaving Toronto to "go West Young Woman" and pursue this degree in B . C . , especially Dr. Anne Autor who invited me, my parents and family,  Dr. Marlene Rabinovitch, Dr. Hersch Rosenberg,  Helen Ives, Dr. Paul Adonis Hamel, Dr. D.G. Butler, and Michelle Mullen.  -1INTRODUCTION: Oxygen free radicals are known to be involved i n the etiology of disease  states  induced by the toxic  pesticides  and chemotherapeutic  recognized  that  important  endogenously  i n responses  tissue damage.  effects  agents.  generated  to injury  of radiation, xenobiotics,  I t i s now oxygen  free  becoming radicals  such as inflammation  better also are  and post-ischemic  The enzyme system xanthine dehydrogenase: xanthine oxidase,  which i n the oxidase form i s capable of generating oxygen free r a d i c a l s i n the oxidation of hypoxanthine  and xanthine to urate, i s considered to play  an important role i n free r a d i c a l induced reperfusion injury, as well as i n several other disease states (Table 1). The pathogenesis of several forms of pulmonary injury have been linked to oxygen free r a d i c a l formation. thesis  investigates  the presence  dehydrogenase: xanthine  oxidase  and  characteristics  of  in the lung with p a r t i c u l a r  This  xanthine interest i n  whether oxygen free r a d i c a l s produced by xanthine oxidase could be important in  inflammatory  lung disease.  In order to best introduce the topic of  xanthine oxidase in the pulmonary microvasculature i t w i l l be necessary to provide a b r i e f introduction to free r a d i c a l biology i n general and explain why t h i s area i s of p a r t i c u l a r concern i n pulmonary disease.  Oxygen Free Radicals: Overview In  1878 Paul  Bert  showed  that  the element  considered to be the very sustenance of aerobic l i f e , the brain, lungs and other organs of animals.  oxygen,  long  could be damaging to  This discovery introduced a  very paradoxical concept that was not well accepted.  I t was almost another  -2TABLE I  DISEASES FOR WHICH XANTHINE OXIDASE MEDIATION HAS BEEN SUGGESTED ACUTE PANCREATITIS ' 1  STREPTOZYTOCIN INDUCED DIABETES MELLITUS ISCHEMIC BOWEL DISEASE GASTRIC ULCERS  2  2  NECROTIZING ENTERITIS CARCINOGENESIS  1  2  3  PRODUCTION OF CLASTOGENIC FACTORS RHEUMATOID ARTHRITIS  3  4  4  GOUT XANTHINURIA STROKE  4  4  DISSEMINATED INTRAVASCULAR COAGULOPATHY  4  ADULT RESPIRATORY DISTRESS SYNDROME  5  TRANSIENT RENAL ISCHEMIA LEADING TO ACUTE RENAL FAILURE ACUTE TUBULAR NECROSIS POST-KIDNEY MYCARDIAL  TRANSPLANTATION  INFARCTION WITH REPERFUSION  6  6  ?  As p e r . 1. Sanfy H, S a r r MG, B u l k l e y GB, Cameron J L . , S u p p l . 548, V o l . 126, A c t a P h y s i o l . Scand. 109-119, 1986. 2. Parks DA, Granger DN, G a s t r o i n t e s t . L i v e r P h y s i o l . 8:G285-G289, 1983. 3. E m e r i t I , Khan SH, C e r u t t i P. J . F r e e Rad. B i o l . Med. 1:51-57, 1985a. 4. Robbins R, C o t r a n R, Kumar P. P a t h o l o g i c B a s i s o f D i s e a s e , 1985. 5. Saugstad OD. Ped. Pulmonary 1:167-175, 1985. 6. Baker GL, C o r r y RJ, Autor AP. Ann. Surg. 202:628-641, 1985. 7. Hearse DJ, Manning AS, Downing JM, Y e l l o n DM. Acta Physiol. Scand. S u p p l . 548:65-78, 1986.  -3century  before Rebecca Gerschman f i r s t  suggested  that  the mechanism of  oxygen poisoning  may have a common mechanism with radiation damage, based on  the  that  observation  i r r a d i a t i o n of oxygenated  formation of highly reactive oxygen species  solutions  caused the  known as oxygen free radicals  (1). A free r a d i c a l i s an atom or molecule which incorporates electron  into i t s outermost o r b i t (2).  These species  chemical r e a c t i v i t y due to t h e i r a b i l i t y to abstract molecules. as  have a very high  an electron from other  Because of i t s electronic structure, ground state oxygen exists  a d i r a d i c a l , O^, unlike  state.  an unpaired  most elements which e x i s t  i n the s i n g l e t  In the absence of a c a t a l y s t , because of Hund's Rule oxygen is most  e a s i l y reduced by four sequential univalent electron additions rather than a single concerted four electron reduction.  Sequential  create  and e s t a b l i s h the potential for  the  short  l i v e d reactive  incomplete reduction  Figure  intermediates,  reductions  of oxygen, thus producing oxygen free r a d i c a l s (See  1)(3). In  aerobic  many biochemical  organisms, free radicals are an e s s e n t i a l component to processes.  The c a t a l y t i c action  dependent upon the generation of free r a d i c a l s . of ATP by the mitochondrial radical  univalent  intermediates  electron transport  i n the Q cycle  of enzymes  i s often  For example, the production chain requires  organic free  and at NADH dehydrogenase (4).  However, a paradox exists due to the highly reactive nature of free radicals which makes them toxic unless kept i n check.  -4REACTIONS OF REACTIVE OXYGEN SPECIES  0  2  05•  +05-  0 -  + Fe +X  One e l e c t r o n r e d u c t i o n o f oxygen  +  2  + Fe  2 +  2  _^ 0  2  0 2  + H 0  Reduction  2  2  + 0  Dismutation o f supero x i d e t o form hydrogen p e r o x i d e and oxygen  2  + Fe2+X  HO-  X  Reaction:  F i g u r e 1.  The pKa o f t h i s r e a c t i o n i s 4.8  2  _^ H 0  + 2H+  3  2  Net  <>2'  -> H 0  + H  2  Description  + le"  05-  H 0  Reactions  Reduction o f i r o n c o n t a i n i n g compounds such as f e r r i t i n  + OH" + F e  iron  2  v0 catalyst  Reactions  2  3 +  X  + HO-  o f Oxygen  Generation of hyroxyl f r e e r a d i c a l s from hydrogen p e r o x i d e i n the presence o f reduced i r o n ; t h e Fenton R e a c t i o n  + OH  -  Net r e a c t i o n f o r the g e n e r a t i o n o f h y d r o x l f r e e r a d i c a l s from supero x i d e and hydrogen p e r o x i d e  -5The  cell  intracellular of  0^  pH,  is  oxygen  produces  and  free  0^  o f 0^"  thus  whose  rates  reaction.  These  include  highly  acid,  heme  reactive  different 1).  0^').  r a p i d l y dismutate the  of r e a c t i o n compounds  proteins  with  several  (designated  limiting  substrates  ascorbic  to  r a d i c a l s (Figure  superoxide  two m o l e c u l e s  (H^O^)  exposed  most  such  (5) ( 6 ) .  oxidant  referred  t o as t h e Fenton r e a c t i o n ( a c c o r d i n g  hydroxyl  radical  step  this  oxidation  generation is  free  i s termed  abstract  The i n i t i a t i o n  react  with  the  2°2  i  t  with dismutation  c, q u i n o n e s , s  l f  e  *  s  n  °t  i n the  i r o n the e x t r e m e l y  to Figure with  peroxide  However, H^O^  i n a reaction  1, R e a c t i o n  little  until  of  5).  selectivity,  the f r e e  cleavage  of  organic  The at  free  further  radical  o f one o r g a n i c  causing  This  OH'.  r e a c t i o n , thus This  from the  second  free radical  molecules.  r a d i c a l s a r e quenched  by  i s produced  electrons  i n a succession.  organic  molecules  the t a r g e t molecule  f u r t h e r free r a d i c a l s i n a chain  oxidative  another f r e e r a d i c a l radical.  An o r g a n i c  o f more f r e e r a d i c a l m o i e t i e s  ultimate  hydrogen  i s generated  oxidation  o f an e l e c t i o n from  can i t s e l f  termed p r o p a g a t i o n .  continues  compounds  mediated  initiation.  which  cascade p r o p a g a t i n g the  radical  by a b s t r a c t i o n  This  physiologic  to  as f e r r o u s  OH'(6)  reduction  i n t h e range o f 1 x 10^ t o 1 x 1 0 ^ M *S ^ ( 9 ) .  Hydroxyl proceeds  organic  H  (8).  powerful  reaction rates  such  radical  at  as f e r r i c y t o c h r o m e  compounds  t r a n s i t i o n metals  oxidizes  0^'  are c o m p e t i t i v e  o f reduced  free  However,  of  presence  hydroxyl  The one e l e c t r o n  t o produce  ability  and t h i o l s  organic  e x t r a c e l l u l a r and  step  starts a causing  process  e i t h e r by i n t e r a c t i o n w i t h  o r by a m o l e c u l e which does not i n t u r n produce a f r e e  -6In v i v o , systems.  r e a c t i v e oxygen s p e c i e s  These  neutrophils,  include  t h e cytochrome  prostaglandin  purine  nucleotides.  oxygen  r a d i c a l s are a l s o  states,  P450  i n a v a r i e t y o f enzyme  system,  NADPH  oxidase  s y n t h e t a s e and x a n t h i n e o x i d a s e which  In a d d i t i o n  pesticides,  are produced  t o these  generated  from  chemotherapeutic  normal  exposure  agents,  physiologic  catabolizes processes,  to r a d i a t i o n ,  tobacco  smoke  of  hyperoxic  and  aromatic  hydrocarbons ( 1 0 ) . In o r d e r cell  contains  commented not  be  numerous p r o t e c t i v e a n t i o x i d a n t  antioxidant  to  survive  mechanisms  species  s c a v e n g e r s , which  and  The most  dismutase,  which  glutathione  not  i t s oxidizing  employ low  catalyzes  Low  both  molecular  weight  which  (11).  both  referred  molecular and  weight  glutathione  enzymes  the r e d u c t i o n  scavengers a c t by  such  being  remove to  as  further  free  are superoxide  of superoxide;  catalyze  These  catalytically  compounds  o f the a n t i o x i d a n t  the dismutation  which  B-carotene  defenses t h e c e l l would  environment"  enzymes  As F r i d o v i c h has  f r e e r a d i c a l s without generating  important  peroxidase,  t o H^O.  a-tocopherol,  in  react with  radicals.  peroxide  mechanisms.  " i f i t were not f o r a group o f a n t i o x i d a n t  able  reactive  t o c o n t r o l p o t e n t i a l l y c y t o t o x i c f r e e r a d i c a l r e a c t i o n s , the  as  catalase of  and  hydrogen  ascorbate,  oxidzied  become f r e e r a d i c a l s themselves. Even w i t h t h i s complex a r r a y o f p r o t e c t i v e mechanisms, i n a  but do  -7CELLULAR FREE RADICAL TARGETS  Consequence  Target  Low M o l e c u l a r Weight  Molecules  Unsaturated & thiol-containing amino a c i d s  Protein denaturation & c r o s s l i n k i n g , enzyme i n h i b i t i o n O r g a n e l l e and c e l l p e r m e a b i l i t y changes  Nucleic bases  acid  Cell  c y c l e changes,  Carbohydrates  Cell  s u r f a c e r e c e p t o r changes  Unsaturated  lipids  Cofactors  mutations  Cholesterol & fatty acid oxidation L i p i d cross-linking Organelle & c e l l p e r m e a b i l i t y changes Deceased n i c o t i n a m i d e & f l a v i n containing cofactor a v a i l a b i l i t y a c t i v i t y , ascorbate o x i d a t i o n , porphyrin oxidation  &  Neurotransmitters  Decreased n e u r o t r a n s m i t t e r a v a i l a b i l i t y (e.g. s e r o t o n i n , epinephrine  Antioxidants  Decreased a v a i l a b i l i t y , i n c l u d e s a - t o c o p h e r o l & B-carotene  Macromolecules Protein DNA Hyaluronic  F i g u r e 2.  acid  Peptide chain s c i s s i o n Strand s c i s s i o n Change i n s y n o v i a l f l u i d  viscosity  E f f e c t s o f oxygen f r e e r a d i c a l s on c e l l u l a r components. Reproduced from Freeman and Crapo 1982, ( 1 0 ) .  -8strongly o x i d i z i n g environment a l l of these antioxidant defenses may to be  inadequate. E s s e n t i a l l y a l l c e l l u l a r constituents are susceptible to  injury will  prove  from free radicals (12)  (Figure 2).  i n t e r a c t with protein, DNA,  glycoprotein  in s e l f  Once produced, free r a d i c a l s  membrane phospholipid,  propagating  reactions  which may  l i p o p r o t e i n and  alter  the  native  structure of both the i n t r a c e l l u l a r components and the e x t r a c e l l u l a r matrix. Because reactive oxygen species w i l l r e a d i l y react with unsaturated and  sulphur-containing  molecules,  tyrosine, phenylalanine, s e n s i t i v e to oxidation  the  acid  h i s t i d i n e , methionine and  residues  required  cysteine are e s p e c i a l l y  for. enzymatic function may  enough to i n t e r f e r e with a c t i v i t y More permanent damage may  be due  oxidation  proteolytic  could  critical  to protein c r o s s l i n k i n g and  implies  be the f i r s t  suggestion  step  has  functions,  such as  b a c t e r i a , a c t i v a t i o n of the  chemotractants and  modified  fragmentation  (15).  been made that  the  I t i s known  more susceptible  in normal protein turnover  that free r a d i c a l s produced during  have multiple  killing  groups makes proteins  breakdown, thus the  modification may This  of  be  (both augmenting or a r r e s t i n g function).  have s t r u c t u r a l and u l t r a s t r u c t u r a l consequences (14)  that  tryptophan,  (13).  S p e c i f i c residues  may  amino  to  oxidative (16)  (17).  inflammatory response  i s o l a t i o n of the  area of damage,  immune system v i a superoxide dependent  i n i t i a t i n g breakdown of damaged s t r u c t u r a l components in  order to f a c i l i t a t e r e p a i r . Highly reactive free r a d i c a l s are also capable of inducing DNA breakage  as  superoxide  demonstrated generating  in v i t r o  system  (18).  with  strand  a xanthine oxidase + xanthine  I f unrepaired  this may  cause point  -9mutations  (19)  and  enzymatically,  chromosomal  and  in several disease  clastogenic effect, The  Sevanian fatty  of  the  and  results  derivatives. production which  lipid  cause  integrated  addition,  in  fatty  Oxidative of  short  proteins,  lipid  secretory  metabolic as  alcohols  integrity serious  Membrane  the  fatty  processes  will  endothelium  circulation  i n the  and lung  acid  loss  of  and  acts  permeability,  is diffusable  and mutate DNA  edema  formation  where gas  In  across  (23).  Microvasculature as  endothelial The  the  a f f e c t e d (12).  a selective diffusion  interstitium, loss  critical  to  surface interactions  a l l be  on the Pulmonary  see  by-products,  of  cell  endothelial  functions  e f f e c t s of oxygen f r e e  pulmonary e n d o t h e l i u m i n c l u d e i n c r e a s e d c a p i l l a r y  macromolecules  detrimental  review  to  polyunsaturated  viscosity,  m a l o n d i a l d e h y d e , which  microvascular  the  of  aldehyde  functions, c e l l  consequences f o r organ f u n c t i o n .  damage on to  through  recent  chain  and  Oxygen S p e c i e s  the  (21).  becomes a u t o c a t a l y t i c i f unchecked due  E f f e c t s of R e a c t i v e  between  a  cleavage  augment p r o t e i n c r o s s l i n k i n g  barrier  i s perpetuated  (For  c e l l membranes, may  Because the  r a d i c a l s generated  especially susceptible  acids.  oxidation.  by-products  are  accumulation  peroxides,  cellular  such  Free  s t a t e s have a l s o been shown t o have a  organelles  (22).  the  further  membrane t r a n s p o r t and  1985  Such a process  of  will  and  polysaturated  Hochstein,  acids  (20).  such t h a t f u r t h e r damage t o DNA  plasma membrane  oxidation  aberrations  (17)(24).  This  exchange f o r the  is  has radical  permeability  particularly  e n t i r e organism  takes  place. Studies oxygen  species  with are  endothelial cells particularly  i n c u l t u r e have  detrimental  to  the  shown t h a t  reactive  e n d o t h e l i a l membrane  -10integrity. diffusion  Shasby et a l . showed an b a r r i e r of  increase  endothelial c e l l s  grown on micropore f i l t e r s  treatment with oxygen free r a d i c a l generating to be to  be  reversible. index  of  Decrease  in thymidine  cellular  dysfunction.  incorporation Hyperoxia  after  This was  H 0 2  shown  l e v e l s and  2 +  into DNA  or  a  is a  treatment  2  been shown to reduce thymidine kinase a c t i v i t y with implied e f f e c t s on  cellular oxidant  protein injury  unequivocably consequences 0^'  systems (25).  associated with c y t o s k e l e t a l changes attributable to C a  sensitive has  in albumin transfer across  acts  production to  (26).  endothelial  established for  the  cells  that  a  cell  exact mechanism of  OH'  clarified,  species  (28).  agent,  The  be  oxygen  (27)  reducing  extremely powerful o x i d i z i n g agent.  the  remains to  reactive  endothelial  p r e f e r e n t i a l l y as  While  At  i t is lethal  physiologic  whereas  species  have  lethal  HO'  is  pH, an  is however very short  l i v e d and does not cross c e l l membranes but rather w i l l react with membrane macromolecules before i t reaches the cytosol. transition  metals  such  dismutated  into ^ 0 ^  as  will  iron,  0,"  which  generate OH'  However, in the presence of is  produced  and  (Fig. 1 Reaction 6).  then Iron  has  been shown to be e s s e n t i a l for superoxide free r a d i c a l induced injury (29). Thus, while OH' producing lethal  HO'  oxidant  stimulated  i s the via  the  most strongly H^O^  injury to  neutrophils  dependent  endothelial  (31).  This  hydroxyl r a d i c a l s which are not.  pulmonary damage have been  linked  pathway cells  is  uncharged and f r e e l y d i f f u s a b l e across and  o x i d i z i n g species,  important  means of (30)  injury resultant  presumably  because  in from *  s  the c e l l membrane, unlike superoxide The  to  and  is  the  pathogenesis of several forms of  oxygen free  r a d i c a l formation.  -11Hyperoxia The  has been l o n g e s t a b l i s h e d  pulmonary pathology  enhanced a u t o x i d a t i o n reduced  state,  mechanisms.  resultant  of c e l l u l a r  as w e l l  from  (31)  (32) In  of c e l l u l a r  antioxidant  damage t o e n d o t h e l i a l  from i n the  defense  cells  has been  t o f u r t h e r n e u t r o p h i l - m e d i a t e d damage  i n t e r a c t i o n between n e u t r o p h i l s  the extensive d e s t r u c t i o n  with hyperoxic  inflammatory  cells  insult  i n the lung  and e n d o t h e l i a l  cells  o f t h e pulmonary m i c r o v a s c u l a t u r e  i s likewise  combined w i t h  an i n f i l t r a t e o f  (34), thus p e r p e t u a t i n g f r e e r a d i c a l damage  t o s u p e r o x i d e generated by NADPH-oxidase o f n e u t r o p h i l s  0^' i n t h e f o l l o w i n g NADPH + 2 0 The  to originate  (33). vivo,  associated  due  adhesive  h y p e r o x i a appears  as e x h a u s t i o n  Interestingly, oxidative  increasing  o f pulmonary damage ( 3 8 ) .  components which a r e n o r m a l l y found  shown t o augment t h e i r s u s c e p t i b i l i t y by  as a cause  produces  reaction:  +  2  which  NADPH-oxidase ^  NADP+ + 2 0 • + H+ 2  e f f e c t s o f oxygen f r e e r a d i c a l s are p a r t i c u l a r l y d e t r i m e n t a l i n t h e l u n g  (35) which may r e a c t alveolar visible toxins  epithelial  superoxide reduced  i n t e g r i t y (36).  damage occur paraquat  by m i c r o v a s c u l a r leakage  i n t h e pulmonary c a p i l l a r y  redox  i r o n t o produce Oxygen  The e a r l i e s t  ( 4 0 ) , and n i t r o f u r a n t o i n  through  radicals  c y c l i n g , while  pulmonary  mortality  occurs  and d i s r u p t i o n o f  stages o f m i c r o s c o p i c a l l y bed ( 3 7 ) . The pulmonary  (41) a c t by t h e g e n e r a t i o n o f bleomycin  interacts  v i a binding  superoxide (39). generated  by a c t i v a t e d  (PMN's) a r e o f p a r t i c u l a r s i g n i f i c a n c e chronic  of proteins  injury  (42).  i n cases  i n the pathogenesis  I t has been  o f acute  polymorphonuclear  estimated  non-cardiogenic  that  leukocytes  o f acute and up t o 50%  pulmonary  edema,  -12otherwise known as adult  respiratory d i s t r e s s syndrome (ARDS) (43). The  neutrophil has been strongly (45)  implicated i n the pathogenesis of ARDS (44)  (46) (47). The consquences of edema due to increased  permeability  microvascular  (48) (49) i n the lung are e s p e c i a l l y detrimental  f i l l i n g of i n t e r s t i t i a l  because the  spaces with f l u i d interferes with the normal process  of gas exchange upon which the entire organism i s dependent.  Oxygen Free Radical Damage Associated  with Ischemia-Reperfusion Injury i n  Other Tissues Oxygen derived  free r a d i c a l s are now thought to play an important  r o l e i n another form of damage, described injury. heart,  as post-ischemia  I t has been well documented i n other skin, l i v e r ,  or reperfusion  organ systems (kidney, gut,  pancreas and brain) that reperfusion of a previously  ischemic  tissue produces, paradoxically, more damage than that caused by  ischemia  alone  (50) (51) (52). The i n i t i a l  appears to be the microvascular transcapillary  filtration,  s i t e of reperfusion  endothelium (53).  interstitial  injury  This r e s u l t s i n increased  edema and i n f l u x of inflammatory  c e l l s which can lead to the loss of the s p e c i a l i z e d functions c h a r a c t e r i s t i c of the t i s s u e , such as acute tubular necrosis i n the kidney (54) or loss of contractility  i n the myocardium (55).  be  by the reperfusion of the tissue with oxygenated blood.  introduced  Oxygen free r a d i c a l s are thought to This  mechanism has been confirmed by several studies which show that use of superoxide radical (56)  dismutase  and catalase  scavengers w i l l  (57).  The  enzyme  ameliorate xanthine  or dimethylsulphoxide the detrimental oxidase  (DMSO) as free  e f f e c t s of reperfusion  (ECO 1232  oxidoreductase)  -13produces  O^"  xanthine  and  inhibitors  and  H  2^2  ^  hypoxanthine.  xanthine  confirmed  specific  of  oxidase  reperfusion  injury.  oxidase  permeability  oxidation  Because o f the was  xanthine  substrate, prevents  xanthine  xanthine  ^he  suggested  oxidase the  its  as  xanthine  P e r f u s i o n of  (58).  allopurinol acts  xanthine  (60) lung  as  is likewise  a  shown  (54)  injury  similar  increase vascular function  (61)  has  shown  endothelium and  i n c r e a s e s the  cells  thus m a g n i f i y i n g damage due  interesting  speculation that  development o f ARDS ( 6 3 ) . initiating in  derived  other  factor tissues,  i n the (see  free  radical  susceptibility  xanthine  damage  the  oxidase  may  of xanthine  following  section  (62)  Since i t  pulmonary  of n e u t r o p h i l s  r e a c t i o n s , t h i s l e a d s t o the play  S i n c e an e p i s o d e o f t r a n s i e n t activation  to  t o adhesion  t o inflammatory  to  containing  i n s i m i l a r p a t h o l o g i c f i n d i n g s t o acute pulmonary edema. oxygen  (59), a  with a mixture  solution to  now  reperfusion injury  a p p a r e n t l y v i a compromised e n d o t h e l i a l c e l l  that  the  binding  resulting been  of  (55)  a tightly  organs  creates with  source  T h i s p r o p o s a l has  development o f the  the  above d e s c r i b e d  the m o l e c u l a r  which  (33)  substrates  of the  manner p e r f u s i o n o f s p e c i f i c  plus  plus  of  effects  in reperfusion injury  syndrome.In an analogous of  n  by many s t u d i e s showing t h a t  inhibitor  competitive  ^ B  oxidase  oxygen r a d i c a l s produced been  u r  a  role  in  i s c h e m i a may  o x i d a s e mediated  on  the  biochemistry  be  an  pathology  of  XO)(93)  ( F i g u r e 4 ) , under what c i r c u m s t a n c e s c o u l d the l u n g be c o n s i d e r e d " i s c h e m i c " ? ARDS was when  i t was  septic  first  observed  r e c o g n i z e d as a d i s c r e t e to  occur  subsequent  to  syndrome i n 1967  by Ashbaugh  t r a u m a t i c , hemorrhagic  shock and g i v e n the name "shock l u n g " ( 6 4 ) , a l t h o u g h  and  infections,  -14-  Decreased perfusion Decreased O 2 ATP — ^ AMP  Xanthine  — ^ adenosine inosine  dehydrogenase  1.  r e v e r s i b l e t h i o l oxidation  2.  Ca^+ mediated proteolysis  hypoxanthine  Hypoxanthine  +2O2  +  H2O  xanthine oxidase  xanthine  +  20^'  2H+  xanthine oxidase  increase i n O 2  Reperfusion:  u r i c acid + Oj'  Result:  O^- + F e  and  205- + 2H+ H 0 2  Figure 4.  2  +  3 +  Fe2+(X)  •> 0  2  + Fe  ^ 0  2  + H 0 2  2 +  2  •> HO- + OH" + F e ( X ) 3+  Reactions of Xanthine Oxidase. Proposed sequence for superoxide production i n reperfused post-ischemic tissue and subsequent generation of the powerful oxidizing agent OH-  -15pancreatitis, implicated.  inhalants A l l of  and  these  factors  Interestingly,  microthrombi  ammiotic  are  These  fluid  could  bypass  resembles instance of time of  a l s o been ARDS  at  the  pulmonary  (67)  blood  Clinically,  an  There can  a' pneumothorax  and  by  fat,  the  i n the  gas  flow  will  early  create  gas,  In  lung.  assay  Several  i n j u r y which is  supply  another  for a period  a  complication  lung  injury  pulmonary  i n combination w i t h  vascular  heparin  can  increase  leukocyte  that  has  been noted t h a t  uptake been  free radical  in  a l l of these s t u d i e s f o r ARDS the  the  lung  associated induced and  decreased (64).  with  ARDS  damage t o  lipid  activate neutrophils  seem to i m p l i c a t e h y p e r f u s i o n  a s s o c i a t i o n with  reperfusion  injury  in has  established.  (72)  demonstrated to be  and  Cardiopulmonary  some models  c h e m o a t t r a c t a n t s which s e q u e s t e r  neutrophils  air  development of ARDS.  of i t s blood  model i t has  t r a n s i e n t leukopenia  While  exchange.  l e v e l . S u r g i c a l bypass  (66).  also  (68).  X a n t h i n e oxidase  (73).  to  were  i n secondary pulmonary  s u p e r o x i d e dismutase  lung experimental  been a c c e p t a b l y  activity  pulmonary  due  ischemia  result  i s some s p e c u l a t i o n  3)(71).  activating not  focal  l u n g would be d e p r i v e d  of  In the dog  (Figure  microemboli  ultrastructural  p r e v e n t i t s development  bilayers  compromise  drowning  are n e c e s s a r y f o r the development of i n c r e a s e d  permeability  (70).  near  U n i l a t e r a l pulmonary edema i s a l s o known t o be  re-expansion  leukocytes  of  shown t o  of when the (65).  and  and  also o c c a s i o n a l l y followed  l e a d t o areas  has  ingestants  and  has  immunological  localized  recent  been found  i n the means  in microvascular  lung  of  (73). but  not  s e v e r a l s p e c i e s both The  enzyme  has  by  been  l a r g e v e s s e l endothelium  s t u d i e s have been d e s i g n e d to examine whether  inhibition  -16of  xanthine  oxidase  with  allopurinol  could prevent  the  development of  pulmonary edema due to a v a r i e t y of i n s u l t s such as air-embolism, re-expansion of  pneumothorax and hypoxia  (72)  (73)  studies have not yet been resolved.  (74).  Conflicting results  of these  Nevertheless, the a c t i v i t y of xanthine  oxidase i n the lung must be further investigated in order to elucidate whether t h i s enzyme may  play a similar role i n inflammatory lung disease to that which  has been demonstrated  in ischemia-reperfusion injury (63).  Biochemical Aspects of Xanthine Xanthine oxidase (XO) of  the flavoproteins.  Oxidase  i s one of the most well characterized and complex  I t was  f i r s t isolated and studied extensively in bovine  milk, where i t i s found i n great abundance that methylene blue was milk. denoted  This enzyme was  reduced  In 1902 Schardinger f i r s t  by formaldehyde  i n the presence  designated Schardinger's enzyme u n t i l  xanthine oxidase.  Xanthine oxidase contains FAD,  in the molar r a t i o s of 1:4:1  observed  of fresh  i t was  later  iron and molybdenum  as well as inorganic sulphide in two subunits of  approximately 150,000 molecular weight.  Xanthine oxidase i s known to have a  r e l a t i v e l y broad substrate s p e c i f i c i t y , high a c t i v i t y , easy p u r i f i c a t i o n s t a b i l i t y as well as low s p e c i f i t y for the electron acceptor. hypoxanthine  and  xanthine, xanthine  oxidase w i l l  purines, pyrimidines, pterins and aldehydes. substrate  electrons to acceptors such  f e r r i c y a n i d e and a r t i f i c i a l function of XO  appears  In addition to  also oxidize many other  Xanthine oxidase transfers the  as molecular  oxygen, cytochrome c,  dyes such as methylene blue (77).  to be  and  as the r a t e - l i m i t i n g  The  biological  enzyme in nucleic  acid  degradation through which a l l purines are channelled for terminal oxidation.  -17-  ATP .  Dehydrogenase  Superoxide-Activated  AMP  Chemoattractant  Adenosine  HypoxaMhine * Xarrlhinc Oxidase Reoxy genet ion  H 0  cr  ?  O2 *~  2  Fe  MPO HOCI  RNH •  s  •OH  Circulating Neutrophils  Extravasated Neutrophils  <3  Neutrophil Activators  Chemoa'traclanis  RNHCI  -» T I S S U E D A M A G E  Figure 3  A c t i v a t i o n o f n e u t r o p h i l s by oxygen f r e e r a d i c a l d e r i v e d c h e m a t r a c t a n t s . T i s s u e damage due t o the g e n e r a t i o n o f h y d r o x y l f r e e r a d i c a l s and h y p o h a l i d e s i n though t o cause l i p i d peroxidation, generating short chain f a t t y a c i d d e r i v a t i v e s which w i l l a c t i v a t e n e u t r o p h i l s l e a d i n g t o f u r t h e r oxygen f r e e - r a d i c a l damage. Taken from Grisham, Hernandez and Granger 1986 ( 6 0 ) .  -18In v i t r o hypoxanthine as  an electron  by-product  and xanthine are oxidized to urate with molecular oxygen  acceptor by  the oxidase  i s thus generated  (Figure 4).  form.  A superoxide  free  This reaction proceeds  radical by  order enzyme k i n e t i c s and i s product i n h i b i t e d by urate (78) (79). oxidase has long been u t i l i z e d i n conjunction with hypoxanthine one  of  the best  enzymatic  Allopurinol, competitive  sources  a drug used  of oxygen  free  in the treatment  radicals  first Xanthine  or xanthine as available.  of gout which acts by  i n h i b i t i o n of the enzyme i s a known s p e c i f i c i n h i b i t o r  (80) of  XO useful for mechanistic studies (81) as well as allowing for regulation of the enzyme i n vivo (75)  (59)  (82).  For extensive general review of the  mechanism and structure of xanthine oxidase see Bray et a l . (83). The o r i g i n a l established source of xanthine oxidase was Early on i t was  recognized that xanthine oxidase was  and more tissues than milk (84).  bovine milk.  found in most species,  In the tissue xanthine oxidase i s found in  highest concentration in l i v e r and small i n t e s t i n e , with variable l e v e l s i n other  tissues  (72).  Xanthine  heart, lung and kidney (89).  remarkably  well  the  conserved,  amino acid  sequence of xanthine  even in Drosophila melanogaster  oxidase i s (85).  the enzyme was  localized  in culture.  (88).  exclusively to microvascular  endothelial c e l l s using sensitive immunofluoresent these have yet to be assayed  It is  (86) and jejunum,  i s absent from cultured human f i b r o b l a s t s and Hela c e l l s  However, recently  (85)  Xanthine oxidase i s found  i n a v a r i e t y of human tissues most notably l i v e r  (87), but  in l i v e r ,  (85), but i s v i r t u a l l y undetectable i n serum  Brain and cornea have the lowest a c t i v i t y .  in many species and  found  oxidase has been demonstrated  techniques, (73) (87) and  The enzyme i s absent from large  -19v e s s e l endothelium  (73).  When t h e enzyme was f i r s t in  two forms,  an e l e c t r o n and of  a dehydrogenase  different  cannot  transfer  activities  methods o f i s o l a t i o n  liver  i t was found  to exist  capability  of u t i l i z i n g  NAD  form.  dehydrogenase component t h e enzyme  a superoxide  radical  e l e c t r o n s t o NAD .  as  by-product  i g n o r e d because  the p r o t e o l y t i c  4  The s i g n i f i c a n c e  +  had p r e v i o u s l y been  d i d n o t prevent  enzyme t o i t s o x i d a s e  Normally,  (XDH) w i t h  from  a c c e p t o r which does not produce  t h e o x i d a s e which these  isolated  degradation  early o f the  The m i l k enzyme has now been shown t o have some  (91) (92) when i s o l a t e d by n o n - p r o t e o l y t i c methods. i s thought  to exist  almost  exclusively  i n the  dehydrogenase form i n v i v o . Factors  such  as a n a e r o b i o s i s , s t o r a g e  homogenate and s o l v e n t treatment from  t h e dehydrogenase  conversion t o oxidase different  structural  a r e known t o cause  t o oxidase  alterations  retain  enzymatic  thiol  c o n t a i n i n g groups  activity  o f whole  indicating  o f xanthine (93).  of xanthine  tissue  c o n v e r s i o n o f t h e enzyme  (93). Interestingly,  i s reversible,  still  a t -20°C  that  some  o f the  t h e r e a r e a t l e a s t two  dehydrogenase  possible  which  F u r t h e r work showed t h a t r e d u c t i o n o f dehydrogenase by d i t h i o t h r e i t o l  could  p r e v e n t c o n v e r s i o n o f t h e enzyme t o t h e o x i d a s e form caused by a n a e r o b i o s i s , storage  a t -20°C  trypsin  treatment  irreversible mechanisms cleavage process  and s o l v e n t s .  (94).  Conversion  or i n c u b a t i o n w i t h whole t i s s u e These d a t a thus  of conversion,  an  support  irreversible  of a polypeptide e s s e n t i a l due  caused  to modification  c o n f o r m a t i o n a l change.  of  by p r o t e o l y s i s homogenates  the p r o p o s a l t h a t process  resulting  a t 37°C was t h e r e a r e two  due t o p r o t e o l y t i c  f o r binding with  thiols  such as  NAD  in a  +  and a  second  reversible  -20In  the i n t e s t i n e  converts  irreversibly  conversion be  o f mammals  and v e r y r a p i d l y  i n 16s ( 9 5 ) , g i v i n g  rise  a c t e d upon by a s p e c i f i c p r o t e a s e  mucosa  (96) which  heart with  xanthine  i s low o r absent  60 min c o n v e r s i o n  dehydrogenase  t o the o x i d a s e  form  predominantly showing  complete  t o the s p e c u l a t i o n t h a t t h e enzyme may i n high concentration i n the i n t e s t i n a l i n other t i s s u e s .  Other  tissues  such as  time may be s u s c e p t i b l e o n l y t o n o n - s p e c i f i c  p r o t e o l y s i s (94). Electrophoretic irreversible weight  from  previous  conversion  observation  prevented  that  mobility  dehydrogenase c o n v e r t s  i n the r a t l i v e r  Soybean  trypsin  protease to  rat  model  are that  i s also  hypoxanthine, breakdown events  a r e homogenized, t h e  tissues  of limited  a chelator of C a  o f note  the s u b s t r a t e  product  2 +  ( 9 9 ) . Use o f p r o t e a s e this  proteolytic  activates  of calmodulin  show  different  proteolysis  divalent  inhibitors  such as  conversion  a Ca -calmodulin 2 +  with  w i t h soybean t r y p s i n that  ischemia  cannot  f o r xanthine  o f ATP i n t h e l u n g  occurring during  an  incidence  mediated  be c o n v e r t e d back  inhibitor  form  cause  and i n t h e plasma could  also i n the  (100). a rise in  oxidase/dehydrogenase  of ischemia  (100).  trifluorperazine  o r hypoxia  which  and o t h e r  c o n v e r s i o n o f t h e enzyme t o t h e o x i d a s e  ileum, as does treatment It  i n molecular  (90) i n agreement w i t h t h e  (101) which a c t s on t h e enzyme which then  irreversible  changes  that  When  ischemia  Inhibition  shown  enzyme  as a r e s u l t  prevent  have  and i n t e s t i n e  o f EDTA,  inhibitor  dehydrogenase.  prevents  liver (98).  by a d d i t i o n  suggestions  and l i v e r  t o 130,000 D a l t o n s  t o oxidase  cations  Current  i n intestine  o f t h e enzyme c o i n c i d e s w i t h  150,000 D a l t o n s  electrophoretic  was  studies  as a  (102) (103). encompass  The both  -21conversion of the enzyme to i t s oxygen dependent form and concommitant increase  i n substrate.  reperfusion  With the reintroduction of molecular oxygen upon  of the tissue with oxygenated  blood, the necessary  electron  acceptor i s supplied f o r the reaction to proceed to the production of urate with superoxide r a d i c a l by-products being generated. also been shown to be capable of mobilizing superoxide effect  dependent  of free  and independent  radical  iron  Xanthine oxidase has  from f e r r i t i n  mechanisms, thus  by both  potentiating the  damage by generating the more highly  reactive  hydroxyl r a d i c a l s (71), (Figure 4). This e f f e c t has been demonstrated blocked by 70% by superoxide dismutase, while u r i c acid removal  to be  does not  a f f e c t the mobilization when a system of bovine milk xanthine oxidase and horse spleen f e r r i t i n  i s employed (104).  During a period of ischemia ATP l e v e l s are diminished, as documented by NMR  spectroscopy studies using an ischemic r a t kidney model that from the  onset of ischemia u n t i l reperfusion was i n i t a t e d after 45 min the a , B , and y ATP peaks v i r t u a l l y disappeared while the inorganic phosphate peak rose by four f o l d (105). when  energy  dependent  Loss of c e l l u l a r ATP promotes an i n f l u x of calcium  channels  which maintain  the lower  intracellular  concentration of this cation are inactivated. Since xanthine oxidase i s known to be an active enzymatic oxygen free radicals and this enzyme has been demonstrated it  i s necessary  to investigate  dehydrogenase/oxidase  source of  in the lung (73)  the conversion properties of xanthine  in the lung in order to determine whether t h i s enzyme  may play a part in the etiology of pulmonary edema attributed to reactive oxygen species (49).  -22-  Perfusion/02  xanthine dehydrogenase  Then: Ca dependent proteolysis activated by ischemia + +  xanthine ^ xanthine + 2 0 ' + 2 H oxidase \ xanthine \ oxidase  hypoxanthine + 2(>2 + H2O  +  2  u r i c acid + 0 2  Reperfusion  ^ 2O2  Then: 0 and:  + Fe+  2  205*  + 2H+  H 0  + Fe+ (X)  2  Figure 4.  ^ 0 5 -+ Fe  2  ^ 2  2  0  2  +  H 0 2  + 3  2  ^HO- + OH~ + F e ( X )  Reactions of XO i n Ischemia  +3  and Reperfusion  (14)  -  -23PURPOSE OF THESE STUDIES 1. To determine the presence of xanthine dehydrogenase/oxidase i n human, p i g and r a t lung and to measure i t s l e v e l of a c t i v i t y . 2. To compare enzyme content and a c t i v i t y i n various organs within the same species using immunoreactivity, g e l electrophoresis and s p e c i f i c enzyme assays. 3. To assess the r e l a t i v e content of the enzyme i n the dehydrogenase or the oxidase form under standard conditions of enzyme p u r i f i c a t i o n . 4. To study factors which might affect conversion from the dehydrogenase to the  oxidase form i n the lung and to determine under what conditions the  conversion can be i n h i b i t e d or reversed.  -24METHODS  Methods of Detection of Xanthine Dehydrogenase - Oxidase A c t i v i t y Several d i f f e r e n t assays for XDH-XO have been developed and each has advantages  and disadvantages  which  earliest  assay was developed  function  of the enzyme i n oxidizing  (106).  contribute  to i t s usefulness.  i n 1914 and employed hypoxanthine  the normal  The  i n vivo  or xanthine to urate  The formation of urate i n the presence of enzyme and substrate was  determined  by measuring  the increase i n absorbance -3  extinction  coefficient  f o r urate of 9.6 x 10  at 295 nm using a molar -1  cm  The assay carried  out with oxygen as an electron acceptor measures oxidase a c t i v i t y . of NAD  +  activity.  as the second  electron  acceptor measures  total  Addition  XDH plus XO  The technique was modified by Morell (106), who found removal of  endogenous substrate increased a c t i v i t y .  There are two major drawbacks to  this method, the f i r s t being that i t i s not very sensitive and the second being that most organisms urate  to a l l a n t o i n  lower than man contain uricase which  and thus production of urate cannot  degrades  be accurately  measured without i n h i b i t i o n of uricase a c t i v i t y . Several more sensitive oxidase  activity  have  been  methods  f o r the measurement of xanthine  developed.  These  include  the use of  14 [  C]-labelled  xanthine  (107), use of high pressure l i q u i d chromatography  (108), a manometric assay oxygen uptake  (110).  (109) and an assay u t i l i z i n g sulphite-enhanced  A fluorometric  currently the preferred method because  assay developed by Lowry (111)  is  i t i s extremely sensitive due to the  -25highly fluorescent nature of i t s product and i s also more convenient, since i t does not require isotopes. found  This assay was modified by Glassman (112) who  that removal of endogenous small molecular  inhibitors  increased  determination  the s e n s i t i v i t y  of enzyme a c t i v i t y  enough  weight substrates and  to make  possible the  in a single Drosophila melanogaster.  A  modification of this assay was the f i r s t to make possible the determination of the very low l e v e l s of xanthine oxidase a c t i v i t y i n brain (113).  In an  extensive comparison of several assays, Haining and Legan (114) found the modified fluorescence method (113) to be the optimal technique. Initial determined  studies were carried out with the urate assay u n t i l i t was  to be unsatisfactory f o r our purposes,  contains uricase. and  proved  tissue  The fluorescence method as modified (113) was then used  to be more s e n s i t i v e  contaminating  since porcine  and less  prone to interference from  components.  A synthetic pterin i s employed as the substrate and i s enzymatically oxidized molecular reaction  to a highly fluorescent product, oxygen and methylene blue allowing f o r determination  isoxanthopterin (IXP).  act as electron accepters of oxidase  to dehydrogenase  Both  i n this ratios.  With oxygen as the electron acceptor oxidase a c t i v i t y alone  i s measured.  When methylene blue  oxidase and  i s added as the electron acceptor both  dehydrogenase a c t i v i t y are measured. Determination of XDH-XO A c t i v i t y by the Determination of the Rate of Urate Production (115) 1.  The tissue was excised and trimmed.  Pig l i v e r  and kidney  obtained  fresh from a l o c a l slaughterhouse had capsule membranes removed prior  -26to mincing. airways Major  P i g lung  dissected  bronchi  tissue  was  phosphate  out.  minced  Whole  i n four  buffer  (KPi),  Homogenization was  of  the  homogenized  and  volumes  pH7.4  of  removed  livers  were  large  minced.  out b e f o r e m i n c i n g .  ice cold  containing (PMSF)  and  as  50  O.lmM a  mM  The  potassium  EDTA  protease  and  ImM  inhibitor.  accomplished w i t h a S o r v a l l Omnimixer a t 75% speed second b u r s t s  tissue.  tissue  Cells  on  homogenate was  i c e , depending  were broken  i n an e l e c t r i c a l l y  equipped w i t h a T e f l o n The  membrane  r a t kidney  fluoride  from 3 t o 8 f i f t e e n  content  the p l e u r a l  o f r a t lungs were d i s s e c t e d  phenylmethylsulfonyl  in  had  after  10  on  collagen  passes o f the  d r i v e n P o t t e r - E l j e v h e i m mortar  pestle.  fractionated  by  ultracentrifugation  a t 100,000g  o f o r 60 minutes a t 4 C. A  2 ml  aliquot  G-25  column  rate  of  to  (2 x 25  collect  five  at  following 295  cuvette  nm  was  l o a d e d onto  e q u i l i b r a t e d w i t h 50 mM  fractions  was  0.8  ml/min.  of  450  endogenous  drops  (15.6  ml)  FRAC  used.  a Beckman 35 Recorder  each.  300 used  substrates activity.  peaks.  the p r o d u c t i o n o f u r a t e .  The absorbance change was  Spectrophotometer w i t h  sensitivity  The  This  enzyme  o f two p r o t e i n  determined by measuring  p r o c e d u r e was  a t 25°C.  EDTA.  A Pharmacia  s m a l l m o l e c u l a r weight  a s s o c i a t e d w i t h the f i r s t  using  a Sephadex  KPi + ImM  from the crude homogenate c o n t a i n i n g  XDH-XO a c t i v i t y was The  removed by p i p e t .  equippped w i t h a p r o t e i n d e t e c t i o n u n i t was  removed  inhibitors  A c t i v i t y was  cm)  the b u f f e r  collector  fractionation and  of supernatant f r a c t i o n  flow of  fraction  Supernatant was  range  was  measured  a 1ml q u a r t z  s e t at 0 t o  0.5  absorbance u n i t s , and t h e c h a r t speed was 1 inch/min. mixture contained volume The 15  sample + Na-pyrophosphate  o f 990 u l 4 10 y l o f 10 mM  initial mM  +  as  a  second  dehydrogenase + o x i d a s e to  (0.1M) pH 8.3 i n a t o t a l  xanthine  i n Na-pyrophosphate.  absorbance change measured o x i d a s e  NAD  inhibit  electron  activity.  acceptor  20 uMoles  t h e r e a c t i o n and v e r i f y  that  The r e a c t i o n  activity. measures  Addition of total  a l l o p u r i n o l was added  the urate  production  was  c a t a l y z e d by XDH-XO. Calculation:  A Absorbance 295 nm/min = mU/ml (9.6 x 1 0 - M c m ~ l ) ( s a m p l e s i z e i n ml) 3  -3 where  9.6 x 10  urate. of 1. of  -1 M  -1  -1  cm  i s t h e molar  One u n i t (U) i s d e f i n e d Protein concentration  Lowry  (121).  Enzyme  e x t i n c t i o n c o e f f i c i e n t of  as a change o f absorbance a t 295 nm  o f samples was determined by t h e method  activities  were  expressed  as mU/mg  protein/min.  Determination  o f XDH-XO A c t i v i t y by P r o d u c t i o n  Preparation  o f t i s s u e proceeded  o f IXP (113)  i n t h e same manner as f o r u r a t e  assay  steps 1-3. 4.  An Aminco SPF-125 S p e c t r o f l u o r i m e t e r in  fluorescence  emission  a t an e x c i t a t i o n wave  wave l e n g t h  o f 390 nm.  s e n s i t i v i t y o f 10 mV f u l l 5.  was used t o r e c o r d length  The r e c o r d e r  scale with  t h e changes  o f 345 nm  and an  was o p e r a t e d  at a  t h e c h a r t r a t e s e t a t 1 cm/min.  T i s s u e samples were pre-warmed a t 37°C i n water  bath.  -286.  The reaction mixture  contained:  450 y l of sample 450 y l of KPi (50mM) at 37°C 25 y l of 900 uM pterin was Oxidase a c t i v i t y was  added to s t a r t the reaction.  f i r s t determined  and then 5 y l of a ImM  stock  of methylene blue added as a second electron acceptor to determine the t o t a l XDH 7.  The  + XO  activity.  fluorescent emission at 390 nm was  compared to a standard  prepared with a known sample of IXP dissolved in 50 mM a concentration range of 0.01 intensity  was  found  to 1 yg/ml.  to correspond  One  consistently  curve  KPi pH 7.4 at  unit of to 2.3  relative x 10 ^"yg  IXP/ml. 8.  Initial  rates of enzyme a c t i v i t y were determined  produced.  as yg IXP/ml/min  Protein concentrations of samples were determined  method of Lowry (122).  Enzyme a c t i v i t i e s were expressed  by the  as ug  IXP  produced/minute/mg protein. 9.  The  production of IXP  could be i n h i b i t e d by addition of 20 yMoles  a l l o p u r i n o l , thus confirming that the increase i n emission at 390 was 10.  due to the c a t a l y t i c action of XDH  + XO.  The proportion of XO a c t i v i t y in the tissue samples was a r a t i o of t o t a l enzyme a c t i v i t y : XDH XO rate of conversion.  nm  + XO  expressed as  in order to give a l i n e a r  -29Experiments  U s i n g P u r i f i e d Xanthine  Purification  of Xanthine  x a n t h i n e o x i d a s e was and  samples  polyclonal the  to  use  as The  n o n - p r o t e o l y t i c method  amounts  sources  of  The  liters  o f XO  proteases  purposes. 16  Oxidase  centrifuged  at  an  antigen  purified  (117)  purification of  Waud,  and  p r e p a r a t i o n of  rpm  thus  f o r the  et  was  a  based  on  a l . (116)  Commercially  contaminated  with  c o u l d not  be  used  variable  for  these  follows:  o b t a i n e d from the UBC  f o r 10  tissue  p r e p a r a t i o n of  method chosen  c a r r i e d out as  f r e s h m i l k was 3000  f o r the  are known t o be  procedure was  of raw  A highly  r e q u i r e d f o r a base o f comparison  antibody.  available  1.  and  Oxidase  minutes.  This  D a i r y Barn  resulted  in  and  the  s e p a r a t i o n of 425 ml of cream. 2.  3.  Equal  volumes  mM  HPO^,  4 mM  c o n t a i n i n g 0.01%  This mixture  cooled  pH at 155.25  was  continuous  g  of and  Stirring  continued  was  the pH  stirring.  sodium  EDTA were added a t 80°C.  and  135  10N  ml  of b u t a n o l  NaOH was  a t 4°C  was  added t o m a i n t a i n  the  wool  added  sulphate  o f the m i x t u r e f o r 20  minutes  was  slowly  a d j u s t e d t o 9.0 f o l l o w e d by  added  with  with  10N  NaOH.  centrifugation  at  4°C.  supernatant  glass  7°C  u l t r a p u r e ammonium  stirring  The  to  c y s t e i n e - H C l , 2 mM  9.0.  13,700g and 5.  200  s a l i c y l a t e pH 9.0  added w i t h  4.  of  buttermilk fraction  t o remove f a t g l o b u l e s . to  the  filtrate.  (700 Solid  T h i s was  p r e c i p i t a t e c o l l e c t e d by c e n t r i f u g a t i o n  ml)  was  filtered  through  ammonium s u l p h a t e  stirred  f o r 30 min.  at 13,700 g f o r 20  (135g)  and  the  minutes.  -306.  The p e l l e t  was suspended  containing clarified  1 mM  i n a minimal amount of 50 mM K^HPO^  salicylate  and 0.005% EDTA.  by centrifugation and applied  The suspension was  to a Sephadex G-25 ion  exchange column that was e q u i l i b r a t e d with the same buffer i n order to remove r e s i d u a l butanol.  The protein eluted from the G-25 column  after a volume of 330 ml. 7.  The above protein component was applied to a DEAE - Sephadex A-50 column e q u i l i b r a t e d with 5mM potassium phosphate pH 7.8 containing 0.005% EDTA and ImM s a l i c y l a t e . accident  the cold  I t should  room temperature  dropped  be noted here that by to -10° during the  sample e l u t i o n and the column was p a r t i a l l y frozen.  Despite  problem  the column  the enzyme remained  d i f f e r e n t l y than expected.  active but eluted Because XO contains  group, the enzyme could be detected bright yellow  from  this  a f l a v i n prosthetic  v i s u a l l y by the e l u t i o n of a  flavin-containing protein f r a c t i o n which eluted from  the column with the e q u i l i b r a t i o n buffer rather than a f t e r gradient elution.  The l i n e a r  gradient  between  5mM  and lOOmM  phosphate buffer was not applied therefore. pooled and concentrated 8.  potassium  Active f r a c t i o n s were  using an Amicon PM-30 membrane.  5 ml of sample was chromatographed on a Sephadex G-200 column equilibrated  with  salicylate.  Xanthine  fractions activity assay.  eluted  50 mM K phosphate pH 7.8 + 0.005% EDTA + ImM oxidase  was contained  between 32 ml and 56 ml from column.  was determined using Molecular  activity  both  the urate  assay  i n four Enzyme  and the  IXP  weight determinations were c a r r i e d out using the  -31Phannacia PhastSystem SDS-PAGE. check  purity  of  protein.  This  Pooled  technique was  also used to  f r a c t i o n s were  stored  as  p r e c i p i t a t e s in 50% ammonium sulphate.  Preparation of Polyclonal Antibody to P u r i f i e d Bovine Xanthine Oxidase In order to i d e n t i f y s p e c i f i c a l l y XO in tissue samples an antibody XO was  used against proteins separated  transferred identified  by  blotting  containing  (87).  the  the  to XO  sheets. has  activity  of  XO  was  gel with mercaptoethanol 1.5%  (118).  The gel was  protein bands.  135,000, where XO  XDH  could  also  be  also been shown to cross  One ml of the p a r t i a l l y p u r i f i e d homogenate f r a c t i o n  highest  SDS-dissociating  by SDS-PAGE gel electrophoresis and  nitrocellulose  since polyclonal antibody  react with XDH  of Laemroli  to  to  The  electrophoresed  on  per volume using the method  l i g h t l y stained with Coomassie blue to locate  band containing protein of a molecular  i s known to run, was  cut out and l y o p h i l i z e d  weight of overnight.  Protein was  electroeluted from the gel through an agarose gel (1%) and into  an  length of d i a l y s i s  attached  tubing  (20  cm  weight exclusion of 10,000) using a Buchler conducted f o r two was  days at 4°C,  changed every 24 hours.  20 mA,  in SDS  in length  apparatus.  and  dialyzed for two  was  changed every 24 hours. Purified  electrophoresis buffer which  When a l l the blue stained protein had migrated  protein/ml).  days against 1 1 of d i s t i l l e d H^O.  antigen  One  was  molecular  E l e c t r o e l u t i o n was  into the d i a l y s i s tubing, the tubing containing protein was and  a  removed, knotted D i s t i l l e d water  emulsified in Freunds complete adjuvant (1 mg  ml of emulsion was  injected intramuscularly into a rabbit  -32at  several sites  around  the r a b b i t  prior  used  control.  as  a  facility ad  to i n j e c t i o n Rabbit  caged  weeks  (0.5  times  to c l o t  the  mg  i n one  i n the  ml) (119).  been reached  central  ear  and  artery.  serum  Tissue  to P u r i f e d  Homogenates  and  tissue gel  homogenates  Fresh  Minced  Blotting  with  Western  i n v a r i o u s s p e c i e s and  accomplished (SDS-PAGE)  blotting  and  using  in  SDS  polyclonal  organs  denaturing  conjunction  with  antibody  run  using  the m o d i f i e d  system  f o l l o w i n g method f o r sample p r e p a r a t i o n was  tissue  50mM KPi  Western  raised  XO.  The  sacrifice  was  electrophoresis  using  use.  XO  e l e c t r o p h o r e s i s was  1.  exsanguinated  the  against p u r i f i e d  (121).  e i g h t weeks a  the r a b b i t was collected  was  a t 37°C, c e n t r i f u g e d at lOOOg f o r 20 minutes and  immunoreplication  (118),  After  titer  was  In o r d e r t o l o o k f o r immunoreactive XO  Gel  antigen i n  antibody  (120).  Blood  care  ml)  P o l y c l o n a l Antibody  polyacrylamide  of p u r i f i e d  The  A l i q u o t s were f r o z e n at -20°C f o r l a t e r  of  animal  (120  E l e c t r o p h o r e s i s of  separation  from  s t a n d a r d l a b o r a t o r y chow  injections  u s i n g ELISA t e c h n i q u e had  taken  a sample of non-immune serum t o be  individually  two  at these  collected.  Gel  was  r e c e i v e d booster  c a n n u l a t i o n of  allowed  t o prepare  animal  maximal a n t i b o d y response by  A b l o o d sample of 15 ml was  The  every  measured  body.  of S t . P a u l ' s H o s p i t a l and g i v e n water and  libatum.  saline  the  obtained  of r a t s was  from, s l a u g t e r h o u s e , d i s s e c t e d and  f i n e l y minced  b u f f e r pH7.4 c o n t a i n i n g ImM  tissue  was  rinsed  and  human  filtered  PMSF and through  0.1  lung  of  Laemmli  used: biopies  or  i n f o u r volumes of mM  EDTA at  4°C.  c h e e s e c l o t h t o remove  -33blood.  The r e t e n t a t e  maximum  speed  3  was then homogenized i n a S o r v a l Omnimixer ( 7 5 %  to 8  times  f o r 10  seconds  each  depending  on  c o n n e c t i v e t i s s u e c o n t e n t ) on i c e and then c e l l s were d i s r u p t e d a Potter-Eljveheim 2.  Protein  was  homogenate. buffer  for  acid  Tris  t o between  5 minutes  and  of  mercaptoethanol  centrifuged  6 and 8.  A  samples  were  weight) were used  standard  as d e s c r i b e d  33%  o f 1:5  TCA:  in electrophoresis  0.1% SDS.  hours.  After  were  to give  a  mixture  of  blue,  was  sample  a t 100°C  clarified  t o 100°C table  (Sigma,  by  electrophoresis,  1.5%  x  beta  Prior 5  to  min and  containing  A  60,000 t o 220,000 d e t e r m i n a t i o n and  were  g e l o f 6% PAGE-SDS  Gels were s u b j e c t e d  protein  top c e n t r i f u g e .  range  100 ug samples  buffer  final  added.  f o r m o l e c u l a r weight  electrophoresis  with  were heated  Samples  heated  mixture  above.  o f t h e 5% s t a c k i n g  3  ratio  samples  i n an Eppendorf  molecular  for  2:1  bromphenol  weight  glycine  These  adjusted  lug/ul.  to c l a r i f y  with  ice cold  d e t e r m i n a t i o n s were made by method o f Lowry  molecular  duplicate  of  t o volume  proteins.  volumes  with  electrophoresis,  slots  addition  pH 6.8, 2.5% SDS and 10% g l y c e r o l w i t h t h e pH  Protein  sample  concentration  prepared  the  a t a volume  t o denature  centrifugation. (122)  by  The p e l l e t was then suspended  o f 20 mM  adjusted  f o r 10 passes on i c e .  precipitated  trichloroacetic  using  slab  applied gels  i n the  run i n  0.050M T r i s ,  to a current  0.384 M  o f 70m amp  one g e l i s s t a i n e d  with  Coomassie Blue t o show p r o t e i n bands w h i l e the o t h e r i s p r o c e s s e d f o r immunoreplication.  -34Immunoreplication G e l was 600  ml  Method  soaked  methanol  Immobilon  transfer  supports.  The  proteins w i l l  gelatin. a  Blot  1:500  antibody,  goat  (Biorad). which then  i s then  After  record.  subjected  i s then  polyclonal  washing,  using  the  filter  IgG,  (115)  (diluted pH  8.3  versus  the  at s i t e and  white  compared  used IXP  p t e r i n was  assay  or  added t o s t a r t  glycine,  Biorad  filter  paper  and  f o r 90 min  in  current, negatively  t r a n s f e r e d t o the  according to B i o r a d  binding sites  substrate  antisera  assay  50 mM  (HRP  filter  are  Immuno-Blot  blocked with  of  against  incubated  purified  with  print  binding.  to maintain of  the  to Isoxanthopterin  second peroxidase added  permanent  reaction  and  limits Serial  Assay  of d e t e c t i o n of dilutions  pH  7.a4  f o r IXP  the  urate  of p u r i f i e d  i n the a p p r o p r i a t e b u f f e r (Na  reaction.  XO  B l o t s were  a  antibody  ml  tissues.  (113).  KPi  a  to h o r s e r a d i s h  antibody  3%  w i t h 100  s u b s t r a t e , B i o r a d ) was  f o r presence  to t e s t  1:1000) were suspended f o r urate  strong  i s then  Comparison o f S e n s i t i v i t y of U r a t e Assay  assay  a  covalently linked  black  were  was  Whatman  the g e l and  rabbit  v a r y i n g i n t e n s i t y of r e a c t i o n between  XO  to  processed  a coloured product  Purified  layered with  i n c u b a t e d o v e r n i g h t at room temperature  of  Photographs  of  48g  cathode.  anti-rabbit  photographed  then  sheets  removed from  4 - c h l o r o - l napthol  leaves  was  Non-specific protein  dilution  (anti-XO).  be  Gel  between  When  filter  techniques.  H^O.  s u b j e c t e d t o a c u r r e n t of 1 amp  towards the  Immobilon  of  membrane  buffer.  as they m i g r a t e  Assay  2400 ml  c a s s e t t e was  electrotransfer charged  in  i n e l e c t r o t r a n s f e r b u f f e r of 10 g T r i s ,  assay).  XO  pyrophosphate 25y  moles  -35D i l u t i o n s were as 1.  follows:  1:1000 d i l u t i o n of XO  stock  1 v l i n 1 ml 24.5  2.  1:1  d i l u t i o n of  above  0.5  yg XO  1:1  d i l u t i o n o f above  1:1  d i l u t i o n of above  protein.  u l i n 1ml  12.25  3.  ug XO  u l i n 1ml  6.12  yg XO  0.125  highest  the  spectophotometer  s e n s i t i v i t y range of  Product I n h i b i t i o n of IXP Urate important w i t h the assay  yl  of  50  the  an  u l i n 1 ml  mM  reaction  contained  KPi.  The  rate  a d d i t i o n of 3 nm  spectrofluorimeter  as  a 10  whether IXP  control y l of  and  of r e a c t i o n  i n the  IXP.  would be  were  used  (78)  (79).  the  in  pterin  similarly  a c t i v i t y was the  presence  1:1000 d i l u t i o n  y moles  containing  activity  P u r i f i e d XO  Twenty-five  i n cuvette  buffer  protein.  i n h i b i t o r of XO  p t e r i n f o r X0.  additions  cuvette  reaction. of  no  protein.  at  the  Assay  i s known to be  substrate  buffer  absorbance.  t o determine t h e r e f o r e ,  with  Reaction  and  buffer  protein  0.25  =3.06 ug XO Both  buffer  was  measured by of  c o n t r o l cuvette  was  to  3nm  start  IXP  IXP. in  900  the  compared to  same amounts of enzyme and  was  competitive  of p u r i f i e d XO added  It  rate  p t e r i n with  -36Experintents  i n Porcine  Tissue  Assay f o r XO-XDH A c t i v i t y  Pig abattoir  heart,  the  and l i v e r  i n methods  450 y l K P i b u f f e r  volume o f 900 y l . rate  Tissue  (113).  Each  assay  and 25 y moles  In o r d e r  from a l o c a l  contained  pterin  a t 37°C.  5 y moles methylene  activity.  fresh  i n i c e c o l d KPi b u f f e r where they  The assay was conducted  of reaction,  dehydrogenase  were o b t a i n e d  t o the l a b o r a t o r y  as d e s c r i b e d  extract,  aerobic  kidney  and t r a n s p o r t e d  were p r e p a r e d tissue  lung,  i n Pig  in a  450 y l  total  A f t e r measuring t h e  b l u e was added t o determine  to confirm  the s p e c i f i c i t y  of the  r e a c t i o n f o r XDH-XO 20 uM a l l o p u r n i n o l was added t o i n h i b i t  the a c t i v i t y  I n h i b i t i o n o f P u r i f i e d XO A c t i v i t y by P J R  a t t r i b u t a b l e t o XDH-XO.  Extracts  These t e s t s were c a r r i e d out i n o r d e r t o a s c e r t a i n whether t h e l a c k of  activity  found  interference accurately extracts either  reflect  could  impurities  so t h a t  the a c t u a l  inhibit  KPi b u f f e r  preparations. 1.  from  i n some s e m i - p u r i f i e d  organ  rates  activity.  or buffer  plus  could  of reaction  in vitro,  aliquots  be due t o  measured  I n o r d e r t o determine  p u r i f i e d XO a c t i v i t y  alone  extracts  did  not  i f p i g lung  XO was assayed i n  o f homogenized  tissue  Rates o f r e a c t i o n were compared between t h e f o l l o w i n g :  10 y l o f 1:1000 d i l u t i o n  o f p u r i f i e d XO KPi + 900 y l o f 50 mM K P i  + 0.1 mM EDTA 25 uM p t e r i n . 2.  10 y l o f 1:1000 d i l u t i o n + 900 y l o f p i g lung 25  y moles p t e r i n .  o f p u r i f i e d XO i n 50 mM KPi + 0.1 mM EDTA  e x t r a c t p r e p a r e d as d e s c r i b e d  i n methods.  -37Heat S t a b i l i t y o f A c t i v e F a c t o r s In  order  catalytic  t o determine  activity  i n Pig  Tissue  i f factors  o f p u r i f i e d XO were  homogenate from p i g t i s s u e was i n c u b a t e d The  i n p i g t i s s u e which labile  t o heat,  i n b o i l i n g water  a f f e c t e d the  an a l i q u o t o f f o r 20 minutes.  r a t e s o f r e a c t i o n m i x t u r e s were compared as f o l l o w s :  1.  10 y l o f 1:1000 d i l u t i o n  p u r i f i e d XO + 900 y l o f 50 mM  K P i + 0.1  mM EDTA 25 uM o f p t e r i n . 2.  10 y l o f 1:1000 d i l u t i o n  p u r i f i e d XO + 900 y l o f p i g lung  denatured by b o i l i n g f o r 20 minutes, c o o l e d coagulated proteins  Effect  of P i g Tissue  and c e n t r i f u g e d  extract t o remove  + 25 y moles p t e r i n .  Extract  on  Relative  Fluorescent  Intensity  of  Isoxanthopterin In  order  pig  tissue  the  fluorometric  t o determine whether a component or compounds c o n t a i n e d i n  extract  could  assay  e i t h e r quench o r degrade the p r o d u c t measured i n  (IXP)  a comparative d e t e r m i n a t i o n was conducted.  relative  i n t e n s i t y o f 3nM o f IXP was measured  buffer.  This  was compared  The  i n 900 y l o f 50 mM KPi  t o the r e l a t i v e i n t e n s i t y o f 3nM o f IXP i n t h e  p r e s e n c e o f 900 y l o f p i g lung  homogenate.  The r e a c t i o n  was r e c o r d e d a t  o 37  C f o r 10 minutes.  Assay f o r XO/XDH A c t i v i t y In contained cultured  order  to ascertain  XO/XDH a c t i v i t y , under  i n Cultured  conditions  whether  porcine  P i g Pulmonary A r t e r y large  pulmonary  previously  vessel artery  established  Endothelial Cells  endothelium  i n culture  endothelial cells  i n this laboratory  were  (123).  -38Fresh porcine heart-lung sets were obtained from a l o c a l transported  at  20-22°C  to  excised, rinsed b r i e f l y Gibco  Hanks  penicillin  balanced and  instruments. lengthwise.  199  laboratory.  The  pulmonary  i n phosphate buffered saline  salt  solution  streptomycin  in a  (HBSS) (Gibco) Laminar  Flow  artery  (PBS)  and placed in  supplemented  Hood  was  using  with  sterile  A r t e r i e s were placed on a gauze covered P e t r i plate and Endothelium  was  scraping with a Number 22 s t e r i l e HBSS.  the  abbatoir, and  slit  removed from the luminal surface by gentle  surgical blade  and  sheets of c e l l s placed in  C e l l s were centrifuged and the p e l l e t resuspended  in Medium  (Gibco) supplemented with 20% pig serum and seeded in T75 f l a s k s which  had been previously coated with 1% g e l a t i n on the culture surface.  Cells  were grown in a humidified incubator at 37°C in an atmosphere of 95% a i r : 5% CO^.  After 24  h most of the c e l l s which remained unattatched to the  culture surface were washed free using HBSS.  Most of the unattached  were contaminating f i b r o b l a s t s and smooth muscle c e l l s . confluence.  One  artery provided the c e l l s  were removed with a rubber policeman  C e l l s were grown to  to seed two T-75  average time required to reach confluence was  10 days.  and reseeded  cells  flasks.  The  At confluence c e l l s  at a r a t i o of 1:2.  In  addition to selection by detachment c e l l s at confluence were i d e n t i f i e d by these  characteristics  of  endothelial  cells:  1)  their  characteristic  cobblestone monolayer growth pattern and 2) by the detection of Factor VIII by  immunofluorescence  (124).  Cells  f o r these  experiments  were used  at  Passage 3. For assay, seven T-80 estimated  at between 1.1  flasks were used with, the c e l l number being  x 10  ?  to 1.8  x 10  7  cells  per f l a s k .  After  rinsing  with  HBSS monolayers were removed by  scraping with  policeman and the c e l l s suspended i n 5 ml of ice cold 50 mM mM  EDTA.  Clumps of c e l l s  suspension was mortar  and  2 ml  sonicated for 2s  KPi containing 1  using a syringe.  This  with  a  Sonifier  Cell  Disrupter.  This  A  aliquot  of  the  supernatant  was  run  on  a Sephadex G-25  KPi containing O.lmM EDTA.  column  The e q u i l i b r a t i o n buffer  applied to the column and 15.6 ml fractions were c o l l e c t e d .  which contained the f i r s t the IXP assay method. pterin  cell  fractionated by u l t r a c e n t r i f u g a t i o n at 100,000g f o r 60 min.  e q u i l i b r a t e d with 50 mM was  up  rubber  then homogenized by 20 passes with a Teflon pestle i n a glass  preparation was at 4°C.  were broken  a  + 5uM  protein peak was  900  assayed for XO-XDH a c t i v i t y by  u l of Fraction 2 were assayed with 25 u moles  methylene blue  Fraction 2 was  added to determine  XDH  activity  also concentrated three f o l d using a Spectra-Con  Sample Concentrater (MW  Fraction 2  at 37°C. Disposable  l i m i t 8000) and assayed as above.  I s o l a t i o n of Porcine Pulmonary Microvascular C e l l s Because i t has localization endothelial  been reported on  that XO XDH cells  endothelial c e l l s  (73) we  i s found  the basis of immunocytochemical  in microvascular but not large vessel  attempted  to culture  in order to assay for enzyme a c t i v i t y in v i t r o .  methods of obtaining c a p i l l a r y endothelium (125)  (126)  contamination  (127). from  pulmonary microvascular  However, these large  vessel  for culture have been developed  are unsatisfactory  endothelium.  method of microvascular i s o l a t i o n was  Several  attempted  The  due  to p o t e n t i a l  development of a  new  based on the observation by  -40light  m i c r o s c o p i c examination  pleural found  The methods  t e c h n i q u e s developed  by  of less  elutriation  (131).  cell  often  types  dissociation  used  have  different  centrifugal elutriation particles  i s based  in a centrifugal  are m a i n t a i n e d  i n suspension.  dependent on c e l l  size  on  depends sizes  from t h e l i v e r (130)  i n combination  with  on the f i n d i n g  and d e n s i t i e s .  on t h e d i f f e r e n t  field  was based  o f r a b b i t pulmonary Type I I a l v e o l a r  of t i s s u e  T h i s technique  1 cm from t h e  l e s s than 50 um diameter a r e  of microvessel i s o l a t i o n  f o r the i s o l a t i o n  than  (129) and guinea p i g s i n u s o i d a l e n d o t h e l i a l c e l l s  u s i n g enzymatic  rate  t o a depth  surface of the p i g lung only v e s s e l s (128).  cells  that  centrifugal  that  different  S e p a r a t i o n by  rates of sedimentation of  b a l a n c e d by a c o u n t e r f l o w so t h a t  F r a c t i o n a t i o n o f a mixed c e l l  particles  population i s  and can be accomplished by stepwise i n c r e a s e s i n f l o w  (129) A Beckman E l u t r i a t o r  was used.  Adult porcine peripheral  lung  t i s s u e was prepared i n t h e f o l l o w i n g manner: Porcine Peripheral free  l u n g t i s s u e was o b t a i n e d f r e s h  lung t i s s u e  of pleura.  without  Ca  Fifteen  or Mg  2 +  (less  2  2+ containing  Ca  collagenase  and Mg  and 1000 ug DNAse Large  s u r f a c e ) was d i s s e c t e d  was r i n s e d  T i s s u e was minced  finely  c e l l s were seen  (50ml).  along with  i n c o l d PBS i n cold  PBS  0.25% BSA, 1 mg/ml  T h i s mixture  was  chunks o f t i s s u e were then  gauze and t h e f i l t r a t e  be completed.  g of t i s s u e  (0.1 g/1 each)  phase c o n t r a s t m i c r s c o p e single  t o twenty  slaughterhouse.  2+  37°C f o r 30 minutes. sterile  than 1 cm from p l e u r a l  (100 m l ) .  +  from a l o c a l  retained.  The f i l t r a t e  t o gauge t h e degree  (capillary  fragments)  incubated at  filtered  out w i t h  was examined under a  of digestion.  When c h a i n s o f  the d i s s o c i a t i o n was judged t o  F i f t y ml o f t h e above BSA:PBS: c o l l a g e n a s e m i x t u r e was added  -Aland  incubated for approximately  c e l l s were seen.  The c e l l suspension was  ml screw cap centrifuge tubes. ml  in each tube.  another 30 minutes u n t i l c l u s t e r s of  4-5  then equally divided into six 50  PBS-BSA was  added up to a t o t a l volume of 50  Tubes were centrifuged at 300 g for 10 minutes and the  supernatant discarded.  The  c e l l p e l l e t was  washed twice with PBS-BSA and  centrifuged again as above.  The supernatant was  were pooled and resuspended  in 10 ml of PBS-BSA to be loaded into the  elutriator.  The e l u t r i a t i o n buffer contained:  DNase by weight was  and 0.5% BSA  at 15°C.  discarded and a l l p e l l e t s  HBSS supplemented with  0.05%  During loading the e l u t r i a t o r rotor  spun at 2500 rpm and the flow pump was  set at 5 ml/min.  The following  fractions were c o l l e c t e d : Flow (1)  -13.5 ml/min  150 ml  (2)  -19.5  ml/min  250 ml  (3)  -23.5 ml/min  200 ml  (4)  purge e l u t r i a t o r at zero rpm, - 50 ml/min 100 ml  Fraction 2 was was  Volume  found to contain the endothelial c e l l s .  centrifuged at 300 g for 10 minutes.  The p e l l e t was  This f r a c t i o n  suspended in 5 ml  of culture medium containing 50 ml of endothelial conditioned Medium 199,  50  ml of regular Medium 199, 0.002 g Endothelial Derived Growth Factor (Meloy), 1 ml ,1% heparin, 1 ml Fungizone  (Squibb) and 20 ml porcine serum (Gibco).  C e l l s in 4 ml of the above medium were plated onto g e l a t i n i z e d T-25 These  seeded  flasks were incubated at 37°C f o r 60 minutes.  Unattached  c e l l s were then removed by washing with HBSS and c e l l s resuspended of the above medium.  flasks.  in 5 ml  -42C e l l s from Fraction 2 were noted to grow with the appearance of newly seeded large vessel endothelial c e l l s in culture. grow well enough to reach confluence or passage. became contaminated slaughter.  An  possibly  attempt was  due  However, cultures did not In addition,  cultures  to pulmonary infections in pigs  made to accelerate  before  the growth rate of the  cell  colony by using newborn (5 day old) pig tissue which has been shown to have greater  growth p o t e n t i a l than adult t i s s u e .  In addition, we used homologous  newborn pig sera to supplement culture media since f e t a l bovine serum i s widely used as a supplement because i t contains adult sera.  Since this was  not commercially available the sera was  from the same animal which lung tissue was kg and was  anaethetized  with 1.5  cava,  allowed  c o l l e c t e d and using  c o l l e c t e d into a s t e r i l e  to  clot  thoroughly  at  4°C  prepared as follows: syringe and  v i a the  centrifuged.  Serum was  then aliquoted and  serum in the research  hoped to eliminate  Serum  and  vena was  sterilized  By  as  preparing  c e l l culture f a c i l i t y on s i t e , i t was  the problem of contamination during k i l l i n g of the animal  and  removed of tissue by using  and  serum in the  facilities.  inferior  frozen f o r use  to supplement a r t i f i c a l c e l l culture media by 20%.  tissue and  P i g l e t weighed 2  heat inactivated at 56°C for 30 minutes then cold  a 22 micron f i l t e r .  required  c o l l e c t e d from.  prepared  cc sodium pentobarbital.  The pig serum growth supplement was Blood was  growth factors not found in  s t e r i l e conditions  St. Paul's Hospital  and  by preparing  s u r g i c a l research  and  cell  tissue  culture  -43EXPERIMENTS IN RAT TISSUE  Characterization of IXP Assay i n Tissue Rat kidney was used to test the IXP assay system i n tissue since this is a well studied source of XDH-XO (54). Reaction mixture was composed of: 450 y l r a t kidney extract (119) 450 y l 50 mM KPi + 0.1 mM EDTA 25 y moles pterin f o r oxidase a c t i v i t y 5 y moles methylene blue added to measure dehydrogenase activity 20 y moles a l l o p u r i n o l was added to stop reaction.  Determination of XDH-XO Level i n Normal Rat Lung by IXP Assay Normal  r a t lung  was quickly  according to Markley (113).  excised,  homogenized  Tissue extracts were assayed by IXP method to  determine the baseline l e v e l of XDH-XO a c t i v i t y . 1.  and processed  Reaction mixture contained:  450 y l r a t lung extract 450 y l 50 mM KPi + 0.1 mM EDTA 25 y moles pterin  2.  5 y moles methylene blue was added to complete the reaction and determine XDH a c t i v i t y  3.  20 y moles a l l o p u r i n o l was added to i n h i b i t XDH-XO.  -44Investigations In  order  to  l u n g was readily  into Conversion  of XDH  investigate factors  to  affecting  chosen s i n c e the enzyme was available  (93)  (94),  might  play  ischemic  tissue.  i t was  t i s s u e models.  to  found  examine the  Reversibility  s t a t e s of  (DTT)  t o cause r e d u c t i o n of t h i o l  groups was  or  min  of XDH  using r a t l i v e r  that proteolysis  T h i s was  the  i n the  the r a t  easily detectable in this  and  correlated  o f enzyme c o n v e r s i o n  groups  l u n g e x t r a c t s prepared inhibitors  (BDH) .  100  ug  at'37°C w i t h g e n t l e Samples  use  of  Ca  with  + +  two  attributable  to  dithiothreitol  enzyme and  affect  were  were  of XDH  i n 50 mM  incubated  trypsin  and  1ml  to  reversal  KPi  with  XO pH7.4 i n the absence of EDTA  trypsin  purified  of e x t r a c t were  compared  to  reversibility  the  same e x t r a c t s w i t h o u t  was  examined by  i n c u b a t i n g t r y p s i n - t r e a t e d e x t r a c t s w i t h ImM  60  of enzyme c o n v e r s i o n  by  a reducing DTT  agent  f o r 60  min.  assay.  Reaction mixtures  at  for  trypsinzation.  of  250  beef  agitation.  possibility  at 37°C p r i o r t o  from  incubated  The  1.  t o XO  activity.  protease  pancreas  role  studied with  E f f e c t of T r y p s i n i z a t i o n on C o n v e r s i o n Rat  t o be  enzyme c o n v e r s i o n .  redox  back t o XDH  thiol  conversion  Based on assays p u b l i s h e d i n 1972  decided  in affecting  XO  contained:  y l r a t lung 37°C  initiate  f o r 60 the  homogenate and min.  oxidase  25  y l of  reaction.  650 15  u l KPi yM  50 mM  pterin  5 y l of 1 mM  added t o measure dehydrogenase p l u s o x i d a s e  were  was  incubated  added  to  methylene b l u e acitivity.  was  2.  250  y l r a t lung  incubated stock  a t 37°C  determine was 3.  with  homogenate  with  650 y l KPi 50 mM  100 y l o f a t r y p s i n f o r 60 min.  the o x i d a s e  solution  from  25 y moles o f p t e r i n  a c t i v i t y and then  was  a 10 mg/ml was added t o  5 y l o f methylene  blue  added t o measure dehydrogenase p l u s o x i d a s e a c t i v i t y .  250 y l o f r a t l u n g incubated with 37°C.  This  homogenate w i t h  650 y l o f K P i 50 mM was  100 y l o f t h e above t r y p s i n  sample  was  then  incubated  with  s t o c k f o r 60 min a t 1 mM  DTT f o r an  o additional measure was  60 min a t 37 C.  the oxidase  To t h i s  25 y l p t e r i n  a c t i v i t y and then  added t o determine  was added t o  5 y l o f methylene  blue  t o t a l XDH + XO a c t i v i t y .  E f f e c t o f Endogenous P r o t e a s e s on C o n v e r s i o n o f XDH t o XO In  order  conversion temperature to  15°C  were  o f endogenous incubated  proteases  XDH-XO, w h i l e  to extracts  order t o assess  from  as c o n t r o l .  4°C i n h i b i t s p r o t e a s e  t h e same animal The e x t r a c t s  Samples were  which had not been i n c u b a t e d a t were  incubated  with  ImM DTT i n  i n 50 ym K P i , w i t h no  added EDTA i n c u b a t e d a t 15°C f o r 16 h r . 450  activity.  contained:  450 u l r a t lung e x t r a c t p r e p a r e d  y l KPi pH 7.4 50 mM 37°C  25 y l p t e r i n 5 y l methylene b l u e added  This  (93) has been shown  i f enzyme c o n v e r s i o n was r e v e r s i b l e .  Reaction mixture  on t h e  at 15°C o v e r n i g h t .  was chosen because ambient room temperature  overnight  1.  the e f f e c t  o f XDH t o XO samples  inactivate  compared  t o examine  -462.  450 p u l same r a t l u n g e x t r a c t not i n c u b a t e d KPi pH 7.4 50 mM  a t 15°C + 450 p i  37°C  25 p i p t e r i n 5 p methylene b l u e 3.  added  450 p i same r a t l u n g e x t r a c t x 15°C o v e r n i g h t ) + 450 p i KPi 50 mM pH 7.4 i n c u b a t e d w i t h ImM DTT X60 min a t 37°C 25 p i p t e r i n 5 p i methylene b l u e  E f f e c t of Calcium In  order  conversion chelating calcium assay of  on C o n v e r s i o n t o assess  added  o f XDH t o XO  whether  i n the r a t l u n g ,  calcium  as suggested  agents which have d i f f e r e n t  c h e l a t e r present  might  play  by McCord  a role  (52) two known  a f f i n i t i e s were used.  were used as a c o n t r o l .  method o f Markley (113) s p e c i f i e s  i n enzyme  Samples w i t h no  The XDH XO  fluorescence  the a d d i t i o n o f EDTA as a c h e l a t o r  d i v a l e n t c a t i o n s t o a l l homogenization and sample b u f f e r s .  interest  t o a s c e r t a i n whether  the same  calcium  amount o f EGTA,  a more  I t was o f specific  c h e l a t o r o f c a l c i u m would have a more p r o t e c t i v e e f f e c t on c o n s e r v i n g in  t h e XDH  served  form.  Tissue  as a c o n t r o l .  sulphydryl prepared  e x t r a c t s prepared  enzyme  i n t h e absence o f EDTA o r EGTA  R e v e r s i b i l i t y o f enzyme c o n v e r s i o n  by t h e r e d u c t i o n o f  groups was t e s t e d by the a d d i t i o n o f ImM DTT t o t i s s u e e x t r a c t s  with  EDTA, EGTA and i n the absence o f c a l c i u m c h e l a t o r s f o l l o w e d by  i n c u b a t i o n a t 37°C x 60 min.  Reaction mixtures contained: 1.  450 y l r a t lung extract prepared i n 50 mM KPi 450 y l KPi pH 7.4 25 y l pterin 5 y l methylene blue added  2.  450 y l above extract, 450 y l KPi pH 7.4, incubated with 1 mM DTT at 37°C x 60 min. 25 y l pterin 5yl methylene blue added  3.  450 y l r a t lung extract prepared i n 50 mM KPi with ImM EDTA added to a l l homogenizing, chromatography and sample buffers 450 y l KPi pH 7.4 50 mM + ImM EDTA 25 y l pterin 5 y l methylene blue  4.  450 y above extract with 450 y l KPi pH 7.4, 50 mM + ImM EDTA incubated with 1 mM DTT at 37°C f o r 60 min 25 y l pterin 5 y l methylene blue added  5.  450 y l r a t lung extract prepared i n 50 mM KPi with ImM EGTA added to a l l homogenizing, chromatography and sample buffers 450 y KPi pH 7.4 50 mM + ImM EGTA 25 y l pterin 5 y l methylene blue added  -486.  450 y l above extract with 450 y l KPi ph 7.4  50mM + ImM EGTA  incubated with 1 mM DTT at 37°C f o r 60 min 25 y l pterin 5 y l methylene blue added  E f f e c t of Ischemia on Conversion of XDH We  sought  conversion of XDH  to determine  to XO  whether ischemia might have an e f f e c t  to XO by the use of two models of ischemia.  involved incubation of excised lung tissue in PBS  (A) The  on first  at 37°C in a humidified  incubator under normoxic conditions for 60 min p o s t - s a c r i f i c e , according to the method of Granger (132).  (B) The  second method involved perfusion of  the rat lung v i a a pulmonary artery cannula with an oxygen depleted buffer mixture  of 50 mM  KPi pH  7.4  at 37°C through  which  had been  for 20 min and clamping of the pulmonary artery for 60 min. compared  to rat lung  extracts prepared  cannulated and perfused with cold 50 mM 1 mM  (C) These were  lungs which were rapidly  KPi pH 7.4  containing 1 mM EDTA and  DTT and immediately homogenized in the same buffer containing 1 mM PMSF  as a protease i n h i b i t o r . 1 mM  from  sparged  DTT  Samples were then compared to those incubated with  at 37°C x 60 minutes to examine the r e v e r s i b i l i t y of XDH  to XO  conversion. Sample mixtures contained: 1.  450 y l of rat lung extract prepared by Method (A) with 450 y l of KPi buffer 25 y moles p t e r i n 5 y moles methylene blue  2.  450 y l of rat lung extract (A) with 450 y l of KPi buffer incubated with 1 mM DTT at 37°C f o r 60 min 5 y moles of methylene blue  3.  450 y l of r a t lung extract prepared by method (B) with 450 y l of 50 mM KPi pH 7.4 25 y moles pterin 5 y moles methylene blue added  4.  450 y l of r a t lung extract (B) with 450 y l of 50 mM KPi, pH 7.4 incubated with 1 mM DTT at 37°C 60 min 25 y moles p t e r i n 5 y moles methylene blue added  5.  450 y l of r a t lung extract (C)(EDTA + DTT+) 450 y l of 50 mM KPi pH 7.4 + 1 mM EDTA 25 y moles pterin 5 y moles methylene blue added  Experiments It  i n Human Lung Tissue was  of great  interest  to determine  whether  active  XDH  XO  was  detectable i n human lung t i s s u e , since the enzyme as yet has only been l o c a l i z e d using immunohistological means (72) (73). 6-10  g were obtained  from  Human lung samples of approximately  peripheral lung  pneumectomy or lobectomy f o r bronchogenic  sections of patients  carcinoma.  Samples were  frozen at -70°C upon a r r i v a l to be processed at a l a t e r date.  undergoing immediately  Thawed samples  were minced and homogenized i n cold buffer containing 50 mM KPi pH 7.4 and 1 mM EDTA with 1 mM  PMSF, and processed according to the method of Markley  (113).  -50-  Since a c t i v i t y was much lower than rat lung, the spectrofluorimeter was used on the most s e n s i t i v e s e t t i n g . Reaction mixtures  contained:  900 u l of human lung extract 25 u moles of pterin 5 v moles of methylene blue Since many biopsy  samples were obtained from tissue which was  necrotic, v i a b i l i t y of tissue was tissue  to  oxidize NADH.  NADH  determined  (0.1 mM)  was  already  by assessing the capacity of the added  to human lung  tissue  homogenates and the oxidation of NADH to NAD was measured spectrophotometrically by recording the rate of change of absorbance at 340  nm.  S t a t i s t i c a l Analysis SE was  calculated f o r a l l mean values of enzyme a c t i v i t y .  between experimental  groups and  controls were based on  students  xanthine oxidase a c t i v i t i e s f o r the studies of enzyme conversion.  Comparisons t-test  of  -51RESULTS  EXPERIMENTS WITH PURIFIED XO I s o l a t i o n o f M i l k Xanthine After fractions 86.8 is  mg  fractionation  with  xanthine  (122).  defined  Oxidase  oxidase  Activity  as g i v i n g  of milk p r o t e i n activity  a AA  o f one p e r minute  the  authors  specific  p r o t e i n . One u n i t  a t 295 nm  to f l a v i n  t o be 5.75 which i s h i g h e r than  o f the method (116).  p r o t e i n of  during the  (115).  o f A280 t o A450 ( p r o t e i n  was determined  o b t a i n e d had a t o t a l  o f pooled f r a c t i o n s was 4.78 U/mg  conversion o f xanthine t o u r i c a c i d Ratio  as d e s c r i b e d i n methods pooled  r a t i o ) o f the p u r i f i e d  t h e v a l u e o f 4.8 r e p o r t e d by  These r e s u l t s  a c t i v i t y , due t o more f l a v i n - c o n t a i n i n g  suggest  a higher l e v e l of  active subunits.  SDS-PAGE r e v e a l e d a s i n g l e band o f 135KDa ( F i g u r e 5 ) , the known of  the x a n t h i n e  precipitates protein  dialyzed  /ml.  urate  assay;  assay  (113).  xanthine  oxidase  subunit.  of p r e c i p i t a t e s  o r 69.11 ug IXP produced I n accordance  oxidase  was found  samples were i n c u b a t e d w i t h  with  was  t o be 25.4 mg  54 U/ml/min as determined  /min/mg  protein  by  by f l u o r o m e t r i c  the as per method o f Beckman ( 9 1 ) , m i l k  t o have up t o 10% dehydrogenase 1 mM  size  P r o t e i n c o n c e n t r a t i o n o f ammonium s u l p h a t e  on M i l l i p o r e membranes was determined  Activity  XO  dithiothreitol  activity  when  (DTT) a t 37°C f o r 60 min  p r i o r t o assay.  C h a r a c t e r i z a t i o n of P o l y c l o n a l A n t i s e r a to Xanthine When  a rabbit  was  immunized  with  purified  Oxidase m i l k XO which had been  -52-  F i g u r e 5.  Pharmacia Phast System SDS-PAGE a n a l y s i s of p u r i f i e d b o v i n e m i l k XO on a 8-25% g r a d i e n t g e l s t a i n e d w i t h coomassie b l u e . Lane 1 LMW markers (from top to bottom) p h o s p h o r y l a s e b, 94,000, albumin 67,000, ovalbumin 43,000, c a r b o n i c anhydrase 30,000 t r y p s i n i n h i b i t o r 20,000, l a c t a l b u m i n 14,000. Lanes 2 and 5 c o n t a i n h i g h m o l e c u l a r weight markers (Top t o bottom) f e r r i t i n , 220,000, albumin 67,000, c a t a l a s e 60,000, l a c t a t e hydrogenase 36,000. Lane 3:2 pg o f p u r i f i e d bovine m i l k XO. Lane 4: l p g o f p u r i f i e d bovine m i l k XO (arrow).  -53SDS-dissociated, fold  during  the  antibody  the  8 weeks of  technique.  The  titer  in  with  the  keeping  SDS-dissociated  fact  antigen,  exsanguination,  ml  of immune serum was tests  Blotting, milk  XO  weights antibody tests the  120  using  of  XDH  and  ml  also  using  was  and  serum was  lung  tissue  as  antibody  150,000 Da  with  strongly  a  reactivity proteolytic  at  smaller  bovine  When lung,  rat  kidney,  milk  150,000 and of XO  major fragment of the tissue liver  bands were seen  against  were uncovered. from which  antibody  in  gels  reflecting  60  Western  proteolytic  bovine  molecular  r e s p e c t i v e l y (Figure  6).  fragments  The  of XO.  In  p u r i f i e d bovine serum albumin the  albumin  of  (BSA)  several  (Figure 6). Blotting  containing  t o b o v i n e XO (powdered,  130,000 Da,  100  ug  of  protein  cross reacted with  Safeway  Brand),  well  at  as  at 90,000 Da  (87)  as  (Figure  per bovine  showing  characteristic 7)  which  is a  enxyme.  homogenates and  containing  100  h e a r t were examined by  i n a l l t i s s u e samples  w e l l as at 90,000 Da  ELISA  increased,  raised  collected  reactive against  that antiserum  fragment  was  first  SDS-PAGE  t i s s u e homogenates  and  the  i n c r e a s e d 2000 f o l d  SDS-PAGE of T i s s u e Homosenates w i t h Western  sample r e v e a l e d  by  5000  s t r o n g l y r e a c t i v e with p u r i f i e d  on  s p e c i e s which ran at 67,000 Da  SDS-PAGE of  increased  determined  thus more d e t e r m i n a n t s  t i s s u e homogenates and  antibody  serum  obtained.  separated  reacted  the  as  o f whole b l o o d was  immune  130,000  immune  immunization,  that  anti-XO a n t i s e r u m and  of  a g a i n s t n a t i v e XO  At  In  titer  (Figure 8).  yg  protein  SDS-PAGE, XO  at 150,000 and  of  rat  reactive  130,000 Da  as  -54-  Figure 6.  Western blot of p u r i f i e d bovine milk XO. Electrophoresis of proteins was carried out on 6% SDS-PAGE. Protein was electrotransfered onto Immobilon Membrane ( M i l l i p o r e ) . XO was v i s u a l i z e d by incubation of the Western blot with a :500 d i l u t i o n of polyclonal rabbit anit-bovine XO serum followed by incubation with the second antibody, goat-anti-rabbit IgG, covalently linked to horseradish peroxidase. Addition of substrate produces a purple coloured product bound to the membrane at s i t e of antigen location. Lane 1 high molecular weight stained with amido black. (Top to bottom) myosin 205,000, B-galactosidase 116,000, phosphorylase B 97,000, BSA 66,000, ovalbumin 45,000. Lane 2, 3, XO (arrow). Lane 4, 5, 6 albumin showing r e a c t i v i t y of antibody to albumin ( s t a r ) .  -55-  Figure 7.  Western blot of bovine tissues. 100 ug of redissolved protein p r e c i p i t a t e s were electrophoresed, b l o t t e d and v i s u a l i z e d as previously described (Figure 6). Lane 1 p u r i f i e d XO showing 150 KDa and 135 KDa subunits (arrows) as well as 90 KDa fragment. Lane 2 - bovine lung. Lane 3, BSA. Lane 4 high molecular weight markers stained with amido black. (Top to bottom) myosin 205,000, B-galactosidase 116,000, phosphorylase B 97,400, BSA 66,000, ovalbumin, 45,000.  -56-  Figure 8.  Western blot of rat tissues. 100 ug of redissolved protein precipitates were electrophoresed, blotted and visualized as previously described (Figure 6). Lane 1 shows the results from rat lung homogenate, lane 2 rat liver homogenate, lane 3 rat kidney homogenate. Lane 4 shows purified bovine milk XO with the 150 KDa and 135 KDa subunits as well as 90 KDa fragment.  SDS-PAGE o f 100 kidney  d i d not  ug p r o t e i n show  protein  antisera.  Porcine l i v e r  peripheral  human  oxidase  o f p o r c i n e whole l u n g , p e r i p h e r a l  lung  bands which  reacted positvely  tissue  homogenates  f o r XO showed  a n t i - b o v i n e XO  (Figure 9). no  A n a l y s i s of  d e t e c t a b l e xanthine  ( F i g u r e 10).  Comparison o f S e n s i t i v i t y o f Urate Assay Graded d i l u t i o n s The  reacted with  l u n g and  limits  t o I s o x a n t h o p t e r i n Assay  o f XO were assayed u n t i l  no a c t i v i t y  o f d e t e c t i o n o f the u r a t e assay were found  o f XO p r o t e i n  (assuming  a l l o f the p r o t e i n  c o u l d be measured.  t o be 2.A5  x 10  mg  5  i n the p u r i f i e d XO i s XO).  This  c o r r e s p o n d s t o A.93 x 1 0 ^ molecules o f XO With  t h e IXP  protein,  which  assay  the l i m i t s  corresponds  of d e t e c t i o n g  t o 6.17  x 10  were 3.17  molecules  x 10  o f XO.  -6  mg  X0  The IXP assay  was found t o be i n t h e range o f 100 f o l d more s e n s i t i v e than t h e u r a t e assay.  Product I n h i b i t i o n o f IXP Assay IXP IXP  assay  using  f o r XO was  performed  s u b s t r a t e c o n c e n t r a t i o n s from  shown  to i n h i b i t  shown  i n a Lineweaver-Burke  3.A x 10  -3  both  M.  the enzymatic  i n the presence 1 uM t o 25 uM.  rate of r e a c t i o n  Plot  and absence  (Figure 11).  3 nm  o f 3nM  o f IXP was  i n a c o m p e t i t i v e manner as The Km f o r the r e a c t i o n  The K i f o r the r e a c t i o n was A.A x 10  -10  M.  was  Figure 9.  Western blot of porcine tissues. 100 ug of redissolved protein precipitates were electrophoresed, blotted and visualized as previously described (Figure 6) . Lane 1 high molecular weight markers stained with amido black, top to bottom; myosin 205,000 Da, B-galactosidase 116,000 Da, phosphorylase b 97,400, BSA 66,000, ovalbumin 45,000 Da. Lane 2 pig lung homogenate. Lane 3 contains pig liver homogenate. Lane 4 contains pig kidney homogenate. Lane 5 contains purified bovine milk XO showing the 150 KDa, 135 KDa and 90 KDa subunits (arrows).  -59-  Figure 10. Western Blot of human lung tissue. 100 ug of redissolved protein p r e c i p i t a t e s were electrophoresed, b l o t t e d and v i s u a l i z e d as described (Figure 6). Lane 1; p u r i f i e d bovine milk XO showing 150 KD, 135 DK and 90 KD subunits (arrows), lane 2, human lung homogenate, lane 3, high molecular weight markers stained with amido black (Top to bottom) myosin 205,000 Da, B-galactosidase 116,000 Da, phosphorylase b 97,400 Da, BSA 66,000 Da, ovalbumin 45,000 Da.  -60-  A  I h r l l b l T t U  A  A  UNiMHIUITEl)  100  -.  UU  1/S (MOLES PTERIN)  Figure 1 1 .  Lineweaver-Burke plot showing the competitive p u r i f i e d XO by 3 nm IXP; = 3.4 x 10-3 . M )  10-10  M  .  i n h i b i t i o n of K  4 1  >  4  x  -61EXPERIMENTS IN PORCINE TISSUE  Assay for XO XDH No lung  Activity  i n Pig Tissue  enzyme a c t i v i t y was  detected  homogenates  different  from  spectrofluorimeter data obtained  6  in pig kidney, whole lung or peripheral pigs,  settings were used.  even  when most  Representative  sensitive  tracings taken from  on the enzymatic a c t i v i t y in pig lung homogenates  (Figure  13)  and pig kidney homogenates (Figure 14) revealed  a decreased rather than the  increased  of time which would  expected IXP.  fluorescent f o r the  intensity  as  a function  enzymatic conversion  Pig l i v e r contained  XO XDH  of pterin to fluorescent  be  product,  a c t i v i t y as determined by IXP assay,with a  _3 s p e c i f i c a c t i v i t y of 1.1  Inhibition  x 10  the  previous  decrease i n fluorescence  observation i n the IXP  The  rate of IXP production  the  same amount of p u r i f i e d  lung  found to be  extract was  inhibition  12).  of P u r i f i e d XO A c t i v i t y by Pig Extracts  Based on  buffer was  yg IXP/min/mg protein (Figure  7.5  that pig kidney and assay, an i n h i b i t o r  by p u r i f i e d XO  XO  yg IXP/mg protein/min.  found to be  1.98  sought.  compared to that of Enzyme a c t i v i t y in  Enzyme a c t i v i t y in pig  yg IXP/mg protein/min  in enzyme rate of a c t i v i t y (Figure  caused a  for XO was  in buffer was  in pig lung extract.  lung  showing a  73.5%  15).  Heat S t a b i l i t y of Active Factors in Pig Tissues In order inhibition  to test whether an enzyme or protein might be of XO  a c t i v i t y by pig lung extract, we  involved in the  examined the effect  of  -62-  5  OH  1  0  2  :  1  4  •  1  1  6  8  Time (minutes)  Figure  12.  Representative t r a c i n g of XDH XO a c t i v i t y in pig l i v e r homogenates from n=3 samples. A. A d d i t i o n o f 25 y moles p t e r i n t o i n i t i a t e the a e r o b i c r e a c t i o n . B. A d d i t i o n of 5 y moles methylene blue t o measure the t o t a l XDH + XO a c t i v i t y . C. A d d i t i o n of 20 y moles a l l o p u r i n o l t o i n h i b i t the r e a c t i o n .  10  -63-  F i g u r e 13.  R e p r e s e n t a t i v e t r a c i n g o f XDH XO a c t i v i t y i n p i g l u n g from n=6 samples. A. A d d i t i o n of 25 u moles p t e r i n t o i n i t i a t e the a e r o b i c r e a c t i o n . B. A d d i t i o n of 5 u moles methylene b l u e t o measure t o t a l XDH + XO a c t i v i t y .  -64-  Figure 14.  Representative tracing samples. A. Addition aerobic reaction. B. measure total XDH + XO  of XDH-XO activity in pig kidney from n=6 of 25 u moles pterin to initiate the Addition of 5 u moles methylene blue to activity.  -65-  F i g u r e 15.  I n h i b i t i o n of p u r i f i e d XO a c t i v i t y by p i g l u n g homogenate. A. R e a c t i o n r a t e o f p u r i f i e d bovine XO i n 50 mM KPi + mM EDTA with 25 uM p t e r i n . B. R e a c t i o n r a t e o f same amount o f p u r i f i e d bovine XO i n p i g lung homogenate i n 50 mM KPi + 0.1 mM EDTA w i t h 25 uM p t e r i n showed a 73.5% i n h i b i t i o n of r e a c t i o n rate.  -66incubation  a t 100°C f o r 20 min on a b i l i t y  XO a c t i v i t y . protein/min found of  P u r i f i e d XO a c t i v i t y while  p u r i f i e d XO a c t i v i t y  as compared  to inhibit  i n b u f f e r was found t o be 3.6 ug IXP/mg i n boiled  t o be 2.25 yg IXP/mg p r o t e i n / m i n .  activity,  of p i g tissue extract  This  pig tissue  represented  t o a 73.5% i n h i b i t i o n  extract  was  a 37% i n i b i t i o n  i n non-denatured p i g  homogenate ( F i g u r e 1 6 ) .  E f f e c t of P i g Tissue  E x t r a c t on IXP R e l a t i v e  Because b o i l i n g o f p i g t i s s u e e x t r a c t of XO a c t i v i t y , could 3nM  d i d not t o t a l l y  ablate  inhibition  i t was n e c e s s a r y t o i n v e s t i g a t e whether p i g t i s s u e  quench IXP f l u o r e s c e n c e  IXP i n b u f f e r  amount o f p i g lung  or degrade IXP. The f l u o r e s c e n c e  decreased  immediately  extract.  Following  o f XO-XDH  Activity  by 8% when measured  extract  i n t e n s i t y of i n t h e same  t h e r e a c t i o n f o r 10 minutes showed an  a d d i t i o n a l 3% decrease i n IXP f l u o r e s c e n c e  Measurement  Intensity  (Figure 17).  i n Cultured  Porcine  Pulmonary  Artery  Endothelial Cells Cellular endothelial  extract cells  from  were  seven  assayed  T-80 f l a s k s  f o r XO-XDH a c t i v i t y . 7  cell  numbers  activity  was  concentrated  ranging detected threefold  from  7.8 x 10  i n this  of porcine  This  artery  corresponds to  8 t o 1.3 x 10  extract,  by removal  pulmonary  o f H^O  would be l e s s than t h e l e v e l of d e t e c t i o n  cells.  or i n extract therefore  which  activity,  f o r t h i s assay.  No XO-XDH had been i f present,  -67-  F i g u r e 16.  Heat s t a b i l i t y of a c t i v e f a c t o r s i n p i g t i s s u e . A. Reaction r a t e of p u r i f i e d bovine XO i n 50 mM KPi + 0.1 mM EDTA. B. R e a c t i o n r a t e o f p u r i f i e d bovine XO i n p i g l u n g homogenate denatured by i n c u b a t i o n i n b o i l i n g water f o r 20 min, showing 37% i n h i b i t i o n o f XO a c t i v i t y .  -68-  Figure 17.  E f f e c t of Pig Tissue Extract of Relative Fluorescent Intensity of IXP. Averaged curve from points obtained f o r three r e p l i c a t e s of representative traces. A. Relative i n t e n s i t y of 3 nM IXP dissolved i n 900 u l 50 mM KPi. B. Relative i n t e n s i t y of 3nM IXP dissolved i n 900 u l pig lung homogenate.  -69l 8 o l a t i o n of Porcine Pulmonary Microvascular C e l l s Endothelial c e l l s were determined to be separated from parenchymal c e l l s in  elutriation  microscopy. to  Fraction  2.  After i n i t i a l  These  cells  were  followed  with  phase  seed of adult c e l l s , clumps of 4-6 c e l l s adhered  the gelatin-coated culture f l a s k .  By 5 days l a t e r these had grown into  patches of polyhedral c e l l s interspersed with spindly open networks of c e l l s which had the appearance of newly culture.  isolated  large vessel endothelium in  By 10 days many healthy patches of polyhedral c e l l s with oval  nuclei were apparent growing i n cobblestone arrangements t y p i c a l of large vessel endothelium i n culture.  These were interspersed with "networks" of  spindle shaped c e l l s which gave the appearance of c a p i l l a r y networks. days  large  patches of subconfluent  polyhedral  cells  growing with  c h a r a c t e r i s t i c cobblestone monolayer appearance were apparent.  By 14 a  A l l colonies  were found to be infected with a mycotic contamination at this point and were thus discarded.  The c e l l s were not passaged.  Isolation of microvascular endothelial c e l l s from newborn p i g l e t provided a very low i n i t i a l number of c e l l s . 28 days.  Patches of c e l l s  f i b r o b l a s t s were scraped flask.  growing with the morphological appearance of  from the culture  surface  and rinsed  from the  After 56 days the T-25 f l a s k of newborn p i g l e t c e l l s was confluent  with polyhedral c e l l s cells  E l u t r i a t o r f r a c t i o n 2 was cultured for  i n a cobblestone monolayer, similar to large vessel  i n culture (Figure 18). The c e l l s were passaged into two f l a s k s .  f l a s k was supplemented with newborn p i g l e t with commercial adult pig serum. slightly  faster;  Cells  One  serum, the other supplemented  grown in newborn p i g serum grew  i . e . they reached confluence one day sooner than c e l l s  -70-  Figure 18.  Microvascular endothelial cells isolated by centrifugal elutriation shown in culture. Cells show morphological appearance characteristic of cultured endothelial cells with cobblestone monolayer arrangement and ovoid nucleus. (Magnification 142 x)  grown i n a d u l t p i g serum. patches second  of c e l l s ,  the c u l t u r e s were contaminated  c o n f l u e n c e was  b a c t e r i a o r fungus. Neither glass  adult  in  order  i n order  to  was  of f i b r o b l a s t i c by f i b r o b l a s t s  no evidence  appearing  by the time  o f c o n t a m i n a t i o n by  i m p u r i t y c o l o n i e s were d i s c a r d e d .  piglet  cells  perform  the  c o u l d be  assay  to c o n f i r m t h a t  t h e r e f o r e necessary to r e l y  order to i d e n t i f y In  There  p i g nor newborn  coverslips  I t was  reached.  Because of t h i s  immunolocalization cells.  D e s p i t e manual removal  s u b c u l t u r e d onto  f o r Factor  they were  indeed  VIII endothelial  on m o r p h o l o g i c a l appearance i n  them.  a d d i t i o n , n e i t h e r a d u l t p i g nor newborn p i g l e t  c e l l s were c u l t u r e d i n  l a r g e enough q u a n t i t y use f o r b i o c h e m i c a l a n a l y s i s of XO-XDH a c t i v i t y .  EXPERIMENTS IN RAT TISSUE C h a r a c t e r i z a t i o n o f IXP Assay Whereas same assay  use o f the IXP assay d i d not d e t e c t XO  This r a i s e d  showed XO  activity  activity  homogenates,  To determine  the enzyme  was  m i l k , the  system was e f f e c t i v e  the e f f e c t i v e n e s s  assayed  in purified  i n p i g l u n g and k i d n e y homogenates.  the q u e s t i o n o f whether the assay  t i s s u e homogenates.  tissue  in Tissue  of t h i s  assay  i n crude in tissue  i n r a t k i d n e y homogenates s i n c e  this  i s know t o c o n t a i n the enzyme.  Rat k i d n e y was e x c i s e d and immediately + O.lmM EDTA. IXP/min/mg  The t o t a l  protein  (+ SE  enzyme  homogenized i n i c e c o l d  activity  3 x 10  -3  ).  XO  (XDH  + XO)  activity  was  was  1.5  3.9  50 mM Kpi  x 10  x 10  -2  -3  ug  pg  _3 IXP/min/mg shown 19) .  protein  (+ SE 1 x 10  t o be i n h i b i t e d  ) on n=5  animals.  IXP p r o d u c t i o n was  by the a d d i t i o n o f 20 u moles of a l l o p u r i n o l  (Figure  -72D e t e n n i n a t i o n o f XDH-XO L e v e l Normal r a t lung +  0.1 mM  EDTA.  IXP/min/mg 10  -3  of  was q u i c k l y e x c i s e d  pg IXP/min/mg pterin  Studies  enzyme  -3 (+ SE 3 x 10 ) . n=3 animals.  protein  t o IXP was  allopurinol  and homogenized i n i c e c o l d 50 mM KPi  -2 a c t i v i t y (XDH + XO) was 1.7 x 10 ug _3 (+ SE 4 x 10 ). XO a c t i v i t y was shown t o be 9.3 x  Total  protein  i n Normal Rat Lung by IXP Assay  i n h i b i t e d by t h e a d d i t i o n  Conversion  o f 20 u moles o f  (Figure 20).  o f t h e C o n v e r s i o n o f XDH t o XO  E f f e c t o f T r y p s i n Treatment on XDH t o X0 C o n v e r s i o n Based  on s t u d i e s  in liver  XO  by l i m i t e d p r o t e o l y s i s  ml  o f r a t lung  extract. DTT  to test  groups.  to oxidase all  (86) t h e e f f e c t o f i n c u b a t i n g  was s t u d i e d  and compared  converted to  100 ug t r y p s i n p e r  to control  r a t lung  Both t r y p s i n i z e d and c o n t r o l e x t r a c t s were a l s o i n c u b a t e d w i t h ImM  i n order  thiol  extract  showing t h a t XDH i s i r r e v e r s i b l y  Conversion  activity  XDH+XO/XO was 1.05. extract  involving oxidation of  i s e x p r e s s e d as t h e r a t i o o f t o t a l enzyme  (XDH+XO/XO).  enzyme has been  Trypsinized  f o r the r e v e r s i b l e conversion  converted Control  A ratio  o f (XDH + XO)/XO = 1 i m p l i e s  to the oxidase  extract  activity  form.  + DTT showed  showed XDH+XO/XO o f 1.  In c o n t r o l  that  r a t lung  (XDH + XO)/XO o f 4.92.  Trypsinized  extract  incubated  w i t h DTT showed (XDH + XO)/XO o f 1.73 ( F i g u r e 2 1 ) . E f f e c t o f Entended I n c u b a t i o n a t 15°C on C o n v e r s i o n o f XDH t o XO Rat Analysis  lung  extracts  were  incubated  o f (XDH + XO)/XO r a t i o  without  show complete  EDTA  a t 15°C f o r 16 h.  conversion  (XDH + X0)/X0 = o  1).  Further  incubation  o f these e x t r a c t s  with  ImM DTT f o r 60 min a t 37 C  showed a (XDH + X0)/X0 r a t i o s o f 3.59 ( F i g u r e 2 2 ) .  -73-  -  I  0  2  I  4  i  i  6  8  Time (minutes)  Representative t r a c i n g o f XDH-XO a c t i v i t y i n r a t kidney homogenate, from n=6 samples. A. A d d i t i o n o f 25 u moles p t e r i n to i n i t i a t e the-aerobic r e a c t i o n . B. A d d i t i o n of 5 u moles methylene blue t o measure the t o t a l XDH + XO a c t i v i t y . C. A d d i t i o n of 20 u moles a l l o p u r i n o l t o i n h i b i t the r e a c t i o n .  10  -74-  Time (minutes)  Representative tracing of XDH-XO activity in rat lung from n=6 samples. A. Addition of 25 y moles pterin to initiate the aerobic reaction. B. Addition of 5 y moles methylene blue to measure XDH + XO activity. C. Addition of 20 y moles allopurinol to inhibit the reaction.  -75E f f e c t of Calcium  on C o n v e r s i o n  o f XDH t o XO i n Normoxic Lung  In o r d e r t o t e s t the e f f e c t s o f c a l c i u m on XDH t o XO c o n v e r s i o n , t h e e f f e c t o f EDTA as a c h e l a t o r o f d i v a l e n t c a t i o n s was compared w i t h EGTA, a more s p e c i f i c  c h e l a t o r o f c a l c i u m , u s i n g samples prepared  c h e l a t o r as c o n t r o l s . incubation with  i n the absence o f  The r e v e r s i b i l i t y o f t h e c o n v e r s i o n was t e s t e d by  ImM DTT.  Experiments were performed on normoxic r a t lung  q u i c k l y e x c i s e d and homogenized i n the a p p r o p r i a t e b u f f e r . For purposes o f comparison,  (XDH + XO)/XO o f normoxic r a t l u n g  absence o f c a l c i u m c h e l a t o r s was 1.45. all  (Table I I ) .  With the a d d i t i o n o f 1 mM  i n the EDTA t o  b u f f e r s used f o r t i s s u e homogenization, chromatography o r sample  p r e p a r a t i o n , the r a t i o c a l c u l a t e d f o r the t i s s u e samples, (XDH + XO)/XO was 1.8.  When EGTA was used as more s p e c i f i c c h e l a t o r o f c a l c i u m , t h e r a t i o o f  (XDH + XO)/XO was 1.1 + SE 0.33 ( F i g u r e 2 3 ) . Incubation  o f the above d e s c r i b e d e x t r a c t s w i t h ImM DTT f o r 60 min at  37°C t o reduce t h i o l groups gave the f o l l o w i n g (XDH + XO)/XO  ratios:  C o n t r o l 4.74 EDTA 2.8 EGTA 3.25  E f f e c t of Calcium Rat  on XDH t o XO C o n v e r s i o n  l u n g was e x c i s e d , r i n s e d , and i n c u b a t e d  reproduce t h e c o n d i t i o n s o f warm i s c h e m i a then  i n Ischemic Lung  b l o t t e d t o remove excess  i n PBS a t 37°C t o  as d e s c r i b e d  (132).  Lungs were  PBS and homogenized i n i c e d 50 mM KPi  c o n t a i n i n g e i t h e r ImM EDTA, ImM EGTA o r no c a l c i u m c h e l a t o r as a c o n t r o l . Three animals The  were s t u d i e d f o r each experiment  r a t i o o f (XDH + X0)/X0 o b t a i n e d  absence o f c a l c i u m c h e l a t o r s was 1.5.  (Table I I I ) .  from ischemic  r a t lung  i n the  R a t i o o f (XDH + X0)/X0 f o r ischemic  -76TABLE II  Comparison of XDH and XO A c t i v i t i e s in Normoxic Rat Lung in the Absence and Presence of Calcium Chelators During Sample Preparation  Treatment  XDH + XO (x IO" ) 3  XO (x 10" ) 3  XDH + XO XO  Control  7.1 + 0.1  5.0 + 1.0  1.45  Control +lmM DTT  7.0 + 0.1  1.5* + 0.4  4.74  9.3 + 0.3  1.8  ImM EDTA  16.7 + 0.4  ImM EDTA +lmM DTT  6.7 + 0.2  2.4* + 0.1  2.8  ImM EGTA  6.0 + 0.2  5.2 + 0.1  1.1  ImM EGTA +lmM DTT  7.7 + 0.3  2.4* + 0.7  3.25  Values given are expressed as mean + SE pg IXP/min/mg protein. Rat lungs were assayed by the fluorometric method of Markley (113). Experiments were carried out at 37°C. Experiments with 1 mM DTT were incubated at 37°C for 60 minutes. n=3 f o r each group. * = p<0.05 for comparisons between XO a c t i v i t y with and without DTT f o r each calcium chelating agent.  Control  Figure 21.  Control + DTT  Trypsin  Trypsin + DTT  E f f e c t of Trypsin Treatment on the (XDH + XO)/XO Ratio. Conversion of XDH to XO was tested by incubation of one ml of rat lung extract with 100 ug t r y p s i n as compared to c o n t r o l . Conversion to XO i n the control preparation was 80% r e v e r s i b l e to XDH by incubation with DTT. Conversion to XO i n trypsin-treated preparation was r e v e r s i b l e to XDH to only a s l i g h t extent. Enzyme a c t i v i t y was i r r e v e r s i b l y converted to XO by 95%.  -78-  5-  Control  Figure 22.  Control + DTT  15°C Incubation  15°C Incubation + DTT  E f f e c t of Extended Incubation of 15°C on the (XDH + XO)/XO Ratio. Conversion to XO in control lung homogenate was reversible by 80% to XDH after incubation with DTT. Conversion to XO after 16h incubation of r a t lung extract at 15°C was reversible by 75% to XDH with only a small proportion of the enzyme a c t i v i t y being i r r e v e r s i b l y converted.  -79-  5-  Control  Figure 23.  Control + DTT  EDTA  EDTA + DTT  EGTA  EGTA + DTT  Effect of Calcium on R e v e r s i b i l i t y of the conversion of XDH to XO i n Normoxic Rat Lung. Treatment of r a t lung extracts with EDTA or EGTA d i d not a f f e c t r e v e r s i b i l i t y of XDH to XO conversion tested by incubation with DTT as compared to control.  -80TABLE I I I  Comparison of XDH and XO A c t i v i t i e s i n Ischemic Rat Lung in the Absence and Presence of Calcium Chelators During Sample Preparation  Treatment  XDH + XO (x 10-3)  XO ( -3) X 10  XDH + XO XQ  Control  24.3+10.0  15.9+6.0  1.5  Control +lmM DTT  24.4 + 10.0  7.5* + 3.0  3.2  ImM EDTA  7 .0 + 3.0  3. 1 + 0.4  2.24  ImM EDTA +lmM DTT  8 .3 + 4.0  2. 2* + 1.0  2.3  ImM EGTA  7 •5 + 2.0  5. 7 + 1.0  1.28  ImM EGTA+ +lmM DTT  8 .4 + 3.0  2.0* + 0.6  4.2  Values given are expressed as means + SE of yg IXP/min/mg protein. Rat lungs were assayed by fluorometric method of Markley (113). Experiments were carried out at 37°C. Experiments with 1 mM DTT were incubated at 37°C f o r 60 minutes. n=3 f o r each group. * = p<0.05 f o r comparisons between XO a c t i v i t y with and without DTT f o r each calcium chelating agent.  -81-  6n  5-  Control  Control + DTT  EDTA  EDTA + DTT  EGTA  EGTA + DTT  Figure 24. E f f e c t of Calcium on R e v e r s i b i l i t y of the conversion of XDH to XO in Ischemic Rat Lung. Treatment of r a t lung extracts with EDTA or EGTA showed a s l i g h t increase i n r e v e r s i b i l i t y of the conversion as measured :by incubation with DTT, but t h i s was not s i g n i f i c a n t as determined by XO values.  rat lung with 1 mM EDTA in a l l homogenizing, chromatography and sample buffers was  2.24.  Ratio of ischemic rat lung extract with EGTA was  1.28  (Figure 24). Homogenates prepared from ischemic rat lung were then incubated with DTT f o r 60 min at 37°C. conversion from XO to XDH  ImM  This treatment resulted i n reversal of enzyme such that in ischemic lung extract r a t i o of (XDH +  X0)/X0 was  3.2.  Ratio of (XDH + X0)/X0 was 3.8  in rat lung homogenized with  ImM  EDTA.  In rat lung homogenized with 1 mM EGTA, r a t i o of (XDH + X0)/X0  was  4.2.  E f f e c t of Perfusion with EDTA and DTT Prior to Homogenization Because the effects of EDTA as a chelator of divalent cations on the enzyme converting a c t i v i t y might be masked by the conversion of enzyme proceeding before the protective agents reached a l l c e l l s during homogenization,  the tissue was perfused with the chelator before excision.  The pulmonary artery of r a t lungs was cannulated with PE tubing v i a the apex of the r i g h t v e n t r i c l e and the lungs were perfused with iced 50 mM 7.4 + 1 mM was  Kpi pH  EDTA + 1 mM DTT + 1 mM PMSF immediately after the chest cavity  opened.  The lungs were then removed and rapidly homogenized in 2 ml  more of the same buffer and processed as usual (113). In n=5  r a t lung extracts treated, EDTA and DTT were present in a l l  homogenizing, chromatography and sample buffers. preparation the r a t i o of (XDH + XO)/XO was 3.53  After t h i s method of (Table IV)  E f f e c t of Perfusion with Oxygen Depleted Buffer and 60' Ischemia on XDH XO  to  Conversion In order to maximize the e f f e c t of ischemia rat lungs were cannulated and  -83perfused with a mixture  of 50 mM KPi pH 7.4 at 37°C with no DTT, EDTA or  PMSF i n order clear blood from the lungs. at  the pulmonary artery and kept warm i n the rats chest  minutes. in  Blanched lungs were then clamped  iced  studied. was  cavity  f o r 60  After this period of time, clamp was removed and lungs homogenized 50 mM Kpi pH 7.4 and processed  as usual.  Three animals  were  Ratio of (XDH + XO)/XO i n rat lung extracts treated i n this manner  1.32. A f t e r extracts from these experiments were further incubated with  ImM DTT at 37°C for 60 min r a t i o of XDH + XO/XO increased to 2.53 (Table IV).  Experiments i n Human Lung Tissue Six human lung biopsy samples were assayed these, only three were found  to be capable  three samples contained no XDH-XO a c t i v i t y .  of o x i d i z i n g NADH.  No XDH a c t i v i t y was found.  Of  The other  Of the three which redox cycled  NADH, only one showed measureable XO a c t i v i t y protein/min.  for XDH-XO a c t i v i t y .  of 5.35 x 10~ ug IXP/mg 6  Because of unknown v a r i a b i l i t y i n  tissue removal and transport i t was impossible  to determine whether DTT  would be e f f e c t i v e i n reversing conversion to XO (Figure 25).  -84-  Figure 25. Trace of XO activity in human lung sample found to have enzyme activity. A. Addition of 25 u moles pterin to measure the aerobic rate of reaction. B. Addition of 5 u moles methylene blue. C. Addition of 20 u moles allopurinol to inhibit the reaction.  -85TABLE IV  E f f e c t of Perfusion of lung with EDTA and DTT on Preventing Conversion of XDH to XO  Treatment  XDH + XO (x IO" ) 3  Perfusion with 4°C 1 mM EDTA at 4° + 1 nM DTT in 50 mM KPi. N=5  9.9 + 0.3  XO (x IO" ) 3  13.4 + 0.1  XDH + XO xo  3.53  Perfusion with 37° C 50 mM KPi + 60 min ischemia N=3  18.0 + 10.0  13.6 + 0.7  1.32  Perfusion with 37° C 50 mM KPi + ImM DTT X 60 min N=3  27.4 + 15.0  10.9 + 0.6  2.53  Enzyme a c t i v i t i e s are expressed as mean + SE of ug IXP/min/mg protein. Rat lungs were assayed by fluorometric technique of Markley (113). Assays were conducted at37°C.  -86DISCUSSION: The goal of these studies was  to assess XDH  to XO a c t i v i t i e s in lung  tissues of several species, to compare the a c t i v i t y  in lungs to that in  other tissues and to examine the r e l a t i o n s h i p of XDH  to XO in the lung with  a view to understanding lung disease.  I t was  calcium-calmodulin from XDH  to XO,  the implications of such changes i n  inflammatory  of p a r t i c u l a r interest to examine the hypothesis that  mediated proteolysis may  cause i r r e v e r s i b l e  conversion  and to r e l a t e the findings from the study of other organ  systems such as gut (57)(58) and heart (55) (Figure 26) to the conversion of XDH  to XO  in the lung.  I f this were so, the importance of XO  a c t i v i t y in  contributing to free r a d i c a l mediated damage in pathologic conditions could be applied to the proposal that oxygen free radicals generated be  important  by XO could  in the etiology of ARDS by acting on c e l l u l a r components to  produce chemotactic  factors thus causing PMN  activation (71) i n the lung .  Use of P u r i f i e d Bovine Milk XO in These Studies Based on high y i e l d , a c t i v i t y and apparent purity the use of p u r i f i e d XO  from our  own  laboratory by the method of Waud (91) was  superior to use  of commercially  available XO  for characterization of our  assay system and preparation of a polyclonal antibody. had  appeared  as  electrophoresis at 67,000 Da. A-50 the  column was anti-XO  a  single  i t was  band  at  judged to be  135,000 Da  upon  Because p u r i f i e d  XO  post-isolation  surprising to find strong r e a c t i v t y against albumin  Since the 5 to 50 mM  KPi buffer gradient on the DEAE Sephadex  not conducted due to technical problems, the r e a c t i v i t y of  antisera was  attributed  to the fact  that trimers of albumin  -87which have the the contaminants  same molecular  the XO preparation.  to the highly immunogenic BSA  weight  as XO  subunits were possibly  Thus antibodies were raised in the rabbit  as well as to the XO.  This would be a more  reasonable explanation of the results rather than the p o s s i b i l i t y that the antibody to XO was  cross reactive with BSA.  of the polyclonal antisera with BSA antibody to BSA.  Antigen-antibody  precipitation  would help to eliminate some of the  This p u r i f i c a t i o n not being possible, the dual r e a c t i v i t y  of the antisera was  noted to provide a convenient molecular weight marker  for Western b l o t t i n g . Use  of this antisera in Western b l o t t i n g of SDS-PAGE of redissolved  protein p r e c i p i t a t e s  from bovine, porcine rat and human tissues  indicated  that whereas a l l bovine and rat tissues tested showed immunoreactivity  at  150,000 and 130,000 Da with anti-XO, only pig l i v e r , but not heart, lung or kidney  was  immunoreactive with  anti-XO.  An  extensive l i t e r a t u r e  search  indicates that while p i g l i v e r i s a known source of the enzyme (72) there i s no r e l i a b l e information available on other porcine t i s s u e s . if  these tissues have been unsuccessfully assayed,  porcine milk. as confirmed  bovine XO  or i f XO  i s found in  I t i s c l e a r , however, that bovine and porcine X0 cross-react by the observed  reactivity  of the enzyme i n porcine  homogenates with the antisera to the bovine enzyme. reported  I t i s not known  that XO  Although  in human lung cross-reacts with anti  liver  i t has been  sera s p e c i f i c  to  (73), no human r e a c t i v i t y with the anti bovine XO was observed in  these studies. Use of IXP Assay in these Purified  Experiments  bovine X0 was  system of Markley  (113)  used to characterize the fluorescence assay  f o r the purpose of these studies.  Although  this  -88assay has been reported to be 1000 f o l d more sensitive than the conventional urate assay (115), i n this laboratory i t was when assayed attributable  at  the  to the  limits use  of detection.  of d i f f e r e n t  only 100 f o l d more sensitive This difference  fluorimeters,  since  could be  a l l reagents  purchased were the same as those prescribed by the protocol (116). An  interesting  percentage use  finding  from  these  studies was  that  a  certain  (10%) of p u r i f i e d bovine XO could be converted back to XDH by the  of t h i o l  reducing agents  (96).  This was  fortuitous  since  possible the characterization of the assay f o r use with XDH  i t made  in tissue as  well. The  efficiency  of the fluorometric IXP  assay for XDH-XO has been  recently questioned f o r application ot human tissues (90).  Although pterin  is a synthetic substrate for XO (77), IXP c a t a l y t i c a l l y produced from pterin was  similar to the normal product of the degradation of hypoxanthine, urate  in two ways.  F i r s t , the IXP product inhibited the c a t a l y t i c action of XO at  High concentrations as the product accumulated the product i n h i b i t i o n of XO by urate. tissue.  Uricase which  tissues.  While  degrades  during c a t a l y s i s similar to  Secondly, IXP i s degraded  urate into  allantoin  i s found  in pig  in pig  i t i s unknown whether i t i s indeed uricase which i s acting  on the IXP, t h i s could be tested by investigating the e f f e c t s of uricase i n h i b i t o r s on the degradation of IXP by pig tissue extracts.  I t should be  noted that the e f f e c t s of heat denatured pig tissue homogenate on IXP were not investigated i n these studies. Using the IXP  assay to determine  XDH-XO levels  i n various tissue  homogenates gave r e s u l t s similar to those from SDS-PAGE Western b l o t t i n g .  -89Nevertheless, activity  i n p i g lung.  mammalian activity capable enzyme  i t was s u r p r i s i n g  species (72).  studied  t o date,  compartmentalized  finding  distribution  of a c t i v i t y conflict level  XDH-XO  found  with  localized  t o be found was  confirmed  see i f t h i s  subhuman  t o a l l other some  to allantoin  o f the enzyme throughout localized  hypothesis.  only  l e v e l of  s p e c i e s , which a r e do n o t have the  i n the e n d o t h e l i a l c e l l s ,  b u t r a t h e r show  the t i s s u e s (88).  i n the l i v e r  o f the p i g  Because o f t h e extremely  f o r t h a t method.  i n porcine  lung,  i n microvascular  along  low l e v e l s  f i n d i n g was n o t seen t o be i n  by a n a l y s i s .  Based  with  endothelium  i n c u l t u r e d p o r c i n e pulmonary  investigation to  that  at l e a s t  l a c k o f enzyme  from g e l e l e c t r o p h o r e s i s b u t r a t h e r t h a t i t was below  of sensitivity  only  show  i n human l u n g e x t r a c t s , t h i s  results  activity  which  total  the exception  of urate  t h a t XO i s a p p a r e n t l y  appears t o c o n t r a d i c t t h i s  the  i s certainly  of the f u r t h e r degradation strictly  t h e apparent  I t has been h y p o t h e s i z e d  a more u b i q u i t o u s The  This  t o note  on t h e o v e r a l l  data  suggesting  can be confirmed  t h a t XO i s  XDH-XO a c t i v i t y was n o t expected artery endothelial c e l l s .  I t would be o f i n t e r e s t  u s i n g c u l t u r e d bovine  lack of  t o pursue t h i s  o r lamb pulmonary a r t e r y c e l l s  This l i n e of  i n order  as w e l l i n s p e c i e s which do show XO a c t i v i t y  in the lung. Despite  l a c k o f p o r c i n e pulmonary XO a c t i v i t y  porcine microvascular to assay isolating for  endothelium  f o r XDH-XO a c t i v i t y .  investigation  with  c o u l d not be c u l t u r e d i n s u f f i c i e n t  The author  pulmonary m i c r o v a s c u l a r  cells  i s optimistic  A larger  that  number  t h a t t h i s method o f  c o u l d prove t o be a p r o f i t a b l e  a few more t r i a l s .  would be most b e n e f i c i a l .  i t was u n f o r t u n a t e  initial  seed  Based on c a s u a l o b s e r v a t i o n o f c e l l s  tool  of c e l l s  i n culture,  -90-  c e l l s from neonatal tissue d i d show a greater propensity to divide and reach confluence  than  did cells  from  adult  tissue.  The question  remains  unanswered as to whether colonies of c e l l s o r i g i n a t i n g from neonatal pigs were  initially  pleomorphically  contaminated  with  transformed  fibroblasts  or i f c e l l s  into f i b r o b l a s t - l i k e  cells.  i n culture  Since  distinctly  f i b r o b l a s t i c patches d i d not appear u n t i l 4 to 6 weeks a f t e r the i n i t i a l seeding the second  explanation may be more reasonable since neonatal and  fetal cells  are known to show more p l u r i p o t e n t i a l i t y than adult c e l l s and  fibroblasts  normally  grow much  more  rapidly  than  endothelial  cells.  Overgrowth would occur much more rapidly than was observed, therefore. The use of sera from neonatal pigs was not found benefit  i n these  confluence  experiments  to merit  available.  as observed  to have enough observable  by time  required to reach  i t s separate harvesting as i t i s not commercially  However, greater benefits may be present f o r more established  c e l l colonies, since f e t a l bovine serum i s known to be more b e n e f i c i a l than adult bovine  serum as a c e l l  culture supplement.  Again, i t would be of  i n t e r e s t to pursue t h i s technique of microvascular endothelial i s o l a t i o n i n neonatal peripheral lung of other species such as c a l f or lamb. are deemed eliminate  to be too small f o r the peripheral dissection necessary to contamination  endothelium  by  large  vessel  endothelium.  also does not grow r e a d i l y i n culture  great morphological interest culture w i l l propensity  Rat lungs  form monolayers.  to determine  in vivo.  I t would be of  i f microvascular endothelium in  From our observations they appear to have a  to grow i n regularly  microvasculature  (134).  Rat pulmonary  spaced  open networks reminiscent of the  I t may well be that the contact i n h i b i t i o n of  -91large vessel endothelium  i s exaggerated  i n the microvasculature,  causing  them to branch out in new d i r e c t i o n s before c e n t r a l confluence i s reached.  Comparison of Results from These Studies with Those Previously Cited i n the Literature The xanthine  detrimental effects of exogenous perfused xanthine  oxidase plus  on the pulmonary c i r c u l a t i o n are well documented (33), (60). An  attempt has been made to examine the effects of a l l o p u r i n o l , a xanthine oxidase  i n h i b i t o r on preventing  pulmonary edema with Results from these  both  the development of free radical-mediated  collapsed lung  (63) (74) and embolic  models.  studies showed that a l l o p u r i n o l does have a s l i g h t l y  b e n e f i c i a l e f f e c t , however, the e f f i c a c y of the collapsed lung model i n causing slight  s i g n i f i c a n t edema has been c a l l e d effect  minimal.  into question, and thus only a  of a l l o p u r i n o l would be seen  i f the baseline edema was  Nevertheless, r e l a t i v e l y few studies have addressed  of endogenous xanthine  oxidase  the function  in the lung, despite the f a c t that i t has  been known since 1935 to be present i n the lung of r a t and cow as detected by the urate assay (110).  In this early comparative work by Booth (110), a  reference i s given to the enzyme being unsuccessfully assayed but the author  i n the lung,  indicates h i s uncertainty as to whether the enzyme i s present  or not by the notation, "?", opposite that tissue i n h i s table l i s t i n g XO activities study  using  f o r various species and organs. a more sensitive enzyme assay  The results reported i n this and immunolocalization  indicate  that i f present, the enzyme was below the l e v e l of detection i n p i g lung.  -92-  OXIDASE A Proteolysis  (INTERMEDIATE)  DEHYDROGENASE  A  INACTIVE  ^-  _  OXIDASE  EDTA  F i g u r e 26. C o n v e r s i o n o f Rat L i v e r X a n t h i n e Oxidase. T h i s schema shows the interconversion o f x a n t h i n e o x i d a s e among i t s v a r i o u s p o s s i b l e forms. Broken arrows i n d i c a t e u n c e r t a i n o r a l t e r n a t i v e pathways (87).  -93In  the present study 85-90% of the enzyme was found to be reversible to  the dehydrogenase form. Roy  This i s i n accordance with the work of McCord and  (100), who found 85-90% of the enzyme to be i n the dehydrogenase form  when i l e a l tissue was frozen and processed immediately prevent proteolysis.  i n l i q u i d nitrogen to  I n t e r e s t i n g l y i n the present study, freezing was not  necessary to prevent p r o t e o l y t i c conversion i n the lung.  These r e s u l t s are  also i n c o n f l i c t with McCord and Roy who found that 30 min of ischemia could i r r e v e r s i b l y convert XDH to X0 (100). min  of tissue  endogenous conversion. in  ischemia  proteases  Our findings indicate that up to 60  or 10 hours of incubation designed  i n the lung  d i d not cause  to activate  irreversible  I t was found that while the enzyme appears  the oxidase form after standard i s o l a t i o n procedures  enzyme  to be predominantly from the lung even  when EDTA and EGTA are employed as calcium chelators, t h i o l reduction by DTT w i l l convert i t to the dehydrogenase.  In agreement with this observation i s  the work of Delia Corte and Stirpe (94) who noted that while the r a t lung appears  to be i n the oxidase form i t may be converted back to dehydrogenase  by DTT. The work of Jarash et a l . (73) used  an immunolocalization technique to  v i s u a l i z e XO i n frozen sections examined by l i g h t microscopy microscopy.  and electron  These authors found that the enzyme was l o c a l i z e d i n l a c t a t i n g  bovine e p i t h e l i a l c e l l s and microvascular but not large vessel endothelium. An i n i t i a l objective of t h i s thesis project was to assay porcine peripheral lung sections, which are known to contain only vessels less than 50 um (128)  i n comparison with c e n t r a l lung homogenates.  found  i n microvascular endothelium,  an enrichment  I f indeed XDH-XO i s only of enzyme a c t i v i t y would  -94be  expected  activity Bruder  i n the  i n porcine lung  and  isolated bovine  p e r i p h e r a l lung  Jarash  XO  from  l u n g was  SDS-PAGE,  (87)  this  question  involved  various  the  bovine  molecular  precautions  were taken  proteolytic  activity  i n the  T h i s work i s i n agreement w i t h  use  155  their lung  to  a l a c k of XDH-XO  of  F u r t h e r work by  immunoaffinity SDS-PAGE of  chromatography  XO  purified  from  on g e l e l e c t r o p h o r e s i s as a s i n g l e band on  weight  with  Due  remains unanswered.  tissues.  shown t o s e p a r a t e  with  homogenate.  KDa.  Since  preparation, does not  our  no  anti-proteolic  i t can  a c t on XDH  f i n d i n g s on  be  i n t e r p r e t e d that  t o any  conversion  great  o f XDH  extent. to XO  in  the r a t l u n g .  Conversion  o f XDH  While one an  easily  to XO  of the  i n the Rat  initial  manipulatable  g o a l s of t h i s  endothelial c e l l  obtaining viable microvascular made i t n e c e s s a r y trypsin  derived  shown t h a t XDH  t o XO  to  study  from bovine  ( F i g u r e 21)  Lung  effect while these  an  to  pancreas  on  enzyme c o n v e r s i o n 100%  o f the  extracts  dehydrogenase  by  enzyme was  with  1  mM  conformation.  to XO  difficulties  endogenous overnight  extracts with  will  indeed  in  in  EDTA i t was  reducing  from  agents,  scheme of S t i r p e ( F i g u r e 26).  proteases  i n the  lung  i n c u b a t i o n at 15°C  returned  no  XO  Using  cause c o n v e r s i o n  incubation with t h i o l  i n the oxidase DTT  i n r a t lung e x t r a c t s .  lung  p r o t e o l y s i s of XDH  employ  c u l t u r e system,  enzyme c o n v e r s i o n  which cannot be r e v e r s e d by  attempt  t o study XDH  c u l t u r e s i n a s p e c i e s e x h i b i t i n g pulmonary  i n agreement w i t h the m u l t i p l e c o n v e r s i o n In  p r o j e c t was  form a f t e r  most  of  the  Thus, endogenous p r o t e a s e s  extracts  i t was  16  h,  found  to that  i n c u b a t i o n of  enzyme i n the  to  its  l u n g do  not  -95appear t o be capable Since by  of a f f e c t i n g  low m o l e c u l a r  weight  the i r r e v e r s i b l e  substances  s e p a r a t i o n on chromatographic columns t h e p o s s i b i l i t y  a-l-antitrypsin  oxygen  ( 6 3 ) , which  free-radicals.  control  f o r the p r o t e o l y t i c  leading  t o the hypothesis  The l u n g  has i t s e l f  Soybean  trypsin  activity  o f XDH t o XO.  had been removed from t h e l u n g e x t r a c t  weight p r o t e c t i v e compounds are p r e s e n t . by  conversion  been  molecular  i s known t o be p r o t e c t e d  shown t o be degraded  by  has a l s o been used  as a  inhibitor of t r y p s i n  that a - l - a n t i t r y p s i n  of small  on r a t l i v e r may  serve  XO ( 9 3 ) ,  more  than  one  p r o t e c t i v e f u n c t i o n i n the lung. Studies  with  t h e use o f d i f f e r e n t  in  both  normoxic  and i s c h e m i c  of  d i v a l e n t cations with  c a l c i u m c h e l a t o r s on enzyme  lung t i s s u e  1 mM  indicated that neither chelation  EDTA nor more s p e c i f i c  c h e l a t i o n of calcium  with  1 mM  EGTA had any s i g n i f i c a n t e f f e c t on p r e v e n t i n g  XO.  This  conversion  DTT and thus did that  proteolysis The and  for irreversible  the calcium  conversions  effect  i n lung  length  tissue  i f the l u n g  the  the t h i o l  employed  o f the enzyme. factor  i s indeed  o f time which was r e q u i r e d f o r t i s s u e before  by i s c h e m i c  lung  with  thus  assay  was o f some concern  a l l o w i n g the c o n v e r s i o n  rapid cannulation  vasculature with  o f XDH t o  reducing  agent  proteases  (100)  I t i s possible  i n c o n t r o l o f XDH/XO protected  against  earlier.  time t h e diaphragm was opened. which  conversion  i s not an important  as was suggested  homogenized  would  was however r e v e r s i b l e  conversion  p r o t e o l y s i s o f the enzyme by calcium-mediated  not account  conversion  A series  t o be e x c i s e d , weighed  s i n c e presumably the r a t s  process  t o proceed  from the  o f experiments were were conducted  o f the pulmonary  a r t e r y and p e r f u s i o n o f  a homogenizing b u f f e r c o n t a i n i n g 1 mM  EDTA and 1  -96mM DTT was used i n order to investigate whether having calcium chelators and t h i o l reducing agents perfused result  into the lung as rapidly as possible might  i n some s l i g h t difference i n i r r e v e r s i b l e and reversible  conversion.  When compared to perfused lung without protective agents this was found to be true.  Levels of XDH i n lung perfused with DTT and EDTA were s i g n i f i c a n t l y  higher than XDH levels i n lungs which had no DTT and EDTA present from the onset  of ischemia.  When compared with r e s u l t s from previous  experiments  using calcium chelators, i t i s possible to speculate that there i s a small initial in  pool of XDH which w i l l convert very rapidly and i r r e v e r s i b l y to XO  the lung.  Studies using Granger's model of excision "ischemia" (58) on  the lung do not take this into consideration and only account for conversion which may take place after the i n i t i a l small f r a c t i o n  of t o t a l  f i v e minutes.  enzyme a c t i v i t y .  This i s , however, a  XDH which i s not i r r e v e r s i b l y  acted on during the i n i t i a l period of ischemia to be remarkably r e s i s t a n t to p r o t e o l y t i c conversion even up to 10 hours l a t e r , as shown by incubation of lung extracts at 15°C overnight.  Thus oxidation of t h i o l groups appears  to be a more important mechanism i n the lung (93) (96). No success was achieved upon attempts to separate the e f f e c t s of DTT and EDTA  i n the perfusate  to determine which has the greater  maintaining XDH both conformationally and s t r u c t u r a l l y . necessary order  Further studies are  on both of these factors during the i n i t i a l period of ischemia i n  to draw any firm conclusions as to whether t h i o l  Ca -mediated ++  e f f e c t on  proteolysis i s more important  the dehydrogenase form (XDH).  redox status or  for maintaining  the enzyme i n  -97Consequences of Conversion of XDH  to XO f o r the Organism  Regardless of whether XDH XO,  XO  XO,  or r e v e r s i b l y converted to  i s capable of producing superoxide free r a d i c a l s .  bulk of XDH to  i s permanently  in the lung appears to be subject only to reversible conversion  t h i s conversion appears  microemboli  Indeed, while the  cause  a focal  to take place very e a s i l y and r a p i d l y .  area of ischemia within the lung one  If  could  speculate that rapid yet reversible conversion to XO might take place.  The  same would hold true for microthrombi which are known to be cleared from the c i r c u l a t i o n in the pulmonary microvasculature. What the role of this  rapid conversion might be remains  Jarasch, Bruder and Heid have speculated that XDH may when the endothelium injury oxidant  (Figure 27) defense  unclear.  convert r a p i d l y to XO  i s subject to b a c t e r i a l attack causing endothelial  (132).  Xanthine oxidase may  thus form a f i r s t l i n e of  for the body against microorganisms  in the blood.  In  addition superoxide generated by XO could e l i c i t a chemotactic response for neutrophils through the generation of l i p i d  r a d i c a l chemoattractants (71).  This group supports this hypothesis with the interesting observation that while XO was  v i r t u a l l y non-existent in the serum, even in patients with  extensive vasculopathy, extremely  high levels of endogenous antibodies to  xanthine oxidase were found in the serum of a l l animals studied. of  a l l IgG s p e c i f i c i t y was directed against XO in one study of human samples  (133).  I t i s f e l t that these autoantibodies serve to protect the organism  from massive it  Up to 7%  oxygen free r a d i c a l damage, p r e c i p i t a t i n g the enzyme as soon as  i s released from the c e l l .  by-products  However, since one of the most important  of the generation of superoxide, H 0-,  is freely  diffusable  -98-  Bacteriol Attack C a u s i n g E n d o t h e l i a l C e l l Injury Oxidative  Figure 27. Hypothetical scheme of a role of xanthine oxidase i n defence mechanism of c a p i l l a r y endothelial c e l l s against b a c t e r i a l attack. As per Jarash, Cruder and Heid (122).  -99across  cellular  membranes  i t seems unnecessary  o c c u r f o r oxygen f r e e r a d i c a l s to be free  radicals  endothelial  generated  cells  (28),  activated neutrophils Recent protein  by  activated  leading  on  the  (14,  17)  possible  that  f o c a l patches  generation proteins,  gives  might of  a realistic  role  rise  cause  oxygen  thus  the  of  initiating and  another  first  how  the  area  yet  to  be  XO,  either  appears etiology  to  be  of  in  neutrophils  activate  ischemic and  the  i n one  with  human l u n g  the  or  could  While  whether vice  It  is of  subsequent  in clotting Whether t h i s i s return  versa.  neutrophils indeed  The  study  by  interaction  of c o n t i n u i n g  The  and  hypofusion in  the  have been allopurinol between i n t e r e s t to  i d e n t i f i c a t i o n of  confirming  XDH  endothelial  Many s t u d i e s  i n h i b i t i o n of XO  alike.  in this  some form of  i t is  models.  radicals  cause c o n v e r s i o n of  of  c e r t a i n to be  clinicians sample  protein  clearance  with  modifications  presence  lung  e n d o t h e l i u m are and  XO,  in f i b r i n o l y s i s .  e f f e c t s of  hypoxic  to  i s whether oxygen f r e e  unclear  protective  XDH  neutrophils  neutrophils  and  stage  irreversibly.  along  radical biologists  activity  or  of  causing  explored  i t remains  showing the  various  free  required  ARDS,  changes which published  reversibly  causing  seen.  p r o t e a s e s generated from a c t i v a t e d to  in  approach. to  or  egg.  radicals  lung due  lyse  i t i s XO  c h i c k e n or  free  i n the  to  Oxygen  r e v e r s i b l y c o n v e r t e d enzyme would  t o i t s reduced s t a t e remains t o be Another  whether  interesting  conversion  radicals  the  circulation.  normal p r o t e o l y t i c breakdown and  ischemia  rapid  free  hypothesis  to  of  proverbial oxygen  lysis  have been shown to  question  of  which f a c i l i t a t e s  turnover  microthrombi  to  endothelial  i n t o the  neutrophils  which a c t as the  commentary  oxidation  released  for  XO  immunoreactive  -100findings  (73)  investigated  indicates  that  this  avenue  of  thought  should  be  further  f o r whatever r o l e i t might p l a y i n the e t i o l o g y o f ARDS.  -101REFERENCES: 1. Gershmann R, Fenn WO; G i l b e r t SL, Sylvanus WN, Dwyer P. Oxygen poisoning and x - i r r a d i a t i o n : A mechanism i n common science 119:623-627, 1954. 2. Gerdin B, Marklund S. Oxygen derived free r a d i c a l s . 38(Suppl):61-74, 1983.  Uppsula J Med Sci  3. Clark JM, Lambertson CJ. Pulmonary Oxygen T o x i c i t y a review. Pharmacol Rev 23:38-117, 1971. 4. Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochm J 191:421-427, 1980. 5. Fridovich I. The biology of oxygen r a d i c a l s . 1978.  Science 201:875-880,  6. Fridovich I. Superoxide r a d i c a l : An endogenous toxicant. Pharmacol Toxicol 23:239-257, 1983.  Ann Rev  7. Chance B, Sies H, Bovaris A. Hydrogen peroxide metabolism organs. P h y s i o l o g i c a l Reviews 59:527-605, 1979.  i n mammalian  8. Winterbourn CC. Superoxide-dependent production of hydroxyl r a d i c a l s in the presence of iron s a l t s . Biochem J 182:625-628, 1979. 9. Anbar M, Ueta P.  I n t l J Appl Radiation and isotopes 18:493-523.  10. Freeman B, Crapo JP. 47:412-426, 1982.  Free r a d i c a l s and tissue injury.  Lab Invest  11. Fridovich I. Oxygen r a d i c a l s , hydrogen peroxide and oxygen t o x i c i t y In: Free Radicals i n Biology (ed w. Pryor) Academic Press New York pp 239-277 (1976). 12. Freeman Bruce A, Crapo James D. Biology of disease: tissue injury. 47(5):412-426, 1982.  Free r a d i c a l s and  13. Pryor WA. The role of free r a d i c a l reactions i n b i o l o g i c a l systems In: Free Radicals i n Biology (ed W. Pryor) Academic Press, New York pp 1-49, 1976. 14. Wolff SP, Garner A, Dean RT. Free r a d i c a l s , l i p i d s and protein degradation. TIBS 11:27-31, 1986.  -10215.  Lee SL, Douglas WHJ, Deneke SM, Fanburg BL. U l t r a s t r u c t u r a l changes in bovine pulmonary artery endothelial c e l l s exposed to 80% 0 in v i t r o . In V i t r o 19(9):714-722, 1983. 2  16.  Levine RL, Oliver CN, Fulks RM. Stadtman. Turnover of b a c t e r i a l glutamine synthetase: Oxidative i n a c t i v a t i o n precedes proteolysis. Proc Natl Acad Sci USA 78:2120-2124, 1981.  17.  K l i g i e l SEG, Lee E, McCoy JP, Johnson KH, Variani J . Protein degredation following treatment with hydrogen peroxide. Am J Pathol 115:418-425.  18.  Brown K, Fridovich I. DNA strand s c i s s i o n by enxymically generated oxygen r a d i c a l s . Arch Biochem and Biophys 206(2):414-419, 1981.  19.  Moody CS, Hassan HM. Mutagenicity of oxygen free r a d i c a l s . Natl Acad Sci USA 79:2855-2861, 1982.  20.  Weitberg AB, Weitzman SA, Destremp SM, Latts A, Stossel TP. Stimulated human phagocytes produce cytogenetic changes in cultures mammalian c e l l s . N Engl J Med 308:26-39, 1983.  21.  Emerit I, Khan SH, Cerutti P. Treatment of lymphocyte cultures with a hypoxanthine-xanthine oxidase system induces the formation of transferable clastogenic material. J Free Rad B i o l Med 1:51-57, 1985a.  22.  Sevarian A and Hochstein P. Mechanisms and consequences of l i p i d peroxidation in b i o l o g i c a l systems. Ann Rev Nutr 5:365-390, 1985.  23.  Mukai FH, Goldstein BD. Mutagenicity of malonaldehyde a decomposition product of polyunsaturated f a t t y acids. Science 191:868, 1976.  24.  Del Maestro RF, Thaw HH, B j o r i c J , Parker M, Arfors KE. Free r a d i c a l s as mediators of tissue injury In: Free Radicals i n Medicine and Biology Eds. DH Lewis and RF Del Maestro. Acta Physiol Scand Suppl 492:43-58, 1980.  25.  Shasby DM, Lind SE, Shasby SS, Goldsmith JC, Hunninghake GV. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: Alterations i n c e l l shape and calcium homeostasis. Blood 65:605-614, 1985.  26.  Junod AF, Clement A, Jornot L, Peterson H. D i f f e r e n t i a l e f f e c t s of hyperoxia and hydrogen peroxide on thymidine kinase a c t i v i t i e s of cultured endothelial c e l l s . Biochim Biophys Acta 847:20-24 (1985).  Proc  -10327.  Johnson KJ, Fantone JC, Kaplan J, Ward PA. In vivo damage of rat lungs by oxygen metabolites. J C l i n Invest 67:983-993, 1981.  28.  Autor Anne P, Bonnam Ann C, Thies Robert L. T o x i c i t y of oxygen r a d i c a l s in cultured pulmonary endothelial c e l l s . J Tox Environ Health 13:387-395, 1984.  29.  Smith SM, Grisham MB, Manci EA, Granger DN, Kvietys PR. Gastric mucosal injury in the r a t . Role of iron and xanthine oxidase. Gastroenterology 92:950-956, 1987.  30.  Martin WJ. Neutrophils k i l l pulmonary endothelial c e l l s by a hydrogen peroxide dependent pathway. Am Rev Respir Dis 130:209-213, 1984.  31.  Bowman CM, Butler EN, Repine JE. Hyperoxia damages cultures endothelial c e l l s causing increased neutrophil adherence. Am Respir Dis 128:469-472, 1983.  Rev  32.  Del Maestro RF, Planker M, Arfors KE. Evidence f o r the p a r t i c i p a t i o n of superoxide anion r a d i c a l i n a l t e r i n g the adhesive interaction between granulocytes and endothelium, i n vivo. Int J Microcirc: C l i n Exp 1:105-120, 1982.  33.  Del Maestro RF, Bjork U, Arfors KE. Increase in microvascular permeability induced by enzymatically generated free r a d i c a l s . Microvas Res 22:239-254, 1981.  34.  Crapo JD. Morphologic changes in pulmonary oxygen t o x i c i t y . Physiol 48:721-731, 1986.  35.  Taylor AE, Martin D, Parker JC. The effects of oxygen r a l d i c a l s on pulmonary edema formation. Surgery 94(3):433-438, 1983.  36.  Crapo JD, Barry BE, Foscue HA, Shelburne J . Structural and biochemical changes in rat lungs occuring during exposures to l e t h a l and adaptive dosages of oxygen. Am Rev Respir Dis 122:123-143, 1980.  37.  K i s t l e r GS, Caldwell PRB, Weibel WR. Development of fine s t r u c t u r a l damge to alveolar and c a p i l l a r y l i n i n g c e l l s in oxygen poisoned rat lungs. J C e l l Bio 32:605-628, 1967.  38.  Frank L, Massaro D.  39.  Mason RP. Free radicals in pharmacology and toxicology. Rev 33:189-260, 1982.  40.  Rhodes ML, Zavala DC, Brown D. Hypoxic protection in paraquat poisoning. Lab Invest 35:496-500, 1976.  Oxygen t o x i c i t y .  Am J Med  69:117-126,  Ann  Rev  1980.  Pharmacol  -10441.  Dawson, RB. Pulmonary reactions to nitrofurantoin.  274:522, 1966.  N. Engl. J . Med.  42.  Weiss SJ, LoBuglio AF. An oxygen-dependent mechanism of neutrophil-mediated c y t o t o x i c i t y . Blood 55(6):1020-1024, 1980.  43.  Repine JE, Bowmn RM, Tate RM. 81(Suppl):47-51, 1982.  44.  Thommasen HV. The role of the polymorphonuclear leukocyte i n the pathogenesis of the adult respiratory distress syndrome. In: Master of Science Thesis, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. pp  Neutrophils and lung edema.  Chest  1-197, 1984.  45.  Tate RM, Repine JE. Neutrophils and the adult respiratory d i s t r e s s syndrome. Am Rev Respir Dis 552-559, 1984.  46.  Thommasen HV. The role of the polymorphonuclear leukocyte i n the pathogenesis of the adult respiratory distress syndrome. C l Invest  Med 8(2):185-194, 1985. 47.  Rinaldo JE, Rogers RM. Adult respiratory distress syndrome charging concepts of lung injury and repair. N Engl J Med 900-909, 1982.  48.  Staub NC. Pulmonary edema due to increased microvascular permeability. Am Rev Respir Dis 32:291-312, 1981.  49.  Staub NC. Pulmonary edema due to increased microvascular permeability to f l u i d and protein. C i r c Res 43:143-151, 1978.  50.  Frega NS, DiBona DR, Guenther BA et a l . Kidney Int 10:517-532, 1976.  51.  Guarnieri C, Flamigini F, Caldarera CM. Role of oxygen in the c e l l u l a r damage induced by reoxygenation of hypoxic heart. J Mol C e l l Cardiol 12:797-808, 1980.  52.  McCord JM, Roy RS. The pathophysiology of superoxide: roles i n inflammation and ischemia. Can J Physiol Pharmacol 60:1346-1352,  Ischemic renal injury.  1982.  53.  Granger DN, Hollwarth ME, Parks DA. Ischemic reperfusion injury: Role of oxygen-derived free r a d i c a l s . Acta Physiol Scand Suppl  5148:47-63, 1986.  54.  Baker GL, Autor AP, Corry RJ. E f f e c t of a l l o p u r i n o l on kidneys after ischemia and reperfusion. Current Surgery 42:466-469, 1985.  -10555.  Hearse DJ, Manning AS, Downey JM, Yellon DM. Xanthine oxidase: A c r i t i c a l mediator of myocardial injury during ischemia and reperfusion. Acta Physiol Scand Suppl 548:65-78, 1986.  56.  Baker GL, Corry RJ, Autor AP. Oxygen free r a d i c a l induced damage in kidneys subjected to warm ischemia and reperfusion. Ann Surg 202:628-641, 1985.  57.  Parks DA, Granger DN. Ischemia-induced vascular changes role of xanthine oxidase and hydroxy r a d i c a l s . Gastrointest Liver Physiol 8:G285-G289, 1983.  58.  Granger DN, R u t i l i G, McCord JM. Superoxide r a d i c a l s i n f e l i n e i n t e s t i n a l ischemia. Gastroenterology 81:22-29, 1981.  59.  Crowel JW, Jones CE, Smith EE. E f f e c t of a l l o p u r i n o l on hemorrhagic shock. Am J Physiol 216:774-748, 1969.  60.  Steinberg H, Greenwald RA, Sciubba J , Das DK. The e f f e c t of oxygen derived free r a d i c a l s on pulmonary endothelial c e l l function in the i s o l a t e d perfused r a t lung. Exp Lung Res 3:163-173, 1982.  61.  Johnson KJ, Fantone I I I JC, Kaplan J , Ward P. In vivo damage of rat lungs by oxygen metabolites. J C l i n Invest 67:983-993, 1981.  62.  Steinberg M, Greenwald RA, Sciubba J , Das DK. E f f e c t of oxygen-derived free r a d i c a l s on pulmonary endothelial c e l l function in i s o l a t e d perfused r a t lung. Exp Lung Res 3:163-173, 1982.  63.  Saugstad OD. Oxygen r a d i c a l s and pulmonary damage. Pulmonology 1:167-175, 1985.  64.  Sandritter WC, Hermayer CM, Riede UN, Freudenberg N and Grimm N. The shock lung syndrome: An overview. Pathol Res Prac 162:7-23, 1978.  65.  R a t l i f f NB, Young WG, Hackel DB, Mikat E, Wilson JW. Pulmonary injury secondary to extracerporeal c i r c u l a t i o n . J Thoracic & Calrdiovasc Surg 65:425-432, 1973.  66.  Steckel RJ. U n i l a t e r a l pulmonary edema after pneumothorax. Med 289:621-622, 1973.  67.  F l i c k MR, Perel A, Staub NC. Leukocytes are required f o r increased lung microvascular permeability after microembolization i n sheep. C i r c Res 48:344-351, 1981.  Pediatric  N Engl J  -10668.  F l i c k MR, Hoeyyel JM, Staub NC. Superoxide dismutase with heparin prevent increased lung vascular permeability during a i r emboli i n sheep. J Appl Physiol Respirat Environ Exercise Physiol 55:1284-1291, 1983.  69.  Thommasen HV, Martin BA, Wiggs BR, Quiroga M, Baile EM, Hogg JC. Effect of pulmonary blood flow on leukocyte uptake and release by dog lung. J Appl Physiol: Respirat Environ Exercise Physiol 56(4):966-974, 1984.  70.  Thommasen HV, Russell JA, Boyko WJ, Hogg JC. Transient leucopenia associated with adult respiratory d i s t r e s s syndrome. The Lancet 809-812, A p r i l 1984.  71.  Grisham MB, Hernandez LA, Granger DN. Xanthine oxidase and neutrophil i n f l i t r a t i o n i n i n t e s t i n a l ischemia. Am J Physiol 251:G567-G574, 1986.  72.  Parks DA, Granger WD. Xanthine oxidase: Biochemistry d i s t r i b u t i o n and physiology. Acta Physiol Scand Suppl 548:87-89, 1986.  73.  J a r a s c h E-D, Grund C, Bruder G, Heid HW, Keenan TW, Franke WW. Localization of xanthine oxidase in mammary gland epithelium and c a p i l l a r y endothelium. C e l l 25:67-82, 1981.  74.  Bishop MJ, Chi EY, Jordan JP, Chenny FW. Lung reperfusion results i n b i l a t e r a l lung injury and i s not prevented by a l l o p u r i n o l . Am Rev Resp Dis 132-.A275, 1986.  75.  Fukishima M, Kobayashi T, Kubo K, Yoshimura K, Shibamoto T, Hirai K, Kusama S. Effects of a l l o p u r i n o l , A xanthine oxidase i n h i b i t o r on pulmonary a i r embolism-induced lung injury i n awake sheep. Am Rev Resp Dis 132:A275, 1986.  76.  L e s l i e WK, Kinasewitz GT, Groome LJ, Diana JN. E f f e c t of xanthine oxidase i n h i b i t i o n on endothelial permeability during hypoxia. Am Rev Respir Dis 131:A419, 1985.  77.  Krenitsky TA, Tuttle JU, Cattau EL, Wang P. A comparison of the d i s t r i b u t i o n and electron acceptor s p e c i f i c i t i e s of xanthine oxidase and alderyde oxidase. Comp Biochem Physiol 49B:687-703, 1974.  78.  Rajagopalan KU, F r i d o v i t c h I, Handler P. Competitive i n h i b i t i o n of enzyme a c t i v i t y by urea. J B i o l Chemistry 236(4):1059-1065, 1961.  79.  Spector T. I n h i b i t i o n of urate production by a l l o p u r i n o l . Pharmacol 26:355-358, 1977.  Biochem  -10780.  Specter T, H a l l WW, Krenitsky TA. Human and bovine xanthine oxidases. I n h i b i t i o n studies with oxypurinol. Biochem Pharmacol 35(18):3109-3114, 1986.  81.  Hansson R, Jonsson 0, Lundstam S, Pettersson S, Schersten T, Waldenstrom J. E f f e c t s of free r a d i c a l scavengers on renal c i r c u l a t i o n after ischaemia in the rabbit. C l i n Sci 65:605-610, 1983.  82.  Bray RC. (1975) The Enzymes 299-419, Academic Press, N.Y.  83.  Morgan EJ. The d i s t r i b u t i o n of xanthine oxidase. 20:1282,1291, 1926.  84.  Jarasch ED, Bruder G, Heid HW. Significance of xanthine oxidase in c a p i l l a r y endothelial c e l l s . Acta Physiol Scand 548:39-46, 1986.  85.  Krenitsky TA, Spector T, H a l l W. Xanthine oxidase from human l i v e r : P u r i f i c a t i o n and characterization. Arch of Biochem & Biophys 247(1):108-119, 1986.  86.  Morisson B, Desjacques P, Baltassat P. Measurement of xanthine oxidase a c t i v i t y i n some human tissues. Enzyme 29:32-43, 1983.  87.  Bruder G, Heid H, Jarasch E-D, Keenan TW, Mather IH. Characteristics of membrane-bound and solulable forms of xanthine oxidase from milk and endothelial c e l l s of c a p i l l a r i e s . Biochemica et Biophysica Acta 701:357-369, 1982.  88.  Brunschede H, Krooth RS. Studies on the xanthine oxidase a c t i v i t y of mammalian c e l l s . Biochem Genetics 8:341-350, 1973.  89.  Khalidi UAS, Chaglassiar TH. The species d i s t r i b u t i o n of xanthine oxidase. Biochem J 97:318-320, 1965.  90.  Simmonds HA, Goday A, Morris GS. Superoxide r a d i c a l s , immunodeficiency and xanthine oxidase a c t i v i t y : Man i s not a mouse. C l i n i c a l Science 68:561-565, 1985.  91.  Waud WR, Brady FO, Wiley RD, Rajagopalan KV. A new p u r i f i c a t i o n procedure for bovine milk xanthine oxidase: E f f e c t of proteolysis on the subunit structure. Arch of Biochem & Biophys 169:695-701, 1975.  92.  Nishino T, Nishino T, Tsushima K. P u r i f i c a t i o n of highly active milk xanthine oxidase by a f f i n i t y chromatography on sepharose 4B/Folate Gel. FEBS Letters 131(2):369, 1981.  (ed. Boyer PD>  3rd ed. Vol 12 pp  Biochem J  -10893.  Stripe F, Delia CE. The regulation of rat l i v e r xanthine oxidase: conversion in v i t r o of the enzyme a c t i v i t y from dehydrogenase (Type D) to oxidase (Type 0). J B i o l Chemistry 244(14):3855-3863, 1969.  94.  D e l i a CE, Stripe F. The regulation of rat l i v e r xanthine oxidase. Biochem J 126:738-745, 1972.  95.  Roussos GG. Xanthine oxidase from bovine small i n t e s t i n e . Enzymol 12:5-16, 1967.  96.  Clare DA, Blakistone B, Swaisgood HE, Herton HR. Sulphydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase. Arch Biochem Biophys 211:44-47, 1981.  97.  Waud WR, Rajagopalan KV. The mechanism of conversion of rat l i v e r xanthine dehydrogenase from an NAD -dependent form (Type D) to an 02~dependent form (Type 0). Archives of Biochemistry & Biophysics 1720:365-379, 1976.  Meth  +  98.  Sackler ML. Two forms of xanthine oxidoreductasae in rat l i v e r . Histochem Cytochem 14:326, 1966.  99.  B a t e l l i MG, Lorenzoni E, Stripe F. Milk xanthine oxidase type D (Dehydrogenase) and Type 0 (Oxidase) p u r i f i c a t i o n , interconversion and some properties. Biochem J 131:191-198, 1973.  100.  Roy RS, McCord JM. Superoxide and ischemia conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy r a d i c a l s and t h e i r scavenger systems Vol I I , RA Greenwald, G.A. Cohen eds. 1983, E l s e v i e r Amsterdam.  101.  De Maetino GN, Kuers K. Two Ca dependent calmodulin stimulated proteases from rat l i v e r . Fed Proc 40:1738, 1981 (Abstract 1144).  102.  Saugstad Ola Didrik. Hypoxanthine Radiat Res 9:158-161, 1975.  103.  Saugstad OD, Aasen AO. Plasma hypoxanthine concentration in pigs: A prognostic aid in hypoxia. Eur Surg Res 12:123-129, 1980.  104.  Biemond P, Swaak AJG, Beindoff CM, Roster JF. Superoxide-dependent and -independent mechanisms of iron mobilization from f e r r i t i n by xanthine oxidase. Biochem J 239:169-173, 1986.  105.  Personal communication. Nelson G u r l l .  106.  Morell DB. The nature and c a t a l y t i c a c t i v i t i e s of milk xanthine oxidase. Biochem J 51:657-669, 1952.  as a measurement of hypoxia.  Unpublished r e s u l t s .  Dr. Anne Autor, Dr.  -109107.  Dougherty TM. A sensitive assay for xanthine oxidase using commercially available [ C ] xanthine. Anal Biochem 74:604-608, 1976. 74  108.  Schoutsen B, de Tombe P, Harmsen E, Keijzer E, Willem de Jong J . Combined use of radioenzymatic assay and high pressure l i q u i d chromatography f o r the detection of myocardial xanthine oxidase/dehydrogenase. Adv Exp Med B i o l 165(PtB):497-500, 1984.  109.  Westerfeld WW, Richert DA. Determination of xanthin oxidase in rat l i v e r and i n t e s t i n e . J B i o l Chem 199:393-405, 1952.  110.  Booth VH. The i d e n t i t y of xanthine oxidase and the schardinger enzyme. Biochem J 29:1732-405, 1935.  111.  Lowry OH. Techniques for metabolic studies: Xanthine oxidase. Methods in Enxymology 4:380-381, 1957.  112.  Glassman E. Convenient assay of xanthine dehydrog3nase i n single drosophila melanogaster. Science 137:990-991, 1962.  113.  Markley HG, F a i l l a c e LA, Mezey E. Xanthine oxidase a c t i v i t y in brain. Biochem Biophysica Acta 309:23-31, 1973.  114.  Haining JL, Legen JS. Fluorometric assay for xanthine oxidase. Biochem 21:337-343, 1967.  115.  Massey V, Brumby PE, Komai H, Palmer G. Studies on milk xanthine oxidase. J B i o l Chem 244(7):1682-1691, 1969.  116.  Waud WR, Brady FO, Wiley RD, Rajagopalan KU. A new p u r i f i c a t i o n procedure for bovine milk xanthine oxidase: E f f e c t of proteolysis on the subunit structure. Arch Biochem & Biophys 169:695-701, 1975.  117.  Agen A, Wenham DJ, Gordon JL. Stimulation of endothelial c e l l s by protease a c t i v i t y in commercial preparations of xanthine oxidase. Thrombosis Research 35:43-52, 1984.  118.  Laemmli UK. Cleavage of s t r u c t u r a l proteins during the assembly of the head of bacteriophage T$. Nature 227:680-685, 1970.  119.  Baron J . Mammalian adrenal ferredoxin i n the CSCC reaction. Biochem & Biophys 174:226-231, 1976.  120.  V o l l e r A, Bidwell D, B a r t l e t t A. Enzyme immunoassays f o r the immunodiagnosis of v i r a l i n f e c t i o n . In: Manual of C l i n i c a l Immunology. Rose NR, Friedman H ed. Ch. 69, ASM publ.  Anal  Arch  -110121.  Polyacrylamide Gel Electrophoresis. 1984-3. Rahmsilund Uppsala publ.  Revised E d i t i o n .  122.  Lowry OH, Bessy OA, Crawford EJ. 193:263-399, 1949.  123.  Hart DHL, Hobson JE, Walker DC, Autor AP. Antioxidant enzyme content of pulmonary artery endothelial c e l l s : E f f e c t s of subculture. J Free Rad i n B i o l & Med 1:429-435, 1985.  124.  Jaffe EA, Hoyer LW, Nachman RL. Synthesis of antihemophilic factor antigen by cultured human endothelial c e l l s . J C l i n Invest 52:2757-2764, 1973.  125.  Ryan US. Culture of pulmonary endothelial c e l l s on microcarier beads. In: Biology of Endothelial C e l l s . Jaffe EA ed. Martinus N i j h o f f publishers, Boston pp 34-50, 1984.  126.  Bowman PD, Ennis SR, Rarey KE, Betz AL, Goldstein GW. Brain microvessel endothelial c e l l s i n tissue culture: A model f o r study of blood-brain b a r r i e r permeability. Ann Neurol 14:396-402, 1983.  127.  Kern PA, Knedler A, Eckel RH. I s o l a t i o n and culture of microvascular endothelium from human adipose t i s s u e . J C l i n Invest 71:1822-1829, 1983.  128.  Personal communication.  129.  Devereux T, Fouts JR. I s o l a t i o n of pulmonary c e l l s and use in studies of xenobiotic metabolism. In: Meth of Enzymol 77:147-154, WB Jakoby ed. 1981. Academic Press New-York.  130.  Shaw RG, Johnson AR, Schulz WW, Zahlten RN, Combes B. Sinusoidal endothelial c e l l s from normal guinea p i g l i v e r : I s o l a t i o n culture and characterization. Hepatology 4:591-602, 1984.  131.  Knook DL, Sleyester ECh. Separation of kupffer and endothelial c e l l s of the r a t l i v e r by c e n t r i f u g a l e l u t r i a t i o n . Exp C e l l Res 99:444-449, 1976.  132.  Jarsh E-D, Bruder G, Heid HW. S i g n i f i c a n c e of xanthine oxidase i n c a p i l l a r y endothelial c e l l s . Acta Physiol Scand Suppl 548:39-46, 1986.  133.  Bruder G, Jarash E-P, Heid HW. High concentrations of antibodies to xanthine oxidase i n human and animal sera. J C l i n Invest 74:783-794, 1984.  134.  Personal communication.  Pterin oxidase.  Pharmacia  J B i o l Chem  Dr. Peter Dodek.  Dr. Herschel Rosenberg.  

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