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Xanthine oxidase in the lung 1987

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XANTHINE OXIDASE IN THE LUNG by Wendy Lee Wilson B.A., Un i v e r s i t y of Toronto, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA vToAg. t 1987 © W e n d y L. Wilson, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall ' Vancouver, Canada Department of DE-6(3/81) - i - ABSTRACT The generation of oxygen free r a d i c a l s by the c y t o s o l i c enzyme, xanthine oxidase (XO), has been implicated i n post-ischemic or reperfusion damage in several organs. XO catalyzes the conversion of hypoxanthine to urate with the concomitant production of superoxide anion free r a d i c a l (0^') and hydrogen peroxide ^ H 2 ^ 2 ' 0 x y B e n free radical-mediated i n j u r y has also been demonstrated i n inflammatory lung disease. The possible involvement of XO i n oxidative i n j u r y i n the lung has not yet been studied. Therefore, t h i s research project was designed to determine whether XO i s present i n the lung and 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 in porcine, bovine, r a t and human lung and other t i s s u e s . Immunochemical analysis of xanthine oxidase in the t i s s u e s employed on p o l y c l o n a l antibody r a i s e d to bovine milk XO. Proteins were separated by SDS-polyacrylamide gel electrophoresis of t i s s u e homogenates. Proteins were transfered from the gels to n i t r o c e l l u l o s e f i l t e r s by Western b l o t t i n g . A f t e r incubating the f i l t e r s with a a n t i s e r a containing the antibody to the p u r i f i e d bovine XO. XO on the f i l t e r was detected by i t s reaction with an enzyme-conjugated second antibody. XO was immunologically detectable i n bovine lung and milk. Rat lung, kidney and l i v e r a l l showed XO r e a c t i v i t y . XO was detectable i n porcine l i v e r but not detectable i n porcine lung or kidney. Thus, the antibody to bovine XO was c r o s s - r e a c t i v e with porcine and r a t XO. XO p r o t e i n was not immunologically detectable in - i i - human lung p o s s i b l y because the antibody was not cross r e a c t i v e with the bovine antibody. In v i v o , xanthine oxidase e x i s t s predominantly as a dehydrogenase rather than an oxidase. In t h i s form as xanthine dehydrogenase (XDH) the enxyme does not produce e i t h e r 0~" or Ĥ Ô - The a c t i v i t y of both XDH and XO was measured i n several ti s s u e s using a fluorometric assay which uses an a r t i f i c a l substrate, p t e r i n which i s c a t a l y t i c a l l y converted to the fluorescent product isoxanthopterin (IXP). XO a c t i v i t y i n porcine l i v e r was of 1.1 x _3 10 ug IXP/mg protein/min although XO a c t i v i t y was not detectable _2 in porcine lung and kidney, i n rat lung of 1.7 x 10 pg IXP/mg _2 protein/min, r a t kidney of 1.5 x 10 pg IXP/mg protein/min, and _2 rat l i v e r of 2.2 x 10 ug IXP/mg protein/min. Seven human lung biopsy samples were obtained a f t e r lung r e s e c t i o n and i n i t i a l l y tested f o r v i a b i l i t y by determination of NADH oxidase a c t i v i t y and then assayed f o r XO-XDH. Three of these samples showed NADH oxidase a c t i v i t y i n d i c a t i n g t i s s u e v i a b i l i t y , but only one of these three showed measurable XO a c t i v i t y of 5.35 x 1 0 - 6 yg IXP/mg protein/min. I r r e v e r s i b l e conversion of XDH to XO i s thought to be the r e s u l t of l i m i t e d p r o t e o l y s i s by a Ca^ +/calmodulin a c t i v a t e d protease, whereas r e v e r s i b l e conversion of the enzyme occurs by oxidation of c r i t i c a l t h i o l groups. Studies on the rate and nature of the conversion of XDH to XO were conducted in rat lung using the - i i i - fluorescence assay to detect c a t a l y t i c a c t i v i t i e s of both enzyme forms. Incubation of lung homogenates with t r y p s i n f o r 60 min caused i r r e v e r i s b l e conversion of 90% of the XDH to XO. In contrast, incubation of homogenates at 15°C for 10 hours caused conversion of 100% of the XDH to XO. This conversion was r e v e r s i b l e to the extent of 80% by reduction of t h i o l groups with d i t h i o t h r e i t o l (DTT). The e f f e c t s of free C a 2 + on the conversion of XDH to X0 was examined by 2+ using EDTA, a chelator of Ca and other divalent cations; and EGTA, 2+ a more s p e c i f i c chelator of Ca . The presence of these chelating agents during homogenization of eit h e r normoxic or ischemic r a t lung t i s s u e did not i n h i b i t r e v e r s i b l e enzyme conversion. Increased XO a c t i v i t y was r e v e r s i b l e by DTT. In the normoxic r a t lung, homogenates prepared with EDTA and EGTA showed a s i m i l a r conversion of 95% of XDH to XO which was r e v e r s i b l e to 70% with DTT. In the ischemic r a t lung, samples prepared with EDTA and EGTA showed a'conversion of 80% and 95% XDH to XO which was s i m i l a r to con t r o l samples. The extent of r e v e r s i b i l i t y to XDH was 75% with DTT incubation. In add i t i o n , perfusion of rat lungs with EDTA and DTT v i a a pulmonary artery cannula p r i o r to 60 min of ischemia and homogenization d i d not a f f e c t the extent of XDH to XO conversion. These r e s u l t s i n d i c a t e that i r r e v e r s i b l e Ca^ +-mediated p r o t e o l y t i c conversion of XDH to XO does not occur to a great extent in the rat lung during e i t h e r normoxia or ischemia. 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 i v ABBREVIATIONS v i i LIST OF TABLES ix LIST OF FIGURES X ACKNOWLEDGEMENTS , x i i DEDICATION x i i i INTRODUCTION 1 Xanthine oxidase i n the lung 9 Oxygen free r a d i c a l damage associated with ischemia- reperfusion injury i n other tissues 12 Biochemical aspects of xanthine oxidase 16 Purpose of these studies 23 METHODS 24 Methods for detection of xanthine dehydrogenase- oxidase a c t i v i t y 24 Experiments using p u r i f i e d xanthine oxidase 29 Experiments in porcine t i s s u e 36 Experiments in rat ti s s u e 43 Experiments i n human lung t i s s u e 49 - v i - Page STATISTICAL ANALYSIS 50 RESULTS 51 Experiments with p u r i f i e d XO 51 Experiments i n porcine t i s s u e 61 Experiments i n r a t t i s s u e 71 Experiments in human lung tis s u e 83 DISCUSSION 86 Use of p u r i f i e d bovine milk XO i n these studies 86 Use of IXP assay i n these experiments 87 Comparison of r e s u l t s from these studies with those l i s t e d i n the l i t e r a t u r e 91 Conversion of XDH to XO i n the r a t lung 94 Consquences of conversion of XDH to XO for the organism.... 97 REFERENCES 101 - v i i - ABBREVIATIONS ARDS ATP BSA C a 2 + DNA DTT EM(295) EDTA EGTA ELISA FAD 2 2 HBSS HMW HRP IXP KDa KPi M199 MW Adult r e s p i r a t o r y d i s t r e s s syndrome Adenosine triphosphate Bovine serum albumin Divalent calcium cation Deoxyribonucleic acid D i t h i o t h r e i t o l Molar e x t i n c t i o n c o - e f f i c i e n t at 295 nm in gm-moles/1 with a 1cm l i g h t path Ethylenediamine t e t r a a c e t i c a c i d Ethylene g l y c o l t e t r a a c e t i c acid Enzyme-linked-immunosorbant assay F l a v i n adenine dinucleotide Hydrogen peroxide Hanks balanced s a l t s o l u t i o n High molecular weight standards Horseradish peroxidase Isoxanthopterin Kilodaltons Potassiuim phosphate buffer Gibco Medium 199 Molecular weight - v i i i - NAD Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide in the reduced form " Superoxide anion free r a d i c a l OH Hydroxyl free r a d i c a l PBS Phosphate buffered s a l i n e PMN Polymorphonuclear leukocyte PMSF Phenylmethylsulfonylfluoride SE Standard error of the mean SDS-PAGE Sodium dodecyl/sulphate (denaturing) polyacrylamide gel electrophoresis XO Xanthine oxidase XDH Xanthine dehydrogenase XDH-XO Xanthine oxidase-xanthine dehydrogenase t o t a l enzyme a c t i v i t y - i x- LIST OF TABLES Page I Diseases f o r which Xanthine Oxidase Mediation has been suggested 2 II Values f o r XDH + XO and XO in Normoxic Rat Lung a f t e r treatment with Calcium Chelating Agents 76 I I I Values f o r XDH + XO and XO in Ischemic Rat Lung a f t e r treatment with Calcium Chelating Agents 80 IV E f f e c t of Perfusion with EDTA and DTT on the Conversion of XDH to XO 85 -X- LIST OF FIGURES Page 1. Reactions of Reactive Oxygen Species . 4 2. E f f e c t s of Oxygen Free Radicals on C e l l Components 7 3. A c t i v a t i o n of Neutrophils by Oxygen Free Radical Derived Chemotractants 17 4. Reactions of XO in Ischemia and Reperfusion 22 5. SDS-PAGE of P u r i f i e d Bovine XO 52 6. Western Blot of P u r i f i e d XO 54 7. Western Blot of Bovine Tissues 55 8. Western Blot of Rat Tissues 56 9. Western Blot of Porcine Tissues 58 10. Western Blot of Human Lung Tissue 59 11. Lineweaver-Burke Plot 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 Porcine Lung 63 14. XO A c t i v i t y i n Porcine Kidney 64 15. I n h i b i t i o n of P u r i f i e d XO by Pig Tissue 65 16. Heat S t a b i l i t y of Active Factors in Pig Tissue 67 17. E f f e c t of Pig Tissue Extract on Relative Fluorescence 68 18. Microvascular Endothelial C e l l s Isolated by C e n t r i f u g a l E l u t r i a t i o n 70 - x i - Page 19. XO in Rat Kidney 73 20. XO in Rat Lung 74 21. E f f e c t of Trypsin Treatment on (XDH + XO) XO Ratio 77 22. E f f e c t of Overnight Incubation at 15°C on (XDH + XO): XO Ratio 78 23. E f f e c t of Calcium on R e v e r s i b i l i t y of XDH to XO Conversion in Normoxic Rat Lung 79 24. E f f e c t of Calcium on R e v e r s i b i l i t y of XDH to XO Conversion in Ischemic Rat Lung 81 25. XO A c t i v i t y i n Human Lung 84 26. Conversion of Rat L i v e r from XDH to XO 92 27. Proposed Role of XO i n Defence Mechanism of C a p i l l a r y Endothelial C e l l s 98 - x i i - ACKNOWLEDGEMENTS Many people have had important contributions to t h i s project without which none of t h i s would be possi b l e . There are those who contributed t h e i r l e a r n i n g and t e c h n i c a l expertise as well as those whose i n s i g h t support and i n s p i r a t i o n were invaluable. I would l i k e to thank my supervisor, Dr. Anne Autor f o r her teaching, Dr. Margo Moore f o r tea and sympathy and expert guidance, Dr. Peter Dodek f o r h i s us e f u l commentary and c e l l i s o l a t i o n supervision, Michael Breedveld f o r the i s o l a t i o n of XO and comic r e l i e f , Dr. Rob. Thies and Dr. Jinny Hobson f o r t h e i r perspective, Maryette Mar f o r her resourcefulness, c e l l c u l t u r e and tolerance, Dr. Michael Weaver f o r the ELISA technique, Joe Comeau and Barry Wiggs f or computer help, Michelle Mullen who taught me the art in science, Dr. Marlene Rabinovitch my f i r s t mentor, the other Wendy Wilson, Carla Westerbeek-Marres, L o r i e Keenan, Wendy Ba r r e t t , Ingrid Pacey and Bridget 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 . Extra s p e c i a l thanks to Jochen Wolfgang Boehm f o r bunny rabb i t care and libatum. Many thanks to Denise Jackson and Lee Kowk f o r expert typing assistance. DEDICATION I would like to dedicate this to a l l 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. -1- INTRODUCTION: Oxygen free radicals are known to be involved in the etiology of disease states induced by the toxic effects of radiation, xenobiotics, pesticides and chemotherapeutic agents. It is now becoming better recognized that endogenously generated oxygen free radicals also are important in responses to injury such as inflammation and post-ischemic tissue damage. The enzyme system xanthine dehydrogenase: xanthine oxidase, which in the oxidase form is capable of generating oxygen free radicals in the oxidation of hypoxanthine and xanthine to urate, is considered to play an important role in free radical induced reperfusion injury, as well as in several other disease states (Table 1). The pathogenesis of several forms of pulmonary injury have been linked to oxygen free radical formation. This thesis investigates the presence and characteristics of xanthine dehydrogenase: xanthine oxidase in the lung with particular interest in whether oxygen free radicals 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 brief introduction to free radical biology in general and explain why this area is of particular concern in pulmonary disease. Oxygen Free Radicals: Overview In 1878 Paul Bert showed that the element oxygen, long considered to be the very sustenance of aerobic l i f e , could be damaging to the brain, lungs and other organs of animals. This discovery introduced a very paradoxical concept that was not well accepted. It was almost another -2- TABLE I DISEASES FOR WHICH XANTHINE OXIDASE MEDIATION HAS BEEN SUGGESTED ACUTE PANCREATITIS 1' STREPTOZYTOCIN INDUCED DIABETES MELLITUS 1 ISCHEMIC BOWEL DISEASE 2 GASTRIC ULCERS 2 NECROTIZING ENTERITIS 2 CARCINOGENESIS3 PRODUCTION OF CLASTOGENIC FACTORS3 RHEUMATOID ARTHRITIS 4 4 GOUT XANTHINURIA4 STROKE4 DISSEMINATED INTRAVASCULAR COAGULOPATHY4 ADULT RESPIRATORY DISTRESS SYNDROME5 TRANSIENT RENAL ISCHEMIA LEADING TO ACUTE RENAL FAILURE 6 ACUTE TUBULAR NECROSIS POST-KIDNEY TRANSPLANTATION6 MYCARDIAL INFARCTION WITH REPERFUSION? As per. 1. Sanfy H, Sarr MG, Bulkley GB, Cameron JL., Suppl. 548, V o l . 126, Acta P h y s i o l . Scand. 109-119, 1986. 2. Parks DA, Granger DN, Gastrointest. L i v e r P h y s i o l . 8:G285-G289, 1983. 3. Emerit I, Khan SH, C e r u t t i P. J. Free Rad. B i o l . Med. 1:51-57, 1985a. 4. Robbins R, Cotran R, Kumar P. Pathologic Basis of Disease, 1985. 5. Saugstad OD. Ped. Pulmonary 1:167-175, 1985. 6. Baker GL, Corry RJ, Autor AP. Ann. Surg. 202:628-641, 1985. 7. Hearse DJ, Manning AS, Downing JM, Y e l l o n DM. Acta P h y s i o l . Scand. Suppl. 548:65-78, 1986. -3- century 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 observation that irradiation of oxygenated solutions caused the formation of highly reactive oxygen species known as oxygen free radicals (1). A free radical is an atom or molecule which incorporates an unpaired electron into its outermost orbit (2). These species have a very high chemical reactivity due to their a b i l i t y to abstract an electron from other molecules. Because of its electronic structure, ground state oxygen exists as a diradical, Ô , unlike most elements which exist in the singlet state. In the absence of a catalyst, because of Hund's Rule oxygen is most easily reduced by four sequential univalent electron additions rather than a single concerted four electron reduction. Sequential univalent reductions create short lived reactive intermediates, and establish the potential for the incomplete reduction of oxygen, thus producing oxygen free radicals (See Figure 1)(3). In aerobic organisms, free radicals are an essential component to many biochemical processes. The catalytic action of enzymes is often dependent upon the generation of free radicals. For example, the production of ATP by the mitochondrial electron transport chain requires organic free radical intermediates in the Q cycle and at NADH dehydrogenase (4). However, a paradox exists due to the highly reactive nature of free radicals which makes them toxic unless kept in check. -4- REACTIONS OF REACTIVE OXYGEN SPECIES Reactions Description 0 2 + l e " 05- + H + <>2' -> H0 2 One e l e c t r o n reduction of oxygen The pKa of t h i s r eaction i s 4.8 05• + 0 5 - + 2H+ 0 2 - + Fe 3+X H 2 0 2 + F e 2 + X _^ H 2 0 2 + 0 2 _^ 0 2 + Fe2+X HO- + OH" + F e 3 + X Dismutation of super- oxide to form hydrogen peroxide and oxygen Reduction of i r o n - containing compounds such as f e r r i t i n Generation of hyroxyl free r a d i c a l s from hydrogen peroxide i n the presence of reduced i r o n ; the Fenton Reaction iron Net Reaction: 0 2 - + H 2 0 2 v 0 2 + HO- + O H - Net reac t i o n f or c a t a l y s t the generation of hydroxl free r a d i c a l s from super- oxide and hydrogen peroxide Figure 1. Reduction Reactions of Oxygen -5- The c e l l i s exposed to several d i f f e r e n t e x t r a c e l l u l a r and i n t r a c e l l u l a r oxygen free r a d i c a l s (Figure 1). The one el e c t r o n reduction of 0^ produces superoxide (designated 0^'). However, at physiologic pH, two molecules of 0^" r a p i d l y dismutate to produce hydrogen peroxide (H^O^) and 0^ thus l i m i t i n g the a b i l i t y of 0^' to react with substrates whose rates of reaction are competitive with the dismutation r e a c t i o n . These include compounds such as ferricytochrome c, quinones, ascorbic a c i d , heme proteins and t h i o l s (5) (6). H2°2 i t s e l f * s n°t highly r e a c t i v e with most organic compounds (8). However, H^O^ i n the presence of reduced t r a n s i t i o n metals such as ferrous i r o n the extremely powerful oxidant hydroxyl free r a d i c a l OH'(6) i s generated i n a reaction r e f e r r e d to as the Fenton r e a c t i o n (according to Figure 1, Reaction 5). The hydroxyl r a d i c a l o xidizes organic compounds with l i t t l e s e l e c t i v i t y , at re a c t i o n rates i n the range of 1 x 10^ to 1 x 1 0 ^ M *S ^ (9). Hydroxyl free r a d i c a l mediated oxidation of organic molecules proceeds by abstrac t i o n of an e l e c t i o n from the target molecule by OH'. This step i s termed i n i t i a t i o n . An organic free r a d i c a l i s produced from t h i s oxidation which can i t s e l f abstract f u r t h e r electrons causing the generation of more free r a d i c a l moieties i n a succession. This second step i s termed propagation. The i n i t i a t i o n of one organic free r a d i c a l s t a r t s a cascade propagating f u r t h e r free r a d i c a l s i n a chain r e a c t i o n , thus causing the ultimate oxidative cleavage of organic molecules. This process continues u n t i l the free r a d i c a l s are quenched either by i n t e r a c t i o n with another free r a d i c a l or by a molecule which does not in turn produce a free r a d i c a l . - 6 - In v i v o , r e a c t i v e oxygen species are produced in a v a r i e t y of enzyme systems. These include the cytochrome P450 system, NADPH oxidase of neutrophils, prostaglandin synthetase and xanthine oxidase which catabolizes purine nucleotides. In addition to these normal physiologic processes, oxygen r a d i c a l s are also generated from exposure to r a d i a t i o n , hyperoxic states, p e s t i c i d e s , chemotherapeutic agents, tobacco smoke and aromatic hydrocarbons (10). In order to c o n t r o l p o t e n t i a l l y cytotoxic free r a d i c a l reactions, the c e l l contains numerous pro t e c t i v e antioxidant mechanisms. As F r i d o v i c h has commented " i f i t were not for a group of antioxidant defenses the c e l l would not be able to survive i n i t s o x i d i z i n g environment" (11). These antioxidant mechanisms employ both enzymes which c a t a l y t i c a l l y remove re a c t i v e species and low molecular weight compounds r e f e r r e d to as scavengers, which react with free r a d i c a l s without generating f u r t h e r free r a d i c a l s . The most important of the antioxidant enzymes are superoxide dismutase, which catalyzes the dismutation of superoxide; catalase and glutathione peroxidase, which both catalyze the reduction of hydrogen peroxide to H^O. Low molecular weight scavengers such as ascorbate, a-tocopherol , B-carotene and glutathione act by being oxidzied but do not become free r a d i c a l s themselves. Even with t h i s complex array of protective mechanisms, in a -7- CELLULAR FREE RADICAL TARGETS Target Consequence Low Molecular Weight Molecules Unsaturated & t h i o l - c o n t a i n i n g amino acids Nucleic acid bases Carbohydrates Unsaturated l i p i d s Cofactors Neurotransmitters Antioxidants Macromolecules Protein DNA Hyaluronic acid 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 Organelle and c e l l permeability changes C e l l cycle changes, mutations C e l l surface receptor changes Cho l e s t e r o l & f a t t y a c i d oxidation L i p i d c r o s s - l i n k i n g Organelle & c e l l permeability changes Deceased nicotinamide & 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 oxidation, porphyrin oxidation Decreased neurotransmitter a v a i l - a b i l i t y (e.g. serotonin, epinephrine Decreased a v a i l a b i l i t y , includes a-tocopherol & B-carotene Peptide chain s c i s s i o n Strand s c i s s i o n Change in synovial f l u i d v i s c o s i t y Figure 2. E f f e c t s of oxygen free r a d i c a l s on c e l l u l a r components. Reproduced from Freeman and Crapo 1982, (10). -8- strongly oxidizing environment a l l of these antioxidant defenses may prove to be inadequate. Essentially a l l cellular constituents are susceptible to injury from free radicals (12) (Figure 2). Once produced, free radicals w i l l interact with protein, DNA, membrane phospholipid, lipoprotein and glycoprotein in self propagating reactions which may alter the native structure of both the intracellular components and the extracellular matrix. Because reactive oxygen species w i l l readily react with unsaturated and sulphur-containing molecules, the amino acid residues tryptophan, tyrosine, phenylalanine, histidine, methionine and cysteine are especially sensitive to oxidation (13). Specific residues required for. enzymatic function may be modified enough to interfere with activity (both augmenting or arresting function). More permanent damage may be due to protein crosslinking and fragmentation may have structural and ultrastructural consequences (14) (15). It is known that oxidation of c r i t i c a l groups makes proteins more susceptible to proteolytic breakdown, thus the suggestion has been made that oxidative modification may be the f i r s t step in normal protein turnover (16) (17). This implies that free radicals produced during the inflammatory response could have multiple functions, such as isolation of the area of damage, k i l l i n g bacteria, activation of the immune system via superoxide dependent chemotractants and i n i t i a t i n g breakdown of damaged structural components in order to f a c i l i t a t e repair. Highly reactive free radicals are also capable of inducing DNA strand breakage as demonstrated in vitro with a xanthine oxidase + xanthine superoxide generating system (18). If unrepaired this may cause point -9- mutations (19) and chromosomal aberrations (20). Free r a d i c a l s generated enzymatically, and in several disease states have also been shown to have a clastogenic e f f e c t , such that further damage to DNA i s perpetuated (21). The plasma membrane and organelles are e s p e c i a l l y susceptible to oxidation of the polysaturated f a t t y acids. (For a recent review see Sevanian and Hochstein, 1985 (22). Oxidative cleavage of polyunsaturated f a t t y acids r e s u l t s in the accumulation of short chain f a t t y acid d e r i v a t i v e s . Such a process becomes a u t o c a t a l y t i c i f unchecked due to the production of l i p i d peroxides, l i p i d alcohols and aldehyde by-products, which w i l l cause further oxidation. Membrane v i s c o s i t y , permeability, membrane transport proteins, secretory functions, c e l l surface i n t e r a c t i o n s and integrated c e l l u l a r metabolic processes w i l l a l l be a f f e c t e d (12). In addition, such by-products as malondialdehyde, which i s d i f f u s a b l e across c e l l membranes, may augment protein cross l i n k i n g and mutate DNA (23). E f f e c t s of Reactive Oxygen Species on the Pulmonary Microvasculature Because the microvascular endothelium acts as a s e l e c t i v e d i f f u s i o n b a r r i e r between the c i r c u l a t i o n and i n t e r s t i t i u m , loss of e n d o t h e l i a l i n t e g r i t y through the loss of c r i t i c a l e n d o t h e l i a l c e l l functions has serious consequences for organ function. The e f f e c t s of oxygen free r a d i c a l damage on the pulmonary endothelium include increased c a p i l l a r y permeability to macromolecules and edema formation (17)(24). This i s p a r t i c u l a r l y detrimental in the lung where gas exchange for the e n t i r e organism takes place. Studies with endothelial c e l l s i n culture have shown that reactive oxygen species are p a r t i c u l a r l y detrimental to the e n d o t h e l i a l membrane -10- integrity. Shasby et a l . showed an increase in albumin transfer across a diffusion barrier of endothelial cells grown on micropore f i l t e r s after treatment with oxygen free radical generating systems (25). This was shown to be associated with cytoskeletal changes attributable to C a 2 + levels and to be reversible. Decrease in thymidine incorporation into DNA is a sensitive index of cellular dysfunction. Hyperoxia or H 20 2 treatment has been shown to reduce thymidine kinase activity with implied effects on cellular protein production (26). While the exact mechanism of lethal oxidant injury to endothelial cells remains to be c l a r i f i e d , i t is unequivocably established that reactive oxygen species have lethal consequences for the endothelial c e l l (27) (28). At physiologic pH, 0^' acts preferentially as a reducing agent, whereas HO' is an extremely powerful oxidizing agent. The OH' species is however very short lived 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. However, in the presence of transition metals such as iron, 0," which is produced and then dismutated into ^ 0 ^ w i l l generate OH' (Fig. 1 Reaction 6). Iron has been shown to be essential for superoxide free radical induced injury (29). Thus, while OH' is the most strongly oxidizing species, the means of producing HO' via the Ĥ Ô  dependent pathway is important (30) in lethal oxidant injury to endothelial cells and injury resultant from stimulated neutrophils (31). This is presumably because * s uncharged and freely diffusable across the c e l l membrane, unlike superoxide and hydroxyl radicals which are not. The pathogenesis of several forms of pulmonary damage have been linked to oxygen free radical formation. -11- Hyperoxia has been long established as a cause of pulmonary damage (38). The pulmonary pathology r e s u l t a n t from hyperoxia appears to o r i g i n a t e from enhanced autoxidation of c e l l u l a r components which are normally found i n the reduced state, as well as exhaustion of c e l l u l a r antioxidant defense mechanisms. I n t e r e s t i n g l y , oxidative damage to en d o t h e l i a l c e l l s has been shown to augment t h e i r s u s c e p t i b i l i t y to further neutrophil-mediated damage by increasing adhesive i n t e r a c t i o n between neutrophils and en d o t h e l i a l c e l l s (31) (32) (33). In vivo, the extensive destruction of the pulmonary microvasculature associated with hyperoxic i n s u l t i s likewise combined with an i n f i l t r a t e of inflammatory c e l l s in the lung (34), thus perpetuating free r a d i c a l damage due to superoxide generated by NADPH-oxidase of neutrophils which produces 0^' i n the following r e a c t i o n : NADPH-oxidase NADPH + 20 2 + ^ NADP+ + 20 2 • + H+ The e f f e c t s of oxygen free r a d i c a l s are p a r t i c u l a r l y detrimental i n the lung (35) which may react by microvascular leakage of proteins and di s r u p t i o n of alv e o l a r e p i t h e l i a l i n t e g r i t y (36). The e a r l i e s t stages of mi c r o s c o p i c a l l y v i s i b l e damage occur i n the pulmonary c a p i l l a r y bed (37). The pulmonary toxins paraquat (40), and n i t r o f u r a n t o i n (41) act by the generation of superoxide through redox c y c l i n g , while bleomycin i n t e r a c t s v i a binding reduced iron to produce superoxide (39). Oxygen r a d i c a l s generated by activated polymorphonuclear leukocytes (PMN's) are of p a r t i c u l a r s i g n i f i c a n c e in the pathogenesis of acute and chronic pulmonary i n j u r y (42). I t has been estimated that up to 50% mo r t a l i t y occurs i n cases of acute non-cardiogenic pulmonary edema, -12- otherwise known as adult respiratory distress syndrome (ARDS) (43). The neutrophil has been strongly implicated in the pathogenesis of ARDS (44) (45) (46) (47). The consquences of edema due to increased microvascular permeability (48) (49) in the lung are especially detrimental because the f i l l i n g of i n t e r s t i t i a l spaces with f l u i d interferes with the normal process of gas exchange upon which the entire organism is dependent. Oxygen Free Radical Damage Associated with Ischemia-Reperfusion Injury in Other Tissues Oxygen derived free radicals are now thought to play an important role in another form of damage, described as post-ischemia or reperfusion injury. It has been well documented in other organ systems (kidney, gut, heart, skin, l i v e r , 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 site of reperfusion injury appears to be the microvascular endothelium (53). This results in increased transcapillary f i l t r a t i o n , i n t e r s t i t i a l edema and influx of inflammatory cells which can lead to the loss of the specialized functions characteristic of the tissue, such as acute tubular necrosis in the kidney (54) or loss of contractility in the myocardium (55). Oxygen free radicals are thought to be introduced by the reperfusion of the tissue with oxygenated blood. This mechanism has been confirmed by several studies which show that use of superoxide dismutase and catalase or dimethylsulphoxide (DMSO) as free radical scavengers w i l l ameliorate the detrimental effects of reperfusion (56) (57). The enzyme xanthine oxidase (ECO 1232 oxidoreductase) -13- produces Ô " and H2^2 ^ u r^ nB ^he oxidation of i t s substrates xanthine and hypoxanthine. Because of the e f f e c t s of the above described i n h i b i t o r s xanthine oxidase was suggested as the molecular source of the oxygen r a d i c a l s produced in reperfusion i n j u r y (58). This proposal has now been confirmed by many studies showing that a l l o p u r i n o l (54) (55) (59), a s p e c i f i c i n h i b i t o r of xanthine oxidase which acts as a t i g h t l y binding competitive substrate, prevents the development of the reperfusion injury syndrome.In an analogous manner perfusion of s p e c i f i c organs with a mixture of xanthine oxidase plus xanthine (33) (60) creates i n j u r y s i m i l a r to reperfusion i n j u r y . Perfusion of the lung with a s o l u t i o n containing xanthine oxidase plus xanthine i s likewise shown to increase vascular permeability apparently v i a compromised e n d o t h e l i a l c e l l f u n ction (61) (62) r e s u l t i n g i n s i m i l a r pathologic findings to acute pulmonary edema. Since i t has been shown that oxygen derived free r a d i c a l damage to pulmonary endothelium increases the c e l l s s u s c e p t i b i l i t y to adhesion of neutrophils and thus magnifiying damage due to inflammatory reactions, t h i s leads to the i n t e r e s t i n g speculation that xanthine oxidase may play a r o l e i n the development of ARDS (63). Since an episode of t r a n s i e n t ischemia may be an i n i t i a t i n g f a c t o r i n the a c t i v a t i o n of xanthine oxidase mediated pathology in other t i s s u e s , (see the following section on biochemistry of XO)(93) (Figure 4), under what circumstances could the lung be considered "ischemic"? ARDS was f i r s t recognized as a d i s c r e t e syndrome i n 1967 by Ashbaugh when i t was observed to occur subsequent to traumatic, hemorrhagic and septi c shock and given the name "shock lung" (64), although i n f e c t i o n s , -14- Decreased perfusion Decreased O2 Xanthine dehydrogenase ATP — ^ AMP — ^ adenosine inosine hypoxanthine 1. reversible t h i o l oxidation 2. Ca^+ mediated proteolysis Hypoxanthine +2O2 + H2O xanthine oxidase 2H+ Reperfusion: increase in O2 xanthine + 20^' xanthine oxidase uric acid + Oj' Result: Ô - + F e 3 + •> 0 2 + F e 2 + and 205- + 2H+ ^ 0 2 + H 20 2 H 20 2 + Fe2+(X) •> HO- + OH" + Fe 3 +(X) Figure 4. Reactions of Xanthine Oxidase. Proposed sequence for superoxide production in reperfused post-ischemic tissue and subsequent generation of the powerful oxidizing agent OH- -15- p a n c r e a t i t i s , inhalants and ingestants and near drowning were also implicated. A l l of these factors compromise pulmonary gas exchange. I n t e r e s t i n g l y , microthrombi and microemboli due to f a t , gas, a i r and ammiotic f l u i d are also o c c a s i o n a l l y followed by the development of ARDS. These could lead to areas of f o c a l ischemia i n the lung. Cardiopulmonary bypass has also been shown to r e s u l t in secondary pulmonary i n j u r y which resembles ARDS at the u l t r a s t r u c t u r a l l e v e l . S u r g i c a l bypass i s another instance of when the lung would be deprived of i t s blood supply f o r a period of time (65). U n i l a t e r a l pulmonary edema i s also known to be a complication of re-expansion of a' pneumothorax (66). In some models lung i n j u r y leukocytes are necessary for the development of increased pulmonary vascular permeability (67) and superoxide dismutase in combination with heparin can prevent i t s development (68). In the dog lung experimental model i t has been noted that decreased pulmonary blood flow w i l l increase leukocyte uptake in the lung (64). C l i n i c a l l y , an early t r a n s i e n t leukopenia has been associated with ARDS (70). There i s some speculation that free r a d i c a l induced damage to l i p i d b i l a y e r s can create chemoattractants which sequester and a c t i v a t e neutrophils (Figure 3)(71). While a l l of these studies seem to implicate hyperfusion i n a c t i v a t i n g neutrophils for ARDS the a s s o c i a t i o n with reperfusion injury has not been acceptably established. Xanthine oxidase has been found in the lung of several species both by a c t i v i t y assay (72) and immunological means (73). The enzyme has been demonstrated to be l o c a l i z e d in microvascular but not large v e s s e l endothelium (73). Several recent studies have been designed to examine whether i n h i b i t i o n -16- of xanthine oxidase with allopurinol could prevent the development of pulmonary edema due to a variety of insults such as air-embolism, re-expansion of pneumothorax and hypoxia (72) (73) (74). Conflicting results of these studies have not yet been resolved. Nevertheless, the activity of xanthine oxidase in the lung must be further investigated in order to elucidate whether this enzyme may play a similar role in inflammatory lung disease to that which has been demonstrated in ischemia-reperfusion injury (63). Biochemical Aspects of Xanthine Oxidase Xanthine oxidase (XO) is one of the most well characterized and complex of the flavoproteins. It was f i r s t isolated and studied extensively in bovine milk, where i t is found in great abundance In 1902 Schardinger f i r s t observed that methylene blue was reduced by formaldehyde in the presence of fresh milk. This enzyme was designated Schardinger's enzyme u n t i l i t was later denoted xanthine oxidase. Xanthine oxidase contains FAD, iron and molybdenum in the molar ratios of 1:4:1 as well as inorganic sulphide in two subunits of approximately 150,000 molecular weight. Xanthine oxidase is known to have a relatively broad substrate spec i f i c i t y , high activity, easy purification and s t a b i l i t y as well as low specifity for the electron acceptor. In addition to hypoxanthine and xanthine, xanthine oxidase w i l l also oxidize many other purines, pyrimidines, pterins and aldehydes. Xanthine oxidase transfers the substrate electrons to acceptors such as molecular oxygen, cytochrome c, ferricyanide and a r t i f i c i a l dyes such as methylene blue (77). The biological function of XO appears to be as the rate-limiting enzyme in nucleic acid degradation through which a l l purines are channelled for terminal oxidation. -17- ATP A M P . D e h y d r o g e n a s e Superoxide-Activated Chemoattractant Adenosine HypoxaMhine * Xarrlhinc Oxidase Reoxy genet ion cr H ? 0 2 O2 *~ MPO HOCI R N H • s RNHCI Fe <3 •OH Extravasated Neutrophils Circulating Neutrophils Neutrophil Activators Chemoa'traclanis -» T ISSUE D A M A G E Figure 3 A c t i v a t i o n of neutrophils by oxygen free r a d i c a l derived chematractants. Tissue damage due to the generation of hydroxyl free r a d i c a l s and hypohalides i n though to 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 neutrophils leading to fu 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 (60). -18- In vitro hypoxanthine and xanthine are oxidized to urate with molecular oxygen as an electron acceptor by the oxidase form. A superoxide free radical by-product is thus generated (Figure 4). This reaction proceeds by f i r s t order enzyme kinetics and is product inhibited by urate (78) (79). Xanthine oxidase has long been ut i l i z e d in conjunction with hypoxanthine or xanthine as one of the best enzymatic sources of oxygen free radicals available. Allopurinol, a drug used in the treatment of gout which acts by competitive inhibition of the enzyme is a known specific inhibitor (80) of XO useful for mechanistic studies (81) as well as allowing for regulation of the enzyme in vivo (75) (59) (82). For extensive general review of the mechanism and structure of xanthine oxidase see Bray et a l . (83). The original established source of xanthine oxidase was bovine milk. Early on i t was recognized that xanthine oxidase was found in most species, and more tissues than milk (84). In the tissue xanthine oxidase is found in highest concentration in l i v e r and small intestine, with variable levels in other tissues (72). Xanthine oxidase has been demonstrated in l i v e r , heart, lung and kidney (85), but is vi r t u a l l y undetectable in serum (85) (89). Brain and cornea have the lowest activity. Xanthine oxidase is found in many species and the amino acid sequence of xanthine oxidase is remarkably well conserved, even in Drosophila melanogaster (85). It is found in a variety of human tissues most notably l i v e r (86) and jejunum, (87), but is absent from cultured human fibroblasts and Hela cells (88). However, recently the enzyme was localized exclusively to microvascular endothelial cells using sensitive immunofluoresent techniques, (73) (87) and these have yet to be assayed in culture. The enzyme is absent from large -19- vessel endothelium (73). When the enzyme was f i r s t i s o l a t e d from l i v e r i t was found to e x i s t i n two forms, a dehydrogenase (XDH) with c a p a b i l i t y of u t i l i z i n g NAD4 as an electron acceptor which does not produce a superoxide r a d i c a l by-product and the oxidase which cannot t r a n s f e r electrons to NAD+. The s i g n i f i c a n c e of these d i f f e r e n t a c t i v i t i e s had previously been ignored because early methods of i s o l a t i o n d i d not prevent the p r o t e o l y t i c degradation of the enzyme to i t s oxidase form. The milk enzyme has now been shown to have some dehydrogenase component (91) (92) when i s o l a t e d by non-proteolytic methods. Normally, the enzyme i s thought to e x i s t almost e x c l u s i v e l y i n the dehydrogenase form i n vivo. Factors such as anaerobiosis, storage at -20°C of whole t i s s u e homogenate and solvent treatment are known to cause conversion of the enzyme from the dehydrogenase to oxidase (93). I n t e r e s t i n g l y , some of the conversion to oxidase i s r e v e r s i b l e , i n d i c a t i n g that there are at l e a s t two d i f f e r e n t s t r u c t u r a l a l t e r a t i o n s of xanthine dehydrogenase pos s i b l e which s t i l l r e t a i n enzymatic a c t i v i t y (93). Further work showed that reduction of t h i o l containing groups of xanthine dehydrogenase by d i t h i o t h r e i t o l could prevent conversion of the enzyme to the oxidase form caused by anaerobiosis, storage at -20°C and solvents. Conversion caused by p r o t e o l y s i s such as tr y p s i n treatment or incubation with whole t i s s u e homogenates at 37°C was i r r e v e r s i b l e (94). These data thus support the proposal that there are two mechanisms of conversion, an i r r e v e r s i b l e process due to p r o t e o l y t i c cleavage of a polypeptide e s s e n t i a l for binding with NAD+ and a second process due to modification of t h i o l s r e s u l t i n g i n a r e v e r s i b l e conformational change. -20- In the i n t e s t i n e of mammals xanthine dehydrogenase predominantly converts i r r e v e r s i b l y and very r a p i d l y to the oxidase form showing complete conversion i n 16s (95), gi v i n g r i s e to the speculation that the enzyme may be acted upon by a s p e c i f i c protease in high concentration i n the i n t e s t i n a l mucosa (96) which i s low or absent i n other t i s s u e s . Other t i s s u e s such as heart with 60 min conversion time may be susceptible only to non-specific p r o t e o l y s i s (94). Ele c t r o p h o r e t i c studies i n i n t e s t i n e and l i v e r have shown that i r r e v e r s i b l e conversion of the enzyme coincides with changes i n molecular weight from 150,000 Daltons to 130,000 Daltons (90) in agreement with the previous observation that l i v e r and i n t e s t i n e enzyme show d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t y (98). When tissues are homogenized, the dehydrogenase converts to oxidase as a r e s u l t of l i m i t e d p r o t e o l y s i s which was prevented by addition of EDTA, a chelator of C a 2 + and other div a l e n t cations i n the r a t l i v e r model (99). Use of protease i n h i b i t o r s such as Soybean t r y p s i n i n h i b i t o r prevent t h i s p r o t e o l y t i c conversion (100). Current suggestions are that ischemia activates a Ca 2 +-calmodulin mediated protease (101) which acts on the enzyme which then cannot be converted back to dehydrogenase. I n h i b i t i o n of calmodulin with t r i f l u o r p e r a z i n e also prevents i r r e v e r s i b l e conversion of the enzyme to the oxidase form in the rat ileum, as does treatment with soybean t r y p s i n i n h i b i t o r (100). I t i s also of note that ischemia or hypoxia cause a r i s e i n hypoxanthine, the substrate f o r xanthine oxidase/dehydrogenase as a breakdown product of ATP i n the lung and in the plasma (102) (103). The events occurring during an incidence of ischemia could encompass both -21- conversion of the enzyme to its oxygen dependent form and concommitant increase in substrate. With the reintroduction of molecular oxygen upon reperfusion of the tissue with oxygenated blood, the necessary electron acceptor is supplied for the reaction to proceed to the production of urate with superoxide radical by-products being generated. Xanthine oxidase has also been shown to be capable of mobilizing iron from f e r r i t i n by both superoxide dependent and independent mechanisms, thus potentiating the effect of free radical damage by generating the more highly reactive hydroxyl radicals (71), (Figure 4). This effect has been demonstrated to be blocked by 70% by superoxide dismutase, while uric acid removal does not affect the mobilization when a system of bovine milk xanthine oxidase and horse spleen f e r r i t i n is employed (104). During a period of ischemia ATP levels are diminished, as documented by NMR spectroscopy studies using an ischemic rat kidney model that from the onset of ischemia un t i l reperfusion was initated after 45 min the a , B , and y ATP peaks virtu a l l y disappeared while the inorganic phosphate peak rose by four fold (105). Loss of cellular ATP promotes an influx of calcium when energy dependent channels which maintain the lower intracellular concentration of this cation are inactivated. Since xanthine oxidase is known to be an active enzymatic source of oxygen free radicals and this enzyme has been demonstrated in the lung (73) i t is necessary to investigate the conversion properties of xanthine dehydrogenase/oxidase in the lung in order to determine whether this 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 hypoxanthine + 2(>2 + H2O ^ xanthine + 20 2' + 2H + oxidase \ xanthine \ oxidase uric acid + 02 - Reperfusion ^ 2O2 Then: 0 2 + Fe+2 ^ 0 5 - + F e + 3 and: 2 0 5 * + 2H+ ^ 0 2 + H 2 0 2 H 2 0 2 + Fe+2(X) ĤO- + OH~ + Fe + 3(X) (14) Figure 4. Reactions of XO in Ischemia and Reperfusion -23- PURPOSE OF THESE STUDIES 1. To determine the presence of xanthine dehydrogenase/oxidase in human, pig and rat lung and to measure its level of activity. 2. To compare enzyme content and activity in various organs within the same species using immunoreactivity, gel electrophoresis and specific enzyme assays. 3. To assess the relative content of the enzyme in the dehydrogenase or the oxidase form under standard conditions of enzyme purification. 4. To study factors which might affect conversion from the dehydrogenase to the oxidase form in the lung and to determine under what conditions the conversion can be inhibited or reversed. -24- METHODS Methods of Detection of Xanthine Dehydrogenase - Oxidase Activity Several different assays for XDH-XO have been developed and each has advantages and disadvantages which contribute to its usefulness. The earliest assay was developed in 1914 and employed the normal in vivo function of the enzyme in oxidizing hypoxanthine or xanthine to urate (106). The formation of urate in the presence of enzyme and substrate was determined by measuring the increase in absorbance at 295 nm using a molar -3 -1 extinction coefficient for urate of 9.6 x 10 cm The assay carried out with oxygen as an electron acceptor measures oxidase activity. Addition of NAD+ as the second electron acceptor measures total XDH plus XO activity. The technique was modified by Morell (106), who found removal of endogenous substrate increased activity. There are two major drawbacks to this method, the f i r s t being that i t is not very sensitive and the second being that most organisms lower than man contain uricase which degrades urate to allantoin and thus production of urate cannot be accurately measured without inhibition of uricase activity. Several more sensitive methods for the measurement of xanthine oxidase activity have been developed. These include the use of 14 [ C]-labelled xanthine (107), use of high pressure liquid chromatography (108), a manometric assay (109) and an assay u t i l i z i n g sulphite-enhanced oxygen uptake (110). A fluorometric assay developed by Lowry (111) is currently the preferred method because i t is extremely sensitive due to the -25- highly fluorescent nature of its product and is also more convenient, since i t does not require isotopes. This assay was modified by Glassman (112) who found that removal of endogenous small molecular weight substrates and inhibitors increased the sensitivity enough to make possible the determination of enzyme activity 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 levels of xanthine oxidase activity in brain (113). In an extensive comparison of several assays, Haining and Legan (114) found the modified fluorescence method (113) to be the optimal technique. I n i t i a l studies were carried out with the urate assay u n t i l i t was determined to be unsatisfactory for our purposes, since porcine tissue contains uricase. The fluorescence method as modified (113) was then used and proved to be more sensitive and less prone to interference from contaminating components. A synthetic pterin is employed as the substrate and is enzymatically oxidized to a highly fluorescent product, isoxanthopterin (IXP). Both molecular oxygen and methylene blue act as electron accepters in this reaction allowing for determination of oxidase to dehydrogenase ratios. With oxygen as the electron acceptor oxidase activity alone is measured. When methylene blue is added as the electron acceptor both oxidase and dehydrogenase activity are measured. Determination of XDH-XO Activity by the Determination of the Rate of Urate Production (115) 1. The tissue was excised and trimmed. Pig liver and kidney obtained fresh from a local slaughterhouse had capsule membranes removed prior -26- to mincing. Pig lung had the p l e u r a l membrane removed and large airways dissected out. Whole r a t kidney and l i v e r s were minced. Major bronchi of r a t lungs were dissected out before mincing. The ti s s u e was minced in four volumes of ice cold 50 mM potassium phosphate buffer (KPi), pH7.4 containing O.lmM EDTA and ImM phenylmethylsulfonyl f l u o r i d e (PMSF) as a protease i n h i b i t o r . Homogenization was accomplished with a S o r v a l l Omnimixer at 75% speed i n from 3 to 8 f i f t e e n second bursts on i c e , depending on collagen content of the t i s s u e . C e l l s were broken a f t e r 10 passes of the homogenized t i s s u e i n an e l e c t r i c a l l y driven Potter-Eljevheim mortar equipped with a Teflon pestle. The homogenate was f r a c t i o n a t e d by u l t r a c e n t r i f u g a t i o n at 100,000g o for 60 minutes at 4 C. Supernatant was removed by pipet. A 2 ml a l i q u o t of supernatant f r a c t i o n was loaded onto a Sephadex G-25 column (2 x 25 cm) e q u i l i b r a t e d with 50 mM KPi + ImM EDTA. The rate of flow of the buffer was 0.8 ml/min. A Pharmacia FRAC 300 f r a c t i o n c o l l e c t o r equippped with a p r o t e i n detection u n i t was used to c o l l e c t f i v e f r a c t i o n s of 450 drops (15.6 ml) each. This f r a c t i o n a t i o n removed endogenous small molecular weight substrates and i n h i b i t o r s from the crude homogenate containing enzyme a c t i v i t y . A c t i v i t y was associated with the f i r s t of two protein peaks. XDH-XO a c t i v i t y was determined by measuring the production of urate. The following procedure was used. The absorbance change was measured at 295 nm using a Beckman 35 Spectrophotometer with a 1ml quartz cuvette at 25°C. Recorder s e n s i t i v i t y range was set at 0 to 0.5 absorbance u n i t s , and the chart speed was 1 inch/min. The reaction mixture contained sample + Na-pyrophosphate (0.1M) pH 8.3 in a t o t a l volume of 990 u l 4 10 y l of 10 mM xanthine i n Na-pyrophosphate. The i n i t i a l absorbance change measured oxidase a c t i v i t y . Addition of 15 mM NAD+ as a second electron acceptor measures t o t a l dehydrogenase + oxidase a c t i v i t y . 20 uMoles a l l o p u r i n o l was added to i n h i b i t the rea c t i o n and v e r i f y that the urate production was catalyzed by XDH-XO. Ca l c u l a t i o n : A Absorbance 295 nm/min = mU/ml (9.6 x 10- 3M - 1cm~l)(sample size i n ml) -3 -1 -1 where 9.6 x 10 M cm i s the molar e x t i n c t i o n c o e f f i c i e n t of urate. One un i t (U) i s defined as a change of absorbance at 295 nm of 1. Protein concentration of samples was determined by the method of Lowry (121). Enzyme a c t i v i t i e s were expressed as mU/mg protein/min. Determination of XDH-XO A c t i v i t y by Production of IXP (113) Preparation of ti s s u e proceeded i n the same manner as f o r urate assay steps 1-3. 4. An Aminco SPF-125 Spectrofluorimeter was used to record the changes in fluorescence at an e x c i t a t i o n wave length of 345 nm and an emission wave length of 390 nm. The recorder was operated at a s e n s i t i v i t y of 10 mV f u l l scale with the chart rate set at 1 cm/min. 5. Tissue samples were pre-warmed at 37°C i n water bath. -28- 6. 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 added to start the reaction. Oxidase activity was 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 total XDH + XO activity. 7. The fluorescent emission at 390 nm was compared to a standard curve prepared with a known sample of IXP dissolved in 50 mM KPi pH 7.4 at a concentration range of 0.01 to 1 yg/ml. One unit of relative intensity was found to correspond consistently to 2.3 x 10 "̂yg IXP/ml. 8. I n i t i a l rates of enzyme activity were determined as yg IXP/ml/min produced. Protein concentrations of samples were determined by the method of Lowry (122). Enzyme acti v i t i e s were expressed as ug IXP produced/minute/mg protein. 9. The production of IXP could be inhibited by addition of 20 yMoles allopurinol, thus confirming that the increase in emission at 390 nm was due to the catalytic action of XDH + XO. 10. The proportion of XO activity in the tissue samples was expressed as a ratio of total enzyme activity: XDH + XO in order to give a linear XO rate of conversion. -29- Experiments Using P u r i f i e d Xanthine Oxidase P u r i f i c a t i o n of Xanthine Oxidase A highly p u r i f i e d preparation of xanthine oxidase was required for a base of comparison for the ti s s u e and samples and to use as an antigen for the preparation of a p o l y c l o n a l antibody. The p u r i f i c a t i o n method chosen was based on the non-proteolytic method of Waud, et a l . (116) Commercially a v a i l a b l e sources of XO are known to be contaminated with v a r i a b l e amounts of proteases (117) and thus could not be used f o r these purposes. The procedure was c a r r i e d out as follows: 1. 16 l i t e r s of raw fresh milk was obtained from the UBC Dairy Barn and centrifuged at 3000 rpm for 10 minutes. This r e s u l t e d in the separation of 425 ml of cream. 2. Equal volumes of 200 mM HPO^, 4 mM cysteine-HCl, 2 mM sodium s a l i c y l a t e pH 9.0 containing 0.01% EDTA were added at 80°C. 3. This mixture was cooled to 7°C and 135 ml of butanol at 4°C was added with continuous s t i r r i n g . 10N NaOH was added to maintain the pH at 9.0. 4. 155.25 g of ultrapure ammonium sulphate was slowly added with s t i r r i n g and the pH of the mixture adjusted to 9.0 with 10N NaOH. S t i r r i n g continued f o r 20 minutes followed by c e n t r i f u g a t i o n at 13,700g and 4°C. 5. The supernatant buttermilk f r a c t i o n (700 ml) was f i l t e r e d through glass wool to remove f a t globules. S o l i d ammonium sulphate (135g) was added to the f i l t r a t e . This was s t i r r e d for 30 min. and the 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 at 13,700 g for 20 minutes. -30- 6. The pellet was suspended in a minimal amount of 50 mM K̂ HPÔ containing 1 mM salicylate and 0.005% EDTA. The suspension was cl a r i f i e d by centrifugation and applied to a Sephadex G-25 ion exchange column that was equilibrated with the same buffer in order to remove residual 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 equilibrated with 5mM potassium phosphate pH 7.8 containing 0.005% EDTA and ImM salicylate. It should be noted here that by accident the cold room temperature dropped to -10° during the sample elution and the column was partially frozen. Despite this problem the enzyme remained active but eluted from the column differently than expected. Because XO contains a flavi n prosthetic group, the enzyme could be detected visually by the elution of a bright yellow flavin-containing protein fraction which eluted from the column with the equilibration buffer rather than after gradient elution. The linear gradient between 5mM and lOOmM potassium phosphate buffer was not applied therefore. Active fractions were pooled and concentrated using an Amicon PM-30 membrane. 8. 5 ml of sample was chromatographed on a Sephadex G-200 column equilibrated with 50 mM K phosphate pH 7.8 + 0.005% EDTA + ImM salicylate. Xanthine oxidase activity was contained in four fractions eluted between 32 ml and 56 ml from column. Enzyme activity was determined using both the urate assay and the IXP assay. Molecular weight determinations were carried out using the -31- Phannacia PhastSystem SDS-PAGE. This technique was also used to check purity of protein. Pooled fractions were stored as precipitates in 50% ammonium sulphate. Preparation of Polyclonal Antibody to Purified Bovine Xanthine Oxidase In order to identify specifically XO in tissue samples an antibody to XO was used against proteins separated by SDS-PAGE gel electrophoresis and transferred by blotting to nitrocellulose sheets. XDH could also be identified since polyclonal antibody to XO has also been shown to cross react with XDH (87). One ml of the pa r t i a l l y purified homogenate fraction containing the highest activity of XO was electrophoresed on SDS-dissociating gel with mercaptoethanol 1.5% per volume using the method of Laemroli (118). The gel was l i g h t l y stained with Coomassie blue to locate the protein bands. The band containing protein of a molecular weight of 135,000, where XO is known to run, was cut out and lyophilized overnight. Protein was electroeluted from the gel through an agarose gel (1%) and into an attached length of dialysis tubing (20 cm in length and a molecular weight exclusion of 10,000) using a Buchler apparatus. Electroelution was conducted for two days at 4°C, 20 mA, in SDS electrophoresis buffer which was changed every 24 hours. When a l l the blue stained protein had migrated into the dialysis tubing, the tubing containing protein was removed, knotted and dialyzed for two days against 1 1 of d i s t i l l e d Ĥ O. D i s t i l l e d water was changed every 24 hours. Purified antigen was emulsified in Freunds complete adjuvant (1 mg protein/ml). One ml of emulsion was injected intramuscularly into a rabbit -32- at several s i t e s around the body. A blood sample of 15 ml was taken from the rab b i t p r i o r to i n j e c t i o n to prepare a sample of non-immune serum to be used as a c o n t r o l . Rabbit was caged i n d i v i d u a l l y i n the animal care f a c i l i t y of St. Paul's Hospital and given water and standard laboratory chow ad libatum. The animal received booster i n j e c t i o n s of p u r i f i e d antigen in s a l i n e every two weeks (0.5 mg in one ml) (119). The antibody t i t e r was measured at these times using ELISA technique (120). A f t e r eight weeks a maximal antibody response had been reached and the rabbit was exsanguinated by cannulation of the c e n t r a l ear artery. Blood c o l l e c t e d (120 ml) was allowed to c l o t at 37°C, centrifuged at lOOOg for 20 minutes and the serum c o l l e c t e d . Aliquots were frozen at -20°C for l a t e r use. Gel Electrophoresis of Tissue Homogenates and Western B l o t t i n g with Polyc l o n a l Antibody to P u r i f e d XO In order to look f o r immunoreactive XO in various species and organs separation of t i s s u e homogenates was accomplished using SDS denaturing polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with immunoreplication using Western b l o t t i n g and p o l y c l o n a l antibody r a i s e d against p u r i f i e d XO. Gel electrophoresis was run using the modified system of Laemmli (118), (121). The following method for sample preparation was used: 1. Fresh ti s s u e obtained from, slaugterhouse, human lung biopies or s a c r i f i c e of rats was dissected and f i n e l y minced in four volumes of 50mM KPi buffer pH7.4 containing ImM PMSF and 0.1 mM EDTA at 4°C. Minced tis s u e was rinsed and f i l t e r e d through cheesecloth to remove -33- blood. The retentate was then homogenized in a Sorval Omnimixer (75% maximum speed 3 to 8 times f o r 10 seconds each depending on connective t i s s u e content) on ice and then c e l l s were disrupted using a Potter-Eljveheim for 10 passes on i c e . 2. Protein was p r e c i p i t a t e d by the addition of ice cold 33% t r i c h l o r o a c e t i c acid at a volume to volume r a t i o of 1:5 TCA: homogenate. The p e l l e t was then suspended in electrophoresis sample buffer of 20 mM T r i s pH 6.8, 2.5% SDS and 10% g l y c e r o l with the pH adjusted to between 6 and 8. These samples were heated at 100°C for 5 minutes to denature proteins. Samples were c l a r i f i e d by ce n t r i f u g a t i o n . Protein determinations were made by method of Lowry (122) and sample volumes adjusted to give a f i n a l p r otein concentration of l u g / u l . A 2:1 mixture of 1.5% beta mercaptoethanol with bromphenol blue, was added. P r i o r to elec t r o p h o r e s i s , samples were heated to 100°C x 5 min and centrifuged to c l a r i f y in an Eppendorf table top centrifuge. A molecular weight standard mixture (Sigma, range 60,000 to 220,000 molecular weight) were used f o r molecular weight determination and prepared as described above. 100 ug samples were applied i n the sl o t s of the 5% stacking gel of 6% PAGE-SDS slab gels run in duplicate with electrophoresis buffer containing 0.050M T r i s , 0.384 M glycine with 0.1% SDS. Gels were subjected to a current of 70m amp for 3 hours. Af t e r electrophoresis, one gel i s stained with Coomassie Blue to show protein bands while the other i s processed f o r immunoreplication. -34- Immunoreplication Method Gel was soaked in e l e c t r o t r a n s f e r buffer of 10 g T r i s , 48g g l y c i n e , 600 ml methanol in 2400 ml H^O. Gel was then layered with Biorad Immobilon t r a n s f e r membrane between sheets of Whatman f i l t e r paper and supports. The cassette was subjected to a current of 1 amp f o r 90 min in e l e c t r o t r a n s f e r b uffer. When subjected to a strong current, negatively charged proteins w i l l be removed from the gel and transfered to the f i l t e r as they migrate towards the cathode. Immobilon f i l t e r i s then processed according to Biorad Immuno-Blot Assay techniques. Non-specific protein binding s i t e s are blocked with 3% g e l a t i n . B l o t i s then incubated overnight at room temperature with 100 ml of a 1:500 d i l u t i o n of p o l y c l o n a l r a b b i t a n t i s e r a against p u r i f i e d XO (anti-XO). A f t e r washing, the f i l t e r i s then incubated with a second antibody, goat a n t i - r a b b i t IgG, covalently l i n k e d to horseradish peroxidase (Biorad). 4 - c h l o r o - l napthol substrate (HRP substrate, Biorad) was added which leaves a coloured product at s i t e of antibody binding. Blots were then photographed using black and white p r i n t to maintain a permanent record. Photographs were compared for presence of antibody r e a c t i o n and varying i n t e n s i t y of reaction between t i s s u e s . Comparison of S e n s i t i v i t y of Urate Assay to Isoxanthopterin Assay P u r i f i e d XO was used to t e s t the l i m i t s of detection of the urate assay (115) versus the IXP assay (113). S e r i a l d i l u t i o n s of p u r i f i e d XO ( d i l u t e d 1:1000) were suspended in the appropriate buffer (Na pyrophosphate pH 8.3 f o r urate assay or 50 mM KPi pH 7.a4 for IXP assay). 25y moles p t e r i n was added to s t a r t r e a c t i o n . -35- D i l u t i o n s were as follows: 1. 1:1000 d i l u t i o n of XO stock 1 v l i n 1 ml buffer 24.5 yg XO protein. 2. 1:1 d i l u t i o n of above 0.5 u l in 1ml buffer 12.25 ug XO protein 3. 1:1 d i l u t i o n of above 0.25 u l i n 1ml buffer 6.12 yg XO protein. 1:1 d i l u t i o n of above 0.125 u l i n 1 ml buffer =3.06 ug XO p r o t e i n . Both the spectophotometer and spectrofluorimeter were used at the highest s e n s i t i v i t y range of absorbance. Product I n h i b i t i o n of IXP Assay Urate i s known to be an i n h i b i t o r of XO a c t i v i t y (78) (79). I t was important to determine therefore, whether IXP would be s i m i l a r l y competitive with the substrate p t e r i n f o r X0. P u r i f i e d XO a c t i v i t y was measured by IXP assay with no additions as a control and in the presence of 3nm IXP. Reaction cuvette contained 10 y l of 1:1000 d i l u t i o n of p u r i f i e d XO i n 900 y l of 50 mM KPi. Twenty-five y moles p t e r i n was added to s t a r t the r e a c t i o n . The rate of reaction in the control cuvette was compared to rate of reaction in cuvette containing the same amounts of enzyme and p t e r i n with the addition of 3 nm IXP. -36- Experintents i n Porcine Tissue Assay f o r XO-XDH A c t i v i t y i n Pig Tissue Pig heart, lung, kidney and l i v e r were obtained fresh from a l o c a l a b a t t o i r and transported to the laboratory i n ice cold KPi buffer where they were prepared as described in methods (113). Each assay contained 450 y l ti s s u e extract, 450 y l KPi buffer and 25 y moles p t e r i n i n a t o t a l volume of 900 y l . The assay was conducted at 37°C. A f t e r measuring the aerobic rate of rea c t i o n , 5 y moles methylene blue was added to determine the dehydrogenase a c t i v i t y . In order to confirm the s p e c i f i c i t y of the reaction f o r XDH-XO 20 uM a l l o p u r n i n o l was added to i n h i b i t the a c t i v i t y a t t r i b u t a b l e to XDH-XO. 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 PJ R Extracts These tests were c a r r i e d out in order to ascertain whether the lack of a c t i v i t y found i n some semi-purified organ extracts could be due to interference from impurities so that rates of reaction measured d i d not accurately r e f l e c t the actual a c t i v i t y . In order to determine i f pig lung extracts could i n h i b i t p u r i f i e d XO a c t i v i t y i n v i t r o , XO was assayed i n eit h e r KPi buffer alone or buffer plus a l i q u o t s of homogenized t i s s u e preparations. Rates of reaction were compared between the following: 1. 10 y l of 1:1000 d i l u t i o n of p u r i f i e d XO KPi + 900 y l of 50 mM KPi + 0.1 mM EDTA 25 uM p t e r i n . 2. 10 y l of 1:1000 d i l u t i o n of p u r i f i e d XO i n 50 mM KPi + 0.1 mM EDTA + 900 y l of pig lung extract prepared as described i n methods. 25 y moles p t e r i n . -37- Heat S t a b i l i t y of Active Factors i n Pig Tissue In order to determine i f factors i n pig tissue which aff e c t e d the c a t a l y t i c a c t i v i t y of p u r i f i e d XO were l a b i l e to heat, an al i q u o t of homogenate from pig ti s s u e was incubated i n b o i l i n g water f o r 20 minutes. The rates of reaction mixtures were compared as follows: 1. 10 y l of 1:1000 d i l u t i o n p u r i f i e d XO + 900 y l of 50 mM KPi + 0.1 mM EDTA 25 uM of p t e r i n . 2. 10 y l of 1:1000 d i l u t i o n p u r i f i e d XO + 900 y l of pig lung extract denatured by b o i l i n g for 20 minutes, cooled and centrifuged to remove coagulated proteins + 25 y moles p t e r i n . E f f e c t of Pig Tissue Extract on Relative Fluorescent I n t e n s i t y of Isoxanthopterin In order to determine whether a component or compounds contained in pig t i s s u e extract could e i t h e r quench or degrade the product measured i n the fluorometric assay (IXP) a comparative determination was conducted. The r e l a t i v e i n t e n s i t y of 3nM of IXP was measured in 900 y l of 50 mM KPi buffer. This was compared to the r e l a t i v e i n t e n s i t y of 3nM of IXP in the presence of 900 y l of pig lung homogenate. The reaction was recorded at o 37 C for 10 minutes. Assay f o r XO/XDH A c t i v i t y i n Cultured Pig Pulmonary Artery E n d o t h e l i a l C e l l s In order to ascertain whether large vessel endothelium i n culture contained XO/XDH a c t i v i t y , porcine pulmonary artery e n d o t h e l i a l c e l l s were cultured under conditions previously established in t h i s laboratory (123). -38- Fresh porcine heart-lung sets were obtained from a local abbatoir, and transported at 20-22°C to the laboratory. The pulmonary artery was excised, rinsed briefly in phosphate buffered saline (PBS) and placed in Gibco Hanks balanced salt solution (HBSS) (Gibco) supplemented with p e n i c i l l i n and streptomycin in a Laminar Flow Hood using s t e r i l e instruments. Arteries were placed on a gauze covered Petri plate and s l i t lengthwise. Endothelium was removed from the luminal surface by gentle scraping with a Number 22 surgical blade and sheets of cells placed in ste r i l e HBSS. Cells were centrifuged and the pellet resuspended in Medium 199 (Gibco) supplemented with 20% pig serum and seeded in T75 flasks which had been previously coated with 1% gelatin on the culture surface. Cells were grown in a humidified incubator at 37°C in an atmosphere of 95% air: 5% CO^. After 24 h most of the cells which remained unattatched to the culture surface were washed free using HBSS. Most of the unattached cells were contaminating fibroblasts and smooth muscle cel l s . Cells were grown to confluence. One artery provided the cells to seed two T-75 flasks. The average time required to reach confluence was 10 days. At confluence cells were removed with a rubber policeman and reseeded at a ratio of 1:2. In addition to selection by detachment cells at confluence were identified by these characteristics of endothelial c e l l s : 1) their characteristic cobblestone monolayer growth pattern and 2) by the detection of Factor VIII by immunofluorescence (124). Cells for these experiments were used at Passage 3. For assay, seven T-80 flasks were used with, the c e l l number being estimated at between 1.1 x 10 ? to 1.8 x 10 7 cells per flask. After rinsing with HBSS monolayers were removed by scraping with a rubber policeman and the cells suspended in 5 ml of ice cold 50 mM KPi containing 1 mM EDTA. Clumps of cells were broken up using a syringe. This c e l l suspension was then homogenized by 20 passes with a Teflon pestle in a glass mortar and sonicated for 2s with a Sonifier Cell Disrupter. This preparation was fractionated by ultracentrifugation at 100,000g for 60 min. at 4°C. A 2 ml aliquot of the supernatant was run on a Sephadex G-25 column equilibrated with 50 mM KPi containing O.lmM EDTA. The equilibration buffer was applied to the column and 15.6 ml fractions were collected. Fraction 2 which contained the f i r s t protein peak was assayed for XO-XDH activity by the IXP assay method. 900 ul of Fraction 2 were assayed with 25 u moles pterin + 5uM methylene blue added to determine XDH activity at 37°C. Fraction 2 was also concentrated three fold using a Spectra-Con Disposable Sample Concentrater (MW limit 8000) and assayed as above. Isolation of Porcine Pulmonary Microvascular Cells Because i t has been reported on the basis of immunocytochemical localization that XO XDH is found in microvascular but not large vessel endothelial cells (73) we attempted to culture pulmonary microvascular endothelial cells in order to assay for enzyme activity in vitro. Several methods of obtaining capillary endothelium for culture have been developed (125) (126) (127). However, these are unsatisfactory due to potential contamination from large vessel endothelium. The development of a new method of microvascular isolation was attempted based on the observation by -40- l i g h t microscopic examination that to a depth of less than 1 cm from the p l e u r a l surface of the pig lung only vessels less than 50 um diameter are found (128). The methods of microvessel i s o l a t i o n used was based on techniques developed f o r the i s o l a t i o n of rabbi t pulmonary Type II alveolar c e l l s (129) and guinea pig si n u s o i d a l e ndothelial c e l l s from the l i v e r (130) by using enzymatic d i s s o c i a t i o n of ti s s u e in combination with c e n t r i f u g a l e l u t r i a t i o n (131). This technique depends on the f i n d i n g that d i f f e r e n t c e l l types often have d i f f e r e n t sizes and de n s i t i e s . Separation by c e n t r i f u g a l e l u t r i a t i o n i s based on the d i f f e r e n t rates of sedimentation of p a r t i c l e s i n a c e n t r i f u g a l f i e l d balanced by a counterflow so that p a r t i c l e s are maintained i n suspension. F r a c t i o n a t i o n of a mixed c e l l population i s dependent on c e l l s i z e and can be accomplished by stepwise increases i n flow rate (129) A Beckman E l u t r i a t o r was used. Adult porcine per i p h e r a l lung t i s s u e was prepared i n the following manner: Porcine lung t i s s u e was obtained fresh from a l o c a l slaughterhouse. Peripheral lung t i s s u e ( l e s s than 1 cm from p l e u r a l surface) was dissected free of pleura. F i f t e e n to twenty g of ti s s u e was rinsed i n cold PBS without C a 2 + or Mg 2 + (100 ml). Tissue was minced f i n e l y i n cold PBS 2+ 2+ containing Ca and Mg (0.1 g/1 each) along with 0.25% BSA, 1 mg/ml collagenase and 1000 ug DNAse (50ml). This mixture was incubated at 37°C f o r 30 minutes. Large chunks of ti s s u e were then f i l t e r e d out with s t e r i l e gauze and the f i l t r a t e retained. The f i l t r a t e was examined under a phase contrast micrscope to gauge the degree of digestion. When chains of singl e c e l l s were seen ( c a p i l l a r y fragments) the d i s s o c i a t i o n was judged to be completed. F i f t y ml of the above BSA:PBS: collagenase mixture was added - A l - and incubated for approximately another 30 minutes until clusters of 4-5 cells were seen. The c e l l suspension was then equally divided into six 50 ml screw cap centrifuge tubes. PBS-BSA was added up to a total volume of 50 ml in each tube. Tubes were centrifuged at 300 g for 10 minutes and the supernatant discarded. The c e l l pellet was washed twice with PBS-BSA and centrifuged again as above. The supernatant was discarded and a l l pellets were pooled and resuspended in 10 ml of PBS-BSA to be loaded into the elutriator. The elutriation buffer contained: HBSS supplemented with 0.05% DNase by weight and 0.5% BSA at 15°C. During loading the elutriator rotor was spun at 2500 rpm and the flow pump was set at 5 ml/min. The following fractions were collected: Flow Volume (1) -13.5 ml/min 150 ml (2) -19.5 ml/min 250 ml (3) -23.5 ml/min 200 ml (4) purge elutriator at zero rpm, - 50 ml/min 100 ml Fraction 2 was found to contain the endothelial c e l l s . This fraction was centrifuged at 300 g for 10 minutes. The pellet was 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). Cells in 4 ml of the above medium were plated onto gelatinized T-25 flasks. These seeded flasks were incubated at 37°C for 60 minutes. Unattached cells were then removed by washing with HBSS and cells resuspended in 5 ml of the above medium. -42- Cells from Fraction 2 were noted to grow with the appearance of newly seeded large vessel endothelial cells in culture. However, cultures did not grow well enough to reach confluence or passage. In addition, cultures became contaminated possibly due to pulmonary infections in pigs before slaughter. An attempt was made to accelerate the growth rate of the c e l l colony by using newborn (5 day old) pig tissue which has been shown to have greater growth potential than adult tissue. In addition, we used homologous newborn pig sera to supplement culture media since fetal bovine serum is widely used as a supplement because i t contains growth factors not found in adult sera. Since this was not commercially available the sera was prepared from the same animal which lung tissue was collected from. Piglet weighed 2 kg and was anaethetized with 1.5 cc sodium pentobarbital. The pig serum growth supplement was prepared as follows: Blood was collected into a sterile syringe via the inferior vena cava, allowed to clot thoroughly at 4°C and centrifuged. Serum was collected and heat inactivated at 56°C for 30 minutes then cold st e r i l i z e d using a 22 micron f i l t e r . Serum was then aliquoted and frozen for use as required to supplement a r t i f i c a l c e l l culture media by 20%. By preparing tissue and serum in the research and c e l l culture f a c i l i t y on site, i t was hoped to eliminate the problem of contamination during k i l l i n g of the animal and removed of tissue by using sterile conditions and by preparing tissue and serum in the St. Paul's Hospital surgical research and c e l l culture f a c i l i t i e s . -43- EXPERIMENTS IN RAT TISSUE Characterization of IXP Assay in Tissue Rat kidney was used to test the IXP assay system in tissue since this is a well studied source of XDH-XO (54). Reaction mixture was composed of: 450 y l rat kidney extract (119) 450 y l 50 mM KPi + 0.1 mM EDTA 25 y moles pterin for oxidase activity 5 y moles methylene blue added to measure dehydrogenase activity 20 y moles allopurinol was added to stop reaction. Determination of XDH-XO Level in Normal Rat Lung by IXP Assay Normal rat lung was quickly excised, homogenized and processed according to Markley (113). Tissue extracts were assayed by IXP method to determine the baseline level of XDH-XO activity. Reaction mixture contained: 1. 450 yl rat 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 activity 3. 20 y moles allopurinol was added to inhibit XDH-XO. -44- Investigations into Conversion of XDH to XO In order to investigate factors a f f e c t i n g conversion of XDH to XO the rat lung was chosen since the enzyme was found to be e a s i l y detectable i n t h i s r e a d i l y a v a i l a b l e t i s s u e . Based on assays published in 1972 using r a t l i v e r (93) (94), i t was decided to examine the r o l e that p r o t e o l y s i s and C a + + might play i n a f f e c t i n g enzyme conversion. This was c o r r e l a t e d with two ischemic t i s s u e models. R e v e r s i b i l i t y of enzyme conversion a t t r i b u t a b l e to redox states of t h i o l groups was studied with the use of d i t h i o t h r e i t o l (DTT) to cause reduction of t h i o l groups in the enzyme and a f f e c t r e v e r s a l back to XDH a c t i v i t y . E f f e c t of T r y p s i n i z a t i o n on Conversion of XDH to XO Rat lung extracts prepared in 50 mM KPi pH7.4 i n the absence of EDTA or protease i n h i b i t o r s were incubated with t r y p s i n p u r i f i e d from beef pancreas (BDH) . 100 ug t r y p s i n and 1ml of e x t r a c t were incubated f o r 60 min at'37°C with gentle a g i t a t i o n . Samples were compared to the same extracts without t r y p s i n z a t i o n . The p o s s i b i l i t y of r e v e r s i b i l i t y of enzyme conversion by a reducing agent was examined by incubating t r y p s i n - t r e a t e d extracts with ImM DTT f o r 60 min. at 37°C p r i o r to assay. Reaction mixtures contained: 1. 250 y l r a t lung homogenate and 650 u l KPi 50 mM were incubated at 37°C f o r 60 min. 25 y l of 15 yM p t e r i n was added to i n i t i a t e the oxidase r e a c t i o n . 5 y l of 1 mM methylene blue was added to measure dehydrogenase plus oxidase a c i t i v i t y . 2. 250 y l r a t lung homogenate with 650 y l KPi 50 mM was incubated with 100 y l of a t r y p s i n solution from a 10 mg/ml stock at 37°C f o r 60 min. 25 y moles of p t e r i n was added to determine the oxidase a c t i v i t y and then 5 y l of methylene blue was added to measure dehydrogenase plus oxidase a c t i v i t y . 3. 250 y l of r a t lung homogenate with 650 y l of KPi 50 mM was incubated with 100 y l of the above t r y p s i n stock f o r 60 min at 37°C. This sample was then incubated with 1 mM DTT f o r an o a d d i t i o n a l 60 min at 37 C. To t h i s 25 y l p t e r i n was added to measure the oxidase a c t i v i t y and then 5 y l of methylene blue was added to determine t o t a l XDH + XO a c t i v i t y . E f f e c t of Endogenous Proteases on Conversion of XDH to XO In order to examine the e f f e c t of endogenous proteases on the conversion of XDH to XO samples were incubated at 15°C overnight. This temperature was chosen because ambient room temperature (93) has been shown to i n a c t i v a t e XDH-XO, while 4°C i n h i b i t s protease a c t i v i t y . Samples were compared to extracts from the same animal which had not been incubated at 15°C overnight as c o n t r o l . The extracts were incubated with ImM DTT in order to assess i f enzyme conversion was r e v e r s i b l e . Reaction mixture contained: 1. 450 u l ra t lung extract prepared i n 50 ym KPi, with no added EDTA incubated at 15°C for 16 hr. 450 y l KPi pH 7.4 50 mM 37°C 25 y l p t e r i n 5 y l methylene blue added -46- 2. 450 pul same r a t lung extract not incubated at 15°C + 450 p i KPi pH 7.4 50 mM 37°C 25 p i p t e r i n 5 p methylene blue added 3. 450 p i same r a t lung extract x 15°C overnight) + 450 p i KPi 50 mM pH 7.4 incubated with ImM DTT X60 min at 37°C 25 p i p t e r i n 5 p i methylene blue added E f f e c t of Calcium on Conversion of XDH to XO In order to assess whether calcium might play a r o l e i n enzyme conversion i n the r a t lung, as suggested by McCord (52) two known calcium chelating agents which have d i f f e r e n t a f f i n i t i e s were used. Samples with no calcium chelater present were used as a c o n t r o l . The XDH XO fluorescence assay method of Markley (113) s p e c i f i e s the addition of EDTA as a chelator of d i v a l e n t cations to a l l homogenization and sample b u f f e r s . I t was of i n t e r e s t to ascertain whether the same amount of EGTA, a more s p e c i f i c chelator of calcium would have a more pro t e c t i v e e f f e c t on conserving enzyme in the XDH form. Tissue extracts prepared in the absence of EDTA or EGTA served as a c o n t r o l . R e v e r s i b i l i t y of enzyme conversion by the reduction of sulphydryl groups was tested by the addition of ImM DTT to t i s s u e extracts prepared with EDTA, EGTA and in the absence of calcium chelators followed by incubation at 37°C x 60 min. Reaction mixtures contained: 1. 450 yl rat lung extract prepared in 50 mM KPi 450 y l KPi pH 7.4 25 yl pterin 5 yl 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 yl pterin 5yl methylene blue added 3. 450 yl rat lung extract prepared in 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 yl pterin 5 yl 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 for 60 min 25 y l pterin 5 yl methylene blue added 5. 450 yl rat lung extract prepared in 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 yl pterin 5 yl methylene blue added -48- 6. 450 y l above extract with 450 yl KPi ph 7.4 50mM + ImM EGTA incubated with 1 mM DTT at 37°C for 60 min 25 y l pterin 5 y l methylene blue added Effect of Ischemia on Conversion of XDH to XO We sought to determine whether ischemia might have an effect on conversion of XDH to XO by the use of two models of ischemia. (A) The f i r s t involved incubation of excised lung tissue in PBS at 37°C in a humidified incubator under normoxic conditions for 60 min post-sacrifice, according to the method of Granger (132). (B) The second method involved perfusion of the rat lung via a pulmonary artery cannula with an oxygen depleted buffer mixture of 50 mM KPi pH 7.4 at 37°C through which had been sparged for 20 min and clamping of the pulmonary artery for 60 min. (C) These were compared to rat lung extracts prepared from lungs which were rapidly cannulated and perfused with cold 50 mM KPi pH 7.4 containing 1 mM EDTA and 1 mM DTT and immediately homogenized in the same buffer containing 1 mM PMSF as a protease inhibitor. Samples were then compared to those incubated with 1 mM DTT 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 yl of rat lung extract prepared by Method (A) with 450 yl of KPi buffer 25 y moles pterin 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 for 60 min 5 y moles of methylene blue 3. 450 y l of rat 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 rat lung extract (B) with 450 yl of 50 mM KPi, pH 7.4 incubated with 1 mM DTT at 37°C 60 min 25 y moles pterin 5 y moles methylene blue added 5. 450 y l of rat 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 in Human Lung Tissue It was of great interest to determine whether active XDH XO was detectable in human lung tissue, since the enzyme as yet has only been localized using immunohistological means (72) (73). Human lung samples of approximately 6-10 g were obtained from peripheral lung sections of patients undergoing pneumectomy or lobectomy for bronchogenic carcinoma. Samples were immediately frozen at -70°C upon arrival to be processed at a later date. Thawed samples were minced and homogenized in 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 activity was much lower than rat lung, the spectrofluorimeter was used on the most sensitive setting. Reaction mixtures contained: 900 ul 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 already necrotic, v i a b i l i t y of tissue was determined by assessing the capacity of the tissue to oxidize NADH. NADH (0.1 mM) was 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 for a l l mean values of enzyme activity. Comparisons between experimental groups and controls were based on students t-test of xanthine oxidase act i v i t i e s for the studies of enzyme conversion. -51- RESULTS EXPERIMENTS WITH PURIFIED XO I s o l a t i o n of Milk Xanthine Oxidase A f t e r f r a c t i o n a t i o n of milk protein as described i n methods pooled f r a c t i o n s with xanthine oxidase a c t i v i t y obtained had a t o t a l p rotein of 86.8 mg (122). A c t i v i t y of pooled f r a c t i o n s was 4.78 U/mg pr o t e i n . One unit i s defined as gi v i n g a AA of one per minute at 295 nm during the conversion of xanthine to u r i c acid (115). Ratio of A280 to A450 (protein to f l a v i n r a t i o ) of the p u r i f i e d XO was determined to be 5.75 which i s higher than the value of 4.8 reported by the authors of the method (116). These r e s u l t s suggest a higher l e v e l of s p e c i f i c a c t i v i t y , due to more f l a v i n - c o n t a i n i n g active subunits. SDS-PAGE revealed a single band of 135KDa (Figure 5), the known size of the xanthine oxidase subunit. Protein concentration of ammonium sulphate p r e c i p i t a t e s dialyzed on M i l l i p o r e membranes was determined to be 25.4 mg prot e i n /ml. A c t i v i t y of p r e c i p i t a t e s was 54 U/ml/min as determined by urate assay; or 69.11 ug IXP produced /min/mg protein by fluorometric assay (113). In accordance with the as per method of Beckman (91), milk xanthine oxidase was found to have up to 10% dehydrogenase a c t i v i t y when samples were incubated with 1 mM d i t h i o t h r e i t o l (DTT) at 37°C f or 60 min p r i o r to assay. C h a r a c t e r i z a t i o n of Polyclonal Antisera to Xanthine Oxidase When a rabbi t was immunized with p u r i f i e d milk XO which had been -52- Figure 5. Pharmacia Phast System SDS-PAGE analysis of p u r i f i e d bovine milk XO on a 8-25% gradient gel stained with coomassie blue. Lane 1 LMW markers (from top to bottom) phosphorylase b, 94,000, albumin 67,000, ovalbumin 43,000, carbonic anhydrase 30,000 t r y p s i n i n h i b i t o r 20,000, lactalbumin 14,000. Lanes 2 and 5 contain high molecular weight markers (Top to bottom) f e r r i t i n , 220,000, albumin 67,000, catalase 60,000, l a c t a t e hydrogenase 36,000. Lane 3:2 pg of p u r i f i e d bovine milk XO. Lane 4: lpg of p u r i f i e d bovine milk XO (arrow). -53- SDS-dissociated, the antibody t i t e r of immune serum increased 5000 f o l d during the 8 weeks of immunization, as determined by the ELISA technique. The t i t e r against native XO increased 2000 f o l d increased, i n keeping with the f a c t that the antibody was r a i s e d against SDS-dissociated antigen, and thus more determinants were uncovered. At exsanguination, 120 ml of whole blood was c o l l e c t e d from which 60 ml of immune serum was obtained. In t e s t s using immune serum as a f i r s t antibody i n Western B l o t t i n g , anti-XO antiserum was strongly r e a c t i v e with p u r i f i e d bovine milk XO and XDH separated on SDS-PAGE gels r e f l e c t i n g molecular weights of 130,000 and 150,000 Da r e s p e c t i v e l y (Figure 6). The antibody also reacted with smaller p r o t e o l y t i c fragments of XO. In t e s t s using t i s s u e homogenates and p u r i f i e d bovine serum albumin (BSA) the antibody was strongly r e a c t i v e against the albumin of several species which ran at 67,000 Da (Figure 6). SDS-PAGE of Tissue Homosenates with Western B l o t t i n g SDS-PAGE of t i s s u e homogenates containing 100 ug of p r o t e i n per sample revealed that antiserum to bovine XO cross reacted with bovine lung t i s s u e and bovine milk (powdered, Safeway Brand), showing r e a c t i v i t y at 150,000 and 130,000 Da, as well as at c h a r a c t e r i s t i c p r o t e o l y t i c fragment of XO at 90,000 Da (87) (Figure 7) which i s a major fragment of the enxyme. When rat t i s s u e homogenates containing 100 yg protein of r a t lung, kidney, l i v e r and heart were examined by SDS-PAGE, XO r e a c t i v e bands were seen in a l l t i s s u e samples at 150,000 and 130,000 Da as well as at 90,000 Da (Figure 8). -54- Figure 6. Western blot of purified bovine milk XO. Electrophoresis of proteins was carried out on 6% SDS-PAGE. Protein was electrotransfered onto Immobilon Membrane (Millipore). XO was visualized by incubation of the Western blot with a :500 dilution 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 site 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 reactivity of antibody to albumin (star). -55- Figure 7. Western blot of bovine tissues. 100 ug of redissolved protein precipitates were electrophoresed, blotted and visualized as previously described (Figure 6). Lane 1 - purified 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 of 100 ug protein of porcine whole lung, peripheral lung and kidney did not show protein bands which reacted with anti-bovine XO anti s e r a . Porcine l i v e r reacted p o s i t v e l y f o r XO (Figure 9). Analysis of perip h e r a l human lung t i s s u e homogenates showed no detectable xanthine oxidase (Figure 10). Comparison of S e n s i t i v i t y of Urate Assay to Isoxanthopterin Assay Graded d i l u t i o n s of XO were assayed u n t i l no a c t i v i t y could be measured. The l i m i t s of detection of the urate assay were found to be 2.A5 x 10 5 mg of XO protein (assuming a l l of the protein in the p u r i f i e d XO i s XO). This corresponds to A.93 x 1 0 ^ molecules of XO -6 With the IXP assay the l i m i t s of detection were 3.17 x 10 mg X0 g protein, which corresponds to 6.17 x 10 molecules of XO. The IXP assay was found to be in the range of 100 f o l d more s e n s i t i v e than the urate assay. Product I n h i b i t i o n of IXP Assay IXP assay f o r XO was performed both i n the presence and absence of 3nM IXP using substrate concentrations from 1 uM to 25 uM. 3 nm of IXP was shown to i n h i b i t the enzymatic rate of reaction i n a competitive manner as shown in a Lineweaver-Burke Plot (Figure 11). The Km for the reaction was -3 -10 3.A x 10 M. The Ki for the reaction was A.A x 10 M. 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 precipitates were electrophoresed, blotted and visualized as described (Figure 6). Lane 1; purified 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 A A I h r l l b l T t U UNiMHIUITEl) 100 -.UU 1/S (MOLES PTERIN) Figure 1 1 . Lineweaver-Burke plot showing the competitive inhibition of purified XO by 3 nm IXP; = 3 . 4 x 1 0 - 3 M ) . K 4 > 4 x 1 0 - 1 0 M . 1 -61- EXPERIMENTS IN PORCINE TISSUE Assay for XO XDH Activity in Pig Tissue No enzyme activity was detected in pig kidney, whole lung or peripheral lung homogenates from 6 different pigs, even when most sensitive spectrofluorimeter settings were used. Representative tracings taken from data obtained on the enzymatic activity in pig lung homogenates (Figure 13) and pig kidney homogenates (Figure 14) revealed a decreased rather than the increased fluorescent intensity as a function of time which would be expected for the enzymatic conversion of pterin to fluorescent product, IXP. Pig l i v e r contained XO XDH activity as determined by IXP assay,with a _3 specific activity of 1.1 x 10 yg IXP/min/mg protein (Figure 12). Inhibition of Purified XO Activity by Pig Extracts Based on the previous observation that pig kidney and lung caused a decrease in fluorescence in the IXP assay, an inhibitor for XO was sought. The rate of IXP production by purified XO in buffer was compared to that of the same amount of purified XO in pig lung extract. Enzyme activity in buffer was found to be 7.5 yg IXP/mg protein/min. Enzyme activity in pig lung extract was found to be 1.98 yg IXP/mg protein/min showing a 73.5% inhibition in enzyme rate of activity (Figure 15). Heat Stability of Active Factors in Pig Tissues In order to test whether an enzyme or protein might be involved in the inhibition of XO activity by pig lung extract, we examined the effect of -62- 5 OH 1 : 1 • 1 1 0 2 4 6 8 10 Time (minutes) Figure 12. Representative t r a c i n g of XDH XO a c t i v i t y i n pig l i v e r homogenates from n=3 samples. A. Addition of 25 y 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. Addition of 5 y moles methylene blue to measure the t o t a l XDH + XO a c t i v i t y . C. Addition of 20 y moles a l l o p u r i n o l to i n h i b i t the r e a c t i o n . -63- Figure 13. Representative t r a c i n g of XDH XO a c t i v i t y i n pig lung from n=6 samples. A. Addition of 25 u moles p t e r i n to i n i t i a t e the aerobic reaction. B. Addition of 5 u moles methylene blue to measure t o t a l XDH + XO a c t i v i t y . -64- Figure 14. Representative tracing of XDH-XO activity in pig kidney from n=6 samples. A. Addition of 25 u moles pterin to initiate the aerobic reaction. B. Addition of 5 u moles methylene blue to measure total XDH + XO activity. -65- Figure 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 pig lung homogenate. A. Reaction rate of p u r i f i e d bovine XO in 50 mM KPi + mM EDTA with 25 uM p t e r i n . B. Reaction rate of same amount of p u r i f i e d bovine XO in pig lung homogenate in 50 mM KPi + 0.1 mM EDTA with 25 uM p t e r i n showed a 73.5% i n h i b i t i o n of reaction rate. -66- incubation at 100°C f o r 20 min on a b i l i t y of pig tiss u e extract to i n h i b i t XO a c t i v i t y . P u r i f i e d XO a c t i v i t y in buffer was found to be 3.6 ug IXP/mg protein/min while p u r i f i e d XO a c t i v i t y i n bo i l e d pig ti s s u e extract was found to be 2.25 yg IXP/mg protein/min. This represented a 37% i n i b i t i o n of a c t i v i t y , as compared to a 73.5% i n h i b i t i o n in non-denatured pig homogenate (Figure 16). E f f e c t of Pig Tissue Extract on IXP Relative Intensity Because b o i l i n g of pig tiss u e extract d i d not t o t a l l y ablate i n h i b i t i o n of XO a c t i v i t y , i t was necessary to investigate whether pig t i s s u e extract could quench IXP fluorescence or degrade IXP. The fluorescence i n t e n s i t y of 3nM IXP in buffer decreased immediately by 8% when measured i n the same amount of pig lung e x t r a c t . Following the reac t i o n f o r 10 minutes showed an ad d i t i o n a l 3% decrease i n IXP fluorescence (Figure 17). Measurement of XO-XDH A c t i v i t y in Cultured Porcine Pulmonary Artery E n d o t h e l i a l C e l l s C e l l u l a r extract from seven T-80 f l a s k s of porcine pulmonary artery e n d o t h e l i a l c e l l s were assayed f o r XO-XDH a c t i v i t y . This corresponds to 7 8 c e l l numbers ranging from 7.8 x 10 to 1.3 x 10 c e l l s . No XO-XDH a c t i v i t y was detected i n t h i s extract, or in extract which had been concentrated t h r e e f o l d by removal of Ĥ O therefore a c t i v i t y , i f present, would be less than the l e v e l of detection f o r t h i s assay. -67- Figure 16. Heat s t a b i l i t y of active factors i n pig t i s s u e . A. Reaction rate of p u r i f i e d bovine XO in 50 mM KPi + 0.1 mM EDTA. B. Reaction rate of p u r i f i e d bovine XO in pig lung homogenate denatured by incubation in b o i l i n g water for 20 min, showing 37% i n h i b i t i o n of XO a c t i v i t y . -68- Figure 17. Effect of Pig Tissue Extract of Relative Fluorescent Intensity of IXP. Averaged curve from points obtained for three replicates of representative traces. A. Relative intensity of 3 nM IXP dissolved in 900 ul 50 mM KPi. B. Relative intensity of 3nM IXP dissolved in 900 ul pig lung homogenate. -69- l 8 o l a t i o n of Porcine Pulmonary Microvascular Cells Endothelial cells were determined to be separated from parenchymal cells in elutriation Fraction 2. These cells were followed with phase microscopy. After i n i t i a l seed of adult c e l l s , clumps of 4-6 cells adhered to the gelatin-coated culture flask. By 5 days later these had grown into patches of polyhedral cells interspersed with spindly open networks of cells which had the appearance of newly isolated large vessel endothelium in culture. By 10 days many healthy patches of polyhedral cells with oval nuclei were apparent growing in cobblestone arrangements typical of large vessel endothelium in culture. These were interspersed with "networks" of spindle shaped cells which gave the appearance of capillary networks. By 14 days large patches of subconfluent polyhedral cells growing with a characteristic cobblestone monolayer appearance were apparent. A l l colonies were found to be infected with a mycotic contamination at this point and were thus discarded. The cells were not passaged. Isolation of microvascular endothelial cells from newborn piglet provided a very low i n i t i a l number of ce l l s . Elutriator fraction 2 was cultured for 28 days. Patches of cells growing with the morphological appearance of fibroblasts were scraped from the culture surface and rinsed from the flask. After 56 days the T-25 flask of newborn piglet cells was confluent with polyhedral cells in a cobblestone monolayer, similar to large vessel cells in culture (Figure 18). The cells were passaged into two flasks. One flask was supplemented with newborn piglet serum, the other supplemented with commercial adult pig serum. Cells grown in newborn pig serum grew slightly faster; i.e. they reached confluence one day sooner than cells -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 in adult pig serum. Despite manual removal of f i b r o b l a s t i c appearing patches of c e l l s , the cultures were contaminated by f i b r o b l a s t s by the time second confluence was reached. There was no evidence of contamination by b a c t e r i a or fungus. Because of t h i s impurity colonies were discarded. Neither adult pig nor newborn p i g l e t c e l l s could be subcultured onto glass c o v e r s l i p s i n order to perform the assay for Factor VIII immunolocalization i n order to confirm that they were indeed e n d o t h e l i a l c e l l s . I t was therefore necessary to r e l y on morphological appearance i n order to i d e n t i f y them. In a d d i t i o n , neither adult pig nor newborn p i g l e t c e l l s were cultured i n large enough quantity use f o r biochemical analysis of XO-XDH a c t i v i t y . EXPERIMENTS IN RAT TISSUE Ch a r a c t e r i z a t i o n of IXP Assay in Tissue Whereas use of the IXP assay showed XO a c t i v i t y i n p u r i f i e d milk, the same assay did not detect XO a c t i v i t y i n pig lung and kidney homogenates. This r a i s e d the question of whether the assay system was e f f e c t i v e i n crude t i s s u e homogenates. To determine the effectiveness of t h i s assay i n t i s s u e homogenates, the enzyme was assayed i n r a t kidney homogenates since t h i s t i s s u e i s know to contain the enzyme. Rat kidney was excised and immediately homogenized in ice cold 50 mM Kpi -2 + O.lmM EDTA. The t o t a l enzyme a c t i v i t y (XDH + XO) was 1.5 x 10 ug -3 -3 IXP/min/mg protein (+ SE 3 x 10 ). XO a c t i v i t y was 3.9 x 10 pg _3 IXP/min/mg protein (+ SE 1 x 10 ) on n=5 animals. IXP production was shown to be i n h i b i t e d by the addition of 20 u moles of a l l o p u r i n o l (Figure 19) . -72- Detennination of XDH-XO Level i n Normal Rat Lung by IXP Assay Normal r a t lung was quickly excised and homogenized in ice cold 50 mM KPi -2 + 0.1 mM EDTA. T o t a l enzyme a c t i v i t y (XDH + XO) was 1.7 x 10 ug _3 IXP/min/mg protein (+ SE 4 x 10 ). XO a c t i v i t y was shown to be 9.3 x -3 -3 10 pg IXP/min/mg protein (+ SE 3 x 10 ). n=3 animals. Conversion of p t e r i n to IXP was i n h i b i t e d by the addition of 20 u moles of a l l o p u r i n o l (Figure 20). Studies of the Conversion of XDH to XO E f f e c t of Trypsin Treatment on XDH to X0 Conversion Based on studies i n l i v e r showing that XDH i s i r r e v e r s i b l y converted to XO by l i m i t e d p r o t e o l y s i s (86) the e f f e c t of incubating 100 ug t r y p s i n per ml of r a t lung extract was studied and compared to co n t r o l r a t lung e x t r a c t . Both t r y p s i n i z e d and cont r o l extracts were also incubated with ImM DTT i n order to tes t f o r the r e v e r s i b l e conversion involving oxidation of t h i o l groups. Conversion i s expressed as the r a t i o of t o t a l enzyme a c t i v i t y to oxidase a c t i v i t y (XDH+XO/XO). A r a t i o of (XDH + XO)/XO = 1 implies that a l l enzyme has been converted to the oxidase form. In c o n t r o l r a t lung XDH+XO/XO was 1.05. Control extract + DTT showed (XDH + XO)/XO of 4.92. Trypsi n i z e d extract showed XDH+XO/XO of 1. Trypsinized extract incubated with DTT showed (XDH + XO)/XO of 1.73 (Figure 21). E f f e c t of Entended Incubation at 15°C on Conversion of XDH to XO Rat lung extracts were incubated without EDTA at 15°C f o r 16 h. Analysis of (XDH + XO)/XO r a t i o show complete conversion (XDH + X0)/X0 = o 1). Further incubation of these extracts with ImM DTT for 60 min at 37 C showed a (XDH + X0)/X0 r a t i o s of 3.59 (Figure 22). -73- - I I i i 0 2 4 6 8 10 Time (minutes) Representative t r a c i n g of XDH-XO a c t i v i t y i n r a t kidney homogenate, from n=6 samples. A. Addition of 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. Addition of 5 u moles methylene blue to measure the t o t a l XDH + XO a c t i v i t y . C. Addition of 20 u moles a l l o p u r i n o l to i n h i b i t the r e a c t i o n . -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. -75- E f f e c t of Calcium on Conversion of XDH to XO in Normoxic Lung In order to te s t the e f f e c t s of calcium on XDH to XO conversion, the e f f e c t of EDTA as a chelator of divalent cations was compared with EGTA, a more s p e c i f i c chelator of calcium, using samples prepared i n the absence of chelator as controls. The r e v e r s i b i l i t y of the conversion was tested by incubation with ImM DTT. Experiments were performed on normoxic r a t lung quickly excised and homogenized in the appropriate buffer. (Table I I ) . For purposes of comparison, (XDH + XO)/XO of normoxic r a t lung in the absence of calcium chelators was 1.45. With the addition of 1 mM EDTA to a l l buffers used f o r t i s s u e homogenization, chromatography or sample preparation, 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 chelator of calcium, the r a t i o of (XDH + XO)/XO was 1.1 + SE 0.33 (Figure 23). Incubation of the above described extracts with ImM DTT for 60 min at 37°C to reduce t h i o l groups gave the following (XDH + XO)/XO r a t i o s : Control 4.74 EDTA 2.8 EGTA 3.25 E f f e c t of Calcium on XDH to XO Conversion i n Ischemic Lung Rat lung was excised, rinsed, and incubated i n PBS at 37°C to reproduce the conditions of warm ischemia as described (132). Lungs were then b l o t t e d to remove excess PBS and homogenized in iced 50 mM KPi containing e i t h e r ImM EDTA, ImM EGTA or no calcium chelator as a c o n t r o l . Three animals were studied f o r each experiment (Table I I I ) . The r a t i o of (XDH + X0)/X0 obtained from ischemic r a t lung i n the absence of calcium chelators was 1.5. Ratio of (XDH + X0)/X0 for ischemic -76- TABLE II Comparison of XDH and XO Activities in Normoxic Rat Lung in the Absence and Presence of Calcium Chelators During Sample Preparation Treatment XDH + XO XO XDH + XO (x IO"3) (x 10"3) 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 ImM EDTA 16.7 + 0.4 9.3 + 0.3 1.8 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 for each group. * = p<0.05 for comparisons between XO activity with and without DTT for each calcium chelating agent. Control Control + DTT Trypsin Trypsin + DTT Figure 21. Effect 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 trypsin as compared to control. Conversion to XO in the control preparation was 80% reversible to XDH by incubation with DTT. Conversion to XO in trypsin-treated preparation was reversible to XDH to only a slight extent. Enzyme activity was irreversibly converted to XO by 95%. -78- 5- Control Control 15°C 15°C + DTT Incubation Incubation + DTT Figure 22. Effect 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 rat lung extract at 15°C was reversible by 75% to XDH with only a small proportion of the enzyme activity being irreversibly converted. -79- 5- Contro l Contro l EDTA EDTA EGTA EGTA + DTT + DTT + DTT Figure 23. Effect of Calcium on Reversibility of the conversion of XDH to XO in Normoxic Rat Lung. Treatment of rat lung extracts with EDTA or EGTA did not affect 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. -80- TABLE III Comparison of XDH and XO Activities in Ischemic Rat Lung in the Absence and Presence of Calcium Chelators During Sample Preparation Treatment XDH + XO XO XDH + XO (x 10-3) ( X 1 0-3) XQ Control 24.3+10.0 15.9+6.0 1.5 Control 24.4 + 10.0 7.5* + 3.0 3.2 +lmM DTT 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 for 60 minutes. n=3 for each group. * = p<0.05 for comparisons between XO activity with and without DTT for each calcium chelating agent. -81- 6n 5- Contro l Control EDTA EDTA EGTA EGTA + DTT + DTT + DTT Figure 24. Effect of Calcium on Reversibility of the conversion of XDH to XO in Ischemic Rat Lung. Treatment of rat lung extracts with EDTA or EGTA showed a slight increase in re v e r s i b i l i t y of the conversion as measured :by incubation with DTT, but this was not significant 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 ImM DTT for 60 min at 37°C. This treatment resulted in reversal of enzyme conversion from XO to XDH such that in ischemic lung extract ratio 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, ratio of (XDH + X0)/X0 was 4.2. Effect of Perfusion with EDTA and DTT Prior to Homogenization Because the effects of EDTA as a chelator of divalent cations on the enzyme converting activity might be masked by the conversion of enzyme proceeding before the protective agents reached a l l cells during homogenization, the tissue was perfused with the chelator before excision. The pulmonary artery of rat lungs was cannulated with PE tubing via the apex of the right ventricle and the lungs were perfused with iced 50 mM Kpi pH 7.4 + 1 mM EDTA + 1 mM DTT + 1 mM PMSF immediately after the chest cavity was 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 rat lung extracts treated, EDTA and DTT were present in a l l homogenizing, chromatography and sample buffers. After this method of preparation the ratio of (XDH + XO)/XO was 3.53 (Table IV) Effect of Perfusion with Oxygen Depleted Buffer and 60' Ischemia on XDH to XO Conversion In order to maximize the effect of ischemia rat lungs were cannulated and -83- perfused with a mixture of 50 mM KPi pH 7.4 at 37°C with no DTT, EDTA or PMSF in order clear blood from the lungs. Blanched lungs were then clamped at the pulmonary artery and kept warm in the rats chest cavity for 60 minutes. After this period of time, clamp was removed and lungs homogenized in iced 50 mM Kpi pH 7.4 and processed as usual. Three animals were studied. Ratio of (XDH + XO)/XO in rat lung extracts treated in this manner was 1.32. After extracts from these experiments were further incubated with ImM DTT at 37°C for 60 min ratio of XDH + XO/XO increased to 2.53 (Table IV). Experiments in Human Lung Tissue Six human lung biopsy samples were assayed for XDH-XO activity. Of these, only three were found to be capable of oxidizing NADH. The other three samples contained no XDH-XO activity. Of the three which redox cycled NADH, only one showed measureable XO activity of 5.35 x 10~6 ug IXP/mg protein/min. No XDH activity was found. Because of unknown v a r i a b i l i t y in tissue removal and transport i t was impossible to determine whether DTT would be effective in 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. -85- TABLE IV Effect of Perfusion of lung with EDTA and DTT on Preventing Conversion of XDH to XO Treatment XDH + XO XO XDH + XO (x IO"3) (x IO"3) xo Perfusion with 9.9 + 0.3 13.4 + 0.1 3.53 4°C 1 mM EDTA at 4° + 1 nM DTT in 50 mM KPi. N=5 Perfusion with 18.0 + 10.0 13.6 + 0.7 1.32 37° C 50 mM KPi + 60 min ischemia N=3 Perfusion with 27.4 + 15.0 10.9 + 0.6 2.53 37° C 50 mM KPi + ImM DTT X 60 min N=3 Enzyme activities 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. -86- DISCUSSION: The goal of these studies was to assess XDH to XO ac t i v i t i e s in lung tissues of several species, to compare the activity in lungs to that in other tissues and to examine the relationship of XDH to XO in the lung with a view to understanding the implications of such changes in inflammatory lung disease. It was of particular interest to examine the hypothesis that calcium-calmodulin mediated proteolysis may cause irreversible conversion from XDH to XO, and to relate 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. If this were so, the importance of XO activity in contributing to free radical mediated damage in pathologic conditions could be applied to the proposal that oxygen free radicals generated by XO could be important in the etiology of ARDS by acting on cellular components to produce chemotactic factors thus causing PMN activation (71) in the lung . Use of Purified Bovine Milk XO in These Studies Based on high yield, activity and apparent purity the use of purified XO from our own laboratory by the method of Waud (91) was judged to be superior to use of commercially available XO for characterization of our assay system and preparation of a polyclonal antibody. Because purified XO had appeared as a single band at 135,000 Da upon post-isolation electrophoresis i t was surprising to find strong reactivty against albumin at 67,000 Da. Since the 5 to 50 mM KPi buffer gradient on the DEAE Sephadex A-50 column was not conducted due to technical problems, the reactivity of the anti-XO antisera was attributed to the fact that trimers of albumin -87- which have the the same molecular weight as XO subunits were possibly contaminants the XO preparation. Thus antibodies were raised in the rabbit to the highly immunogenic BSA as well as to the XO. This would be a more reasonable explanation of the results rather than the po s s i b i l i t y that the antibody to XO was cross reactive with BSA. Antigen-antibody precipitation of the polyclonal antisera with BSA would help to eliminate some of the antibody to BSA. This purification not being possible, the dual reactivity of the antisera was noted to provide a convenient molecular weight marker for Western blotting. Use of this antisera in Western blotting of SDS-PAGE of redissolved protein precipitates 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 literature search indicates that while pig l i v e r is a known source of the enzyme (72) there is no reliable information available on other porcine tissues. It is not known i f these tissues have been unsuccessfully assayed, or i f XO is found in porcine milk. It is clear, however, that bovine and porcine X0 cross-react as confirmed by the observed reactivity of the enzyme in porcine l i v e r homogenates with the antisera to the bovine enzyme. Although i t has been reported that XO in human lung cross-reacts with anti sera specific to bovine XO (73), no human reactivity with the anti bovine XO was observed in these studies. Use of IXP Assay in these Experiments Purified bovine X0 was used to characterize the fluorescence assay system of Markley (113) for the purpose of these studies. Although this -88- assay has been reported to be 1000 fold more sensitive than the conventional urate assay (115), in this laboratory i t was only 100 fold more sensitive when assayed at the limits of detection. This difference could be attributable to the use of different fluorimeters, since a l l reagents purchased were the same as those prescribed by the protocol (116). An interesting finding from these studies was that a certain percentage (10%) of purified bovine XO could be converted back to XDH by the use of thiol reducing agents (96). This was fortuitous since i t made possible the characterization of the assay for use with XDH in tissue as well. The efficiency of the fluorometric IXP assay for XDH-XO has been recently questioned for application ot human tissues (90). Although pterin is a synthetic substrate for XO (77), IXP catalytically 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 catalytic action of XO at High concentrations as the product accumulated during catalysis similar to the product inhibition of XO by urate. Secondly, IXP is degraded in pig tissue. Uricase which degrades urate into allantoin is found in pig tissues. While i t is unknown whether i t is indeed uricase which is acting on the IXP, this could be tested by investigating the effects of uricase inhibitors on the degradation of IXP by pig tissue extracts. It should be noted that the effects of heat denatured pig tissue homogenate on IXP were not investigated in these studies. Using the IXP assay to determine XDH-XO levels in various tissue homogenates gave results similar to those from SDS-PAGE Western blotting. -89- Nevertheless, i t was s u r p r i s i n g to note the apparent t o t a l lack of enzyme a c t i v i t y i n p i g lung. This i s c e r t a i n l y the exception to a l l other mammalian species studied to date, which show at l e a s t some l e v e l of a c t i v i t y (72). I t has been hypothesized that subhuman species, which are capable of the further degradation of urate to a l l a n t o i n do not have the enzyme s t r i c t l y compartmentalized in the e n d o t h e l i a l c e l l s , but rather show a more ubiquitous d i s t r i b u t i o n of the enzyme throughout the t i s s u e s (88). The f i n d i n g that XO i s apparently l o c a l i z e d only i n the l i v e r of the pig appears to contradict t h i s hypothesis. Because of the extremely low l e v e l s of a c t i v i t y found in human lung extracts, t h i s f i n d i n g was not seen to be in c o n f l i c t with r e s u l t s from gel electrophoresis but rather that i t was below the l e v e l of s e n s i t i v i t y f o r that method. Based on the o v e r a l l lack of XDH-XO a c t i v i t y i n porcine lung, along with data suggesting that XO i s l o c a l i z e d only i n microvascular endothelium XDH-XO a c t i v i t y was not expected to be found i n cultured porcine pulmonary artery e n d o t h e l i a l c e l l s . This was confirmed by ana l y s i s . I t would be of i n t e r e s t to pursue t h i s l i n e of in v e s t i g a t i o n using cultured bovine or lamb pulmonary artery c e l l s i n order to see i f t h i s can be confirmed as well i n species which do show XO a c t i v i t y in the lung. Despite lack of porcine pulmonary XO a c t i v i t y i t was unfortunate that porcine microvascular endothelium could not be cultured i n s u f f i c i e n t number to assay f o r XDH-XO a c t i v i t y . The author i s o p t i m i s t i c that t h i s method of i s o l a t i n g pulmonary microvascular c e l l s could prove to be a p r o f i t a b l e t o o l for i n v e s t i g a t i o n with a few more t r i a l s . A la r g e r i n i t i a l seed of c e l l s would be most b e n e f i c i a l . Based on casual observation of c e l l s i n cultu r e , -90- cells from neonatal tissue did show a greater propensity to divide and reach confluence than did cells from adult tissue. The question remains unanswered as to whether colonies of cells originating from neonatal pigs were i n i t i a l l y contaminated with fibroblasts or i f cells in culture pleomorphically transformed into fibroblast-like c e l l s . Since distinctly fibroblastic patches did not appear unt i l 4 to 6 weeks after the i n i t i a l seeding the second explanation may be more reasonable since neonatal and fetal c e l l s are known to show more pluripotentiality than adult cells and fibroblasts normally grow much more rapidly than endothelial c e l l s . Overgrowth would occur much more rapidly than was observed, therefore. The use of sera from neonatal pigs was not found to have enough observable benefit in these experiments as observed by time required to reach confluence to merit its separate harvesting as i t is not commercially available. However, greater benefits may be present for more established c e l l colonies, since fetal bovine serum is known to be more beneficial than adult bovine serum as a c e l l culture supplement. Again, i t would be of interest to pursue this technique of microvascular endothelial isolation in neonatal peripheral lung of other species such as calf or lamb. Rat lungs are deemed to be too small for the peripheral dissection necessary to eliminate contamination by large vessel endothelium. Rat pulmonary endothelium also does not grow readily in culture (134). It would be of great morphological interest to determine i f microvascular endothelium in culture w i l l form monolayers. From our observations they appear to have a propensity to grow in regularly spaced open networks reminiscent of the microvasculature in vivo. It may well be that the contact inhibition of -91- large vessel endothelium is exaggerated in the microvasculature, causing them to branch out in new directions before central confluence is reached. Comparison of Results from These Studies with Those Previously Cited in the Literature The detrimental effects of exogenous perfused xanthine oxidase plus xanthine on the pulmonary circulation are well documented (33), (60). An attempt has been made to examine the effects of allopurinol, a xanthine oxidase inhibitor on preventing the development of free radical-mediated pulmonary edema with both collapsed lung (63) (74) and embolic models. Results from these studies showed that allopurinol does have a slightly beneficial effect, however, the efficacy of the collapsed lung model in causing significant edema has been called into question, and thus only a slight effect of allopurinol would be seen i f the baseline edema was minimal. Nevertheless, relatively few studies have addressed the function of endogenous xanthine oxidase in the lung, despite the fact that i t has been known since 1935 to be present in the lung of rat and cow as detected by the urate assay (110). In this early comparative work by Booth (110), a reference is given to the enzyme being unsuccessfully assayed in the lung, but the author indicates his uncertainty as to whether the enzyme is present or not by the notation, "?", opposite that tissue in his table l i s t i n g XO activities for various species and organs. The results reported in this study using a more sensitive enzyme assay and immunolocalization indicate that i f present, the enzyme was below the level of detection in pig lung. -92- DEHYDROGENASE INACTIVE ^ - Pr o t e o l y s i s OXIDASE A (INTERMEDIATE) A _ OXIDASE EDTA Figure 26. Conversion of Rat L i v e r Xanthine Oxidase. This schema shows the interconversion of xanthine oxidase among i t s various possible forms. Broken arrows i n d i c a t e uncertain or a l t e r n a t i v e pathways (87). -93- In the present study 85-90% of the enzyme was found to be reversible to the dehydrogenase form. This is in accordance with the work of McCord and Roy (100), who found 85-90% of the enzyme to be in the dehydrogenase form when i l e a l tissue was frozen and processed immediately in liquid nitrogen to prevent proteolysis. Interestingly in the present study, freezing was not necessary to prevent proteolytic conversion in the lung. These results are also in conflict with McCord and Roy who found that 30 min of ischemia could irreversibly convert XDH to X0 (100). Our findings indicate that up to 60 min of tissue ischemia or 10 hours of incubation designed to activate endogenous proteases in the lung did not cause irreversible enzyme conversion. It was found that while the enzyme appears to be predominantly in the oxidase form after standard isolation procedures from the lung even when EDTA and EGTA are employed as calcium chelators, th i o l reduction by DTT w i l l convert i t to the dehydrogenase. In agreement with this observation is the work of Delia Corte and Stirpe (94) who noted that while the rat lung appears to be in 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 visualize XO in frozen sections examined by light microscopy and electron microscopy. These authors found that the enzyme was localized in lactating bovine epithelial cells and microvascular but not large vessel endothelium. An i n i t i a l objective of this thesis project was to assay porcine peripheral lung sections, which are known to contain only vessels less than 50 um (128) in comparison with central lung homogenates. If indeed XDH-XO is only found in microvascular endothelium, an enrichment of enzyme activity would -94- be expected in the p e r i p h e r a l lung homogenate. Due to a l a c k of XDH-XO a c t i v i t y i n porcine lung t h i s question remains unanswered. Further work by Bruder and Jarash (87) involved the use of immunoaffinity chromatography i s o l a t e d XO from various bovine t i s s u e s . SDS-PAGE of XO p u r i f i e d from bovine lung was shown to separate on gel electrophoresis as a s i n g l e band on SDS-PAGE, with molecular weight 155 KDa. Since no a n t i - p r o t e o l i c precautions were taken with t h e i r preparation, i t can be interpreted that p r o t e o l y t i c a c t i v i t y i n the lung does not act on XDH to any great extent. This work i s in agreement with our findings on conversion of XDH to XO in the r a t lung. Conversion of XDH to XO i n the Rat Lung While one of the i n i t i a l goals of t h i s project was to study XDH to XO in an e a s i l y manipulatable e n d o t h e l i a l c e l l culture system, d i f f i c u l t i e s in obtaining v i a b l e microvascular cultures in a species e x h i b i t i n g pulmonary XO made i t necessary to study enzyme conversion in rat lung e x t r a c t s . Using t r y p s i n derived from bovine pancreas on lung extracts with no EDTA i t was shown that (Figure 21) p r o t e o l y s i s of XDH w i l l indeed cause conversion from XDH to XO which cannot be reversed by incubation with t h i o l reducing agents, i n agreement with the m u l t i p l e conversion scheme of Stirpe (Figure 26). In an attempt to employ endogenous proteases in the lung extracts to e f f e c t enzyme conversion by overnight incubation at 15°C i t was found that while 100% of the enzyme was in the oxidase form af t e r 16 h, incubation of these extracts with 1 mM DTT returned most of the enzyme to i t s dehydrogenase conformation. Thus, endogenous proteases in the lung do not -95- appear to be capable of a f f e c t i n g the i r r e v e r s i b l e conversion of XDH to XO. Since low molecular weight substances had been removed from the lung extract by separation on chromatographic columns the p o s s i b i l i t y of small molecular weight p r o t e c t i v e compounds are present. The lung i s known to be protected by a - l - a n t i t r y p s i n (63), which has i t s e l f been shown to be degraded by oxygen f r e e - r a d i c a l s . Soybean t r y p s i n i n h i b i t o r has also been used as a co n t r o l f o r the p r o t e o l y t i c a c t i v i t y of t r y p s i n on r a t l i v e r XO (93), leading to the hypothesis that a - l - a n t i t r y p s i n may serve more than one pro t e c t i v e function i n the lung. Studies with the use of d i f f e r e n t calcium chelators on enzyme conversion in both normoxic and ischemic lung t i s s u e indicated that neither c h e l a t i o n of d i v a l e n t cations with 1 mM 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 preventing conversion of XDH to XO. This conversion was however r e v e r s i b l e with the t h i o l reducing agent DTT and thus p r o t e o l y s i s of the enzyme by calcium-mediated proteases (100) did not account f o r i r r e v e r s i b l e conversion of the enzyme. I t i s possible that the calcium e f f e c t i s not an important f a c t o r i n c o n t r o l of XDH/XO conversions i n lung t i s s u e i f the lung i s indeed protected against p r o t e o l y s i s as was suggested e a r l i e r . The length of time which was required f o r t i s s u e to be excised, weighed and homogenized before assay was of some concern since presumably the rats would by ischemic thus allowing the conversion process to proceed from the time the diaphragm was opened. A series of experiments were were conducted which employed rapid cannulation of the pulmonary artery and perfusion of the lung vasculature with a homogenizing buffer containing 1 mM EDTA and 1 -96- mM DTT was used in order to investigate whether having calcium chelators and thio l reducing agents perfused into the lung as rapidly as possible might result in some slight difference in irreversible and reversible conversion. When compared to perfused lung without protective agents this was found to be true. Levels of XDH in lung perfused with DTT and EDTA were significantly higher than XDH levels in lungs which had no DTT and EDTA present from the onset of ischemia. When compared with results from previous experiments using calcium chelators, i t is possible to speculate that there is a small i n i t i a l pool of XDH which w i l l convert very rapidly and irreversibly to XO in 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 five minutes. This i s , however, a small fraction of total enzyme activity. XDH which is not irreversibly acted on during the i n i t i a l period of ischemia to be remarkably resistant to proteolytic conversion even up to 10 hours later, as shown by incubation of lung extracts at 15°C overnight. Thus oxidation of thi o l groups appears to be a more important mechanism in the lung (93) (96). No success was achieved upon attempts to separate the effects of DTT and EDTA in the perfusate to determine which has the greater effect on maintaining XDH both conformationally and structurally. Further studies are necessary on both of these factors during the i n i t i a l period of ischemia in order to draw any firm conclusions as to whether thiol redox status or Ca + +-mediated proteolysis is more important for maintaining the enzyme in the dehydrogenase form (XDH). -97- Consequences of Conversion of XDH to XO for the Organism Regardless of whether XDH is permanently or reversibly converted to XO, XO is capable of producing superoxide free radicals. Indeed, while the bulk of XDH in the lung appears to be subject only to reversible conversion to XO, this conversion appears to take place very easily and rapidly. If microemboli cause a focal area of ischemia within the lung one 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 circulation in the pulmonary microvasculature. What the role of this rapid conversion might be remains unclear. Jarasch, Bruder and Heid have speculated that XDH may convert rapidly to XO when the endothelium is subject to bacterial attack causing endothelial injury (Figure 27) (132). Xanthine oxidase may thus form a f i r s t line of oxidant defense 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 radical chemoattractants (71). This group supports this hypothesis with the interesting observation that while XO was virtually 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. Up to 7% of a l l IgG specificity was directed against XO in one study of human samples (133). It is f e l t that these autoantibodies serve to protect the organism from massive oxygen free radical damage, precipitating the enzyme as soon as i t is released from the c e l l . However, since one of the most important by-products of the generation of superoxide, H 0-, is freely diffusable - 98 - Bacter io l Attack Caus ing Endothel ial C e l l Injury Oxidat ive Figure 27. Hypothetical scheme of a role of xanthine oxidase in defence mechanism of capillary endothelial cells against bacterial attack. As per Jarash, Cruder and Heid (122). -99- across c e l l u l a r membranes i t seems unnecessary for e n d o t h e l i a l l y s i s to occur f o r oxygen free r a d i c a l s to be released into the c i r c u l a t i o n . Oxygen free r a d i c a l s generated by activated neutrophils have been shown to lyse e n d o t h e l i a l c e l l s (28), leading to the question of whether i t i s XO or a c t i v a t e d neutrophils which act as the proverbial chicken or egg. Recent commentary on the r o l e of oxygen free r a d i c a l s in causing protein oxidation which f a c i l i t a t e s normal p r o t e o l y t i c breakdown and protein turnover (14, 17) gives r i s e to another i n t e r e s t i n g approach. I t i s possible that f o c a l patches of ischemia in the lung due to clearance of microthrombi might cause rapid conversion of XDH to XO, with subsequent generation of oxygen free r a d i c a l s causing modifications i n c l o t t i n g proteins, thus i n i t i a t i n g the f i r s t stage in f i b r i n o l y s i s . Whether t h i s i s a r e a l i s t i c hypothesis and how the r e v e r s i b l y converted enzyme would return to i t s reduced state remains to be seen. Another area yet to be explored i s whether oxygen free r a d i c a l s and proteases generated from activated neutrophils could cause conversion of XDH to XO, e i t h e r r e v e r s i b l y or i r r e v e r s i b l y . While some form of hypofusion appears to be required along with the presence of neutrophils in the e t i o l o g y of ARDS, i t remains unclear whether i t is indeed e n d o t h e l i a l changes which activate neutrophils or vice versa. Many studies have been published showing the p r o t e c t i v e e f f e c t s of i n h i b i t i o n of XO by a l l o p u r i n o l in various ischemic and hypoxic lung models. The i n t e r a c t i o n between neutrophils and the endothelium are c e r t a i n to be of continuing i n t e r e s t to free r a d i c a l b i o l o g i s t s and c l i n i c i a n s a l i k e . 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