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' wq; «r 7' u u: Haul/mumWWW 7‘. b g m as 00550 2772 “BRA“ Y Michigan State University This is to certify that the dissertation entitled The Role of the Platelet and Platelet Mediators in the Pulmonary Hypertensive Response to Monocrotaline Pyrrole presented by Patricia Elaine Ganey has been accepted towards fulfillment of the requirements for Ph.D . degree in Pharmacology and Toxicology '1 Mr A _. 729;) / Major professor Date (7,} Cl. I] 3Q Msu is an Affirmative Action/Equal Opportuniryilnuitution 0-12771 remove this checkout from your record. FINES will ——f be charged if book is returned after the date stamped below. MSU ‘ 3151mm MATERIALS: Place in book drop to LIBRARIES ”- THE ROLE OF THE PLATELET AND PLATELET MEDIATORS IN THE PULMONARY HYPERTENSIVE RESPONSE TO MONOCROTALINE PYRROLE BY Patricia Elaine Ganey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1986 ©1987 PATRICIA ELAINE GANEY All Rights Reserved ABSTRACT The Role of the Platelet and Platelet Mediators in the Pulmonary Hypertensive Response to Monocrotaline Pyrrole by Patricia Elaine Ganey Monocrotaline pyrrole (MCTP) is a metabolite of the plant toxin monocrota- line. Administration of MCTP to rats produces pulmonary vascular injury, pulmonary hypertension (PH), and right ventricular enlargement (RVE). The mechanism by which MCTP causes PH is unknown, however platelets have been implicated in the response. The possibility that the platelet was contributing to MCTP-induced PH by releasing vasoactive mediators, specifically 5-hydroxytryp- tamine (SHT) and thromboxane A2 (TxAZ), two mediators which cause vasocon- striction in the lung, was examined. To determine whether platelet depletion attenuated the development of PH, MCTP-treated rats were co-treated with an anti-platelet serum (PAS) to reduce the circulating platelet count to 10-25% of normal. Treatment with a single dose of MCTP on day 0 produced lung injury, PH, and RVE by day 14. Rats co-treated with PAS so that they were moderately thrombocytopenic from days 6-8 did not develop PH or RVE. Indices of lung injury were not affected by co-treatment with PAS. To examine whether SHT was involved in the response to MCTP, the effect of MCTP on platelet SHT content and the effect of co-treatment with a SHT receptor antagonist on MCTP-induced toxicity was determined. The platelet content of SHT was not affected by MCTP treatment. A dose of the SH'I‘2 Patricia Elaine Ganey receptor antagonist ketanserin which inhibited the SHT—induced shape change in platelets and the SHT-induced vasoconstriction in isolated, perfused lungs, did not attenuate the lung injury or RVE caused by MCTP. These results suggest that SHT is not the sole contributor to PH due to MCTP. To investigate a possible role for the arachidonic acid (AA) metabolite TxA2 in MCTP-induced PH, release of TxAZ was determined in isolated, perfused lungs and in platelets from treated rats. The effect of co-treatment with inhibitors of TXAZ synthesis or activity on MCTP-induced PH was also examined. Treatment with MCTP was associated with an increased release of the TxAz metabolite, Tsz, from isolated lungs perfused with buffer or blood. The increase in release was greater when ltmgs were perfused with blood, suggesting a blood element as a major source of Tsz. Release of a stable metabolite of prostacyclin, an AA metabolite with activities that oppose those of TxAZ, from isolated lungs was not affected by treatment with MCTP _'_u_1 3319.- Generation of Tsz in platelet-rich plasma in response to aggregation by AA was not different for rats treated with MCTP and controls. Co-treatment with either a cyclooxygenase inhibitor (ibuprofen), a thromboxane synthetase inhibitor (Dazmegrel), or a thromboxane receptor antagonist (L-640,035) did not attenuate the development of lung injury, PH, or RVE. Thus, TxAz does not appear to be the sole contributor to MCTP- induced PH. These results indicate that modest depletion of platelets prevents MCTP- induced PH and RVE. Neither of the two platelet-derived mediators examined, SHT or TxAz, appear to be necessary for the development of lung injury, PH, or RVE due to MCTP. 19 [1' ACKNOWLEDGEMENTS I would like to express my sincerest thanks to my major advisor and friend, Dr. Bob Roth, for ideas and encouragement, for insisting on meticulous research, for always believing in me, and for never letting me forget that all of this was fun. I thank the rest of my Guidance Committee, Drs. Theodore M. Brody, Gregory D. Fink, and James W. Aiken, for their time, attention, and helpful advice. My thanks go to Drs. Ron Slocombe and James Reindel for numerous hours and endless patience in assistance with pathology studies, and to Dr. Tom Bell for his expert advice on platelets. I would also like to thank Dr. Scott Walsh for his assistance in setting up the RIA. I feel fortunate to have spent this time among colleagues who have been supportive and encouraging. My thanks go to Katie Sprugel, Leon Bruner, Laurie Carpenter, Lonnie Dahm, and Jim Hewitt for making this experience more enjoyable. I would also like to thank Lynn Georgic, Duane Kreil, Jeff Fagan, and Jim Wagner for their expert technical assistance, careful attention to detail, and just as importantly, for their enthusiasm for the project. I would also like to express thanks to Kaelyn Boner and Nancy Shannon for their contributions to this project. Special thanks go to Diane Hummel, not only for her excellent performance in typing this dissertation, but also for her help over the past four years. Some of the drugs used for this research were supplied as gifts. I would like to thank Janssen Pharmaceutica (Beerse, Belgium) for the gift of ketanserin, Mr. ii Peter Chelune of The Upjohn Company (Kalamazoo, M1) for supplying ibuprofen and U46619, Pfizer Central Research (Sandwich, Kent, England) for the gift of Dazmegrel, and Dr. Ford-Hutchinson of Merck-Frosst (Canada) for supplying L- 640,035. I would also like to acknowledge and thank those who provided financial support for my graduate work. My stipend and travel allowance came from NRSA Training Grant GM07392 and NIH Training Grant HLO7404, and in part from the Hazleton Laboratories Graduate Fellowship Award administered by the Society of Toxicology. My research was supported by US Public Health Service grant ESOZSSI. Finally, my deepest appreciation goes to all of my family, especially to my Mom and Dad, and to Robert, for their support and encouragement, and for their expression of pride in my achievements. And, to Rick, thanks for smiling through it all, and for constantly reminding me of all of the other important things in life. iii TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS INTRODUCTION 1. Pyrrolizidine Alkaloids A. General B. Toxicity to humans C. Toxicity to animals II. Monocrotaline A. General B. Path0physiology 1. Species affected 2. Pharmacokinetics 3. Hepatic toxicity 4. Renal toxicity 5. Cardiac effects 6. Pulmonary toxicity a. Gross changes b. Microscopic changes 1) Changes in endothelial cells 2) Changes in pulmonary vessels 3) Parenchymal changes 4) Summary c. Hemodynamic changes (1. Changes in vascular smooth muscle responsiveness e. Changes in endothelial cell function f. Biochemical changes 1) Lavage fluid protein concentration and lac- tate dehydrogenase activity 2) Polyamines iv xiii xvi WHH gs ~o~o ~o~zo~a~mm dirk TABLE OF CONTENTS (continued) E. g. Changes in respiratory mechanics Metabolism and bioactivation 1. General 2. Monocrotaline pyrrole a. Similarities in pathophysiologic effects of MCT and MCTP b. Differences in pathophysiologic effects of MCT and MCTP c. Bioactivation of MCTP? d. DevelOpment of toxicity Interest in MCT/MCTP toxicity 1. As an environmental toxicant 2. As a pneumotoxic metabolite produced in the liver 3. As a model of human pulmonary vascular disease a. Primary pulmonary hypertension b. Adult respiratory distress syndrome Problems associated with studying this model Mechanisms of Action of MCT/MCTP A. riparian: H.310 J. General The role of angiotensin converting enzyme The role of collagen synthesis The role of immune effectors The role of phagocytic cells The role of reactive oxygen The role of leukotrienes The role of polyamines The role of blood platelets 1. Evidence that platelets may be involved 2. Mechanisms by which platelets may be involved 3. Platelet mediators that may be involved a. 5-Hydroxytryptamine b. Arachidonic acid metabolites c. Platelet-derived growth factor d. Platelet activating factor Summary Specific Aims 22 25 25 26 26 26 26 26 27 28 28 30 30 31 32 33 33 34 35 38 39 39 40 41 41 42 43 43 TABLE OF CONTENTS (continued) MATERIALS AND METHODS I. E. III. ES Animals Diet Restriction Monocrotaline pyrrole (MCTP) A. Synthesis of MCTP B. Treatment with MCTP Assessment of Cardiopulmonary Injury A. Bronchoalveolar lavage B. Pulmonary sequestration of radiolabelled protein as a marker of pulmonary vascular leak C. Lung weight D. Pulmonary artery pressure E. Right ventricular enlargement F. Other indices of injury HistOpathology A. Fixation procedure B. Tissue processing and staining procedure Preparation of Goat Anti-rat Platelet Antibody A. Preparation of pre-immune (control) serum (CS) B. Preparation of antigen: Rat platelet membrane C. Preparation of goat anti-rat platelet serum (PAS) D. Absorption of sera with red blood cells E. Efficacy of PAS '2 m Cell Counting Plate let aggregation A. Blood collection B. Preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) C. Platelet aggregation D. Determination of platelet 5-hydroxytryptamine (SHT) E. Determination of platelet-derived thromboxane B (TxB ) F. Effect of MCTP i_n_ vitro on platelet aggregation and genera- tion of Tsz Isolated, Perfused Lungs A. Surgery B. General perfusion procedure vi 45 45 46 46 46 46 46 47 48 48 48 49 49 49 49 50 50 50 51 51 52 52 53 53 53 53 57 58 58 58 58 TABLE OF CONTENTS (continued) X. Effect of Ketanserin on the Vascular Response to SHT in the Isolated, Perfused Lung XL Prostanoid Release in Buffer-perfused Lungs A. Day 7 after MCTP treatment B. Day 14 after MCTP treatment C. Preparation of arachidonic acid for infusion XII. Prostanoid Release in Blooddperfused Lungs XIII. Determination of Prostanoids by Radioimmunoassay (RIA) A. General procedure B. Preparation of charcoal-stripped plasma C. Extraction of prostanoids XIV. Drug Treatments A. Ketanserin B. Ibuprofen C. Dazmegrel D. L-640,035 XV. Statistical Analysis RESULTS L Dose/Response Relation for MCTP II. Hist0pathology (DevelOpment of MCTP-induced Changes) III. Diet Restriction and MCTP-induced Cardiopulmonary Toxicity A. Effect of diet restriction on MCTP-induced cardiopulmonary toxicity B. Effect of diet restriction on survival of MCTP-treated rats IV. Effect of Thrombocytopenia on MCTP-induced Pulmonary Hyper- tension A. Antiserum characterization B. Effect of severe thrombocytopenia l. Rats made severely thrombocytopenic from days 6-8 a. Effect on MCTP toxicity at day 8 b. Effect on MCTP toxicity at day 14 2. Rats made thrombocytopenic from days 8-10 or days 10-12 vii 60 61 61 62 63 63 64 64 67 67 68 68 68 69 69 69 71 71 74 79 80 85 85 89 96 96 97 97 101 TABLE OF CONTENTS (continued) C. Effect of moderate thrombocytopenia 1. Effect on pulmonary hypertension 2. Platelet rebound Examination of the Role of SHT in MCTP-induced Cardiopulmonary Toxicity A. Determination of platelet SHT B. Effect of co-treatment with ketanserin 1. Confirmation of drug effect a. SHT-induced platelet shape change b. SHT-induced vascular response in isolated, per- fused lungs 2. Effect of ketanserin on MCTP-induced cardiopulmonary toxicity Examination of the Role of Thromboxane in MCTP-induced Cardio- pulmonary Toxicity A. Prostanoid release in isolated, perfused lungs 1. Release in lungs perfused with buffer . a. Day 7 after MCTP treatment b. Arachidonic acid concentration/response relation c. Day 14 after MCTP treatment 2. Release in lungs perfused with blood a. Day 7 after MCTP treatment b. Day 14 after MCTP treatment 9" Generation of TxB in platelet-rich plasma C. Effect on MCTP-mduced pneumotoxicity of drugs which interfere with the synthesis or action of TxAZ 1. Ibuprofen a. Confirmation of drug effect b. Effect of ibuprofen on MCTP—induced cardiopul- monary toxicity 2. Dazmegrel a. Confirmation of drug effect b. Effect of Dazmegrel on MCTP-induced cardiopul- monary toxicity viii 121 121 124 124 124 129 129 135 136 136 136 137 142 151 151 155 160 168 170 170 172 172 174 174 TABLE OF CONTENTS (continued) Page a. Confirmation of drug effect 183 b. Effect of L-640,035 on MCTP-induced cardiopul- monary toxicity 183 DISCUSSION 19 1 I. The Dose/Response Relation for MCTP 191 II. The Development of MCTP-induced Toxicity 191 III. Influence of Diet Restriction on MCTP-induced Cardiopulmonary Toxicity 197 IV. The Effect of Thrombocytopenia on MCTP-induced Pulmonary Hypertension 200 V. The Role of Platelet Mediators in MCTP-induced Cardiopulmonary Toxicity 204 A. 5-Hydroxytryptamine 204 B. Thromboxane A2 208 SUMMARY AND CONCLUSIONS 220 BBLIOGRAPHY 222 ix Bears. 10 ll 12 13 14 LIST OF FIGURES Structures of MCT and MCTP Pathways of arachidonic acid metabolism Typical platelet aggregation curve Diagram of isolated, perfused lung preparation Dose/response relation for MCTP Effect of diet restriction on body weight of MCTP-treated rats Effect of diet restriction on MCTP-induced cardiopulmonary toxicity in rats ‘ Effect of diet restriction on the number of MCTP-treated rats surviving Platelet number in the blood of rats treated with CS or PAS Hematocrit of the blood of rats treated with CS or PAS Effect of severe thrombocytopenia on mean pulmonary artery and right ventricular pressure 8 days after treatment with MCTP Effect of severe thrombocytopenia on mean pulmonary arter and right ventricular pressure 14 days after treatment with MCTP Right ventricular enlargement at day 14 in MCTP-treated rats co-treated with PAS Effect of moderate thrombocytopenia on right ventricular enlargement in MCTP-treated rats 55 59 73 81 83 86 92 94 99 103 105 112 LIST OF FIGURES (Continued) Figge 1 5 l6 17 18 19 20 21 22 23 24 25 26 27 28 Effect of moderate thrombocytopenia on pulmonary artery and right ventricular pressure in MCTP-treated rats Effect of treatment with PAS on platelet number in MCTP- treated and DMF rats 5HT in platelets and PPP from rats treated 14 days earlier with MCTP or DMF 5HT-induced shape change in PRP Effect of ketanserin treatment on 5HT-induced shape change in PRP Effect of co-treatment with ketanserin on the response to 5HT in isolated, perfused lungs from MCTP-treated rats Effect of ketanserin on lung weight, vascular leak, and right ventricular enlargement in MCTP-treated rats Release of 6-Keto PGF and TxB into-effluent perfusion medium from isolated lungs of rats treated 7 days earlier with MCTP Release of 6-Keto PGF from isolated, perfused lungs in response to several concentrations of arachidonic acid Release of 6-Keto PGF and TxB into the effluent of lungs isolated from MCTP-treated rats 14 days after treatment Release of 6-Keto PGF1 a and Tsz in isolated lungs made edematous 6-Keto PGF and TxB in the plasma effluent of isolated lungs from MCTP-treateh rats 7 days after treatment 6-Keto PGF and TxB in the plasma effluent of isolated lungs from MCTP-treatea rats 14 days after treatment Extraction of TxB by isolated ltmgs from rats treated 14 days earlier with M2CTP or DMF xi 119 125 127 130 131 133 139 143 146 150 153 158 162 LIST OF FIGURES (Continued) liars 29 30 31 32 33 TxBZ generated in PRP of MCTP-treated rats The concentration of TxB in PRP and PPP and the concen- tration of 6-Keto PGF in PPP of rats treated with Dazmegrel It! Right ventricular enlargement in MCTP-treated rats follow- ing co-treatment with Dazmegrel Effect of treatment with L-640,035 on the dose/response relationship to U46619 Pulmonary arterial pressure in MCTP-treated rats following co-treatment with L-640,035 xii 175 181 184 189 Table 10 11 12 13 LIST OF TABLES Cross reactivities (at 50% B/Bo) of specific antisera used in radiommunoassays Effect of treatment with various doses of MCTP on body weight gain Development of toxicity due to MCTP Histopathologic changes following treatment with MCTP Liver weight, kidney weight, blood urea nitrogen (BUN) and serum glutamic oxalacetic transaminase (SGOT) activity in diet-restricted, MCTP-treated rats Lavage LDH activity and right ventricular enlargement in rats surviving 41 days after MCTP treatment Effect of treatment with PAS on body weight White blood cell count in the blood of rats treated with CS or PAS Effect of severe thrombocytopenia (Days 6-8) on toxicity 8 days after treatment with MCTP Effect of severe thrombocytopenia (Days 6-8) on toxicity 14 days after treatment with MCTP Effect of severe thrombocytopenia (Days 8-10) on the toxicity of MCTP Effect of severe thrombocytopenia (Days 10-12) on the toxicity of MCTP Effect of moderate thrombocytopenia on MCTP-induced toxicity xiii Page 65 72 75 76 82 88 91 95 98 101 107 108 110 LIST OF TABLES (Continued) Table 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Effect of thrombocytopenia on body weight, lung injury, and right ventricular enlargement in MCTP-treated rats Platelet number in MCTP-treated rats receiving PAS MCTP toxicity in rats used to determine platelet 5HT content Effect of MCTP treatment on platelet number and platelet protein concentration in PRP Effect of ketanserin on body weight of MCTP-treated rats MCTP toxicity at Day 7 in rats used in isolated, buffer- perfused lung studies Inflow perfusion pressure in isolated lungs from rats treated 7 days earlier with MCTP MCTP toxicity at day 14 in rats used in isolated, buffer- perfused lung studies Inflow perfusion pressure in lungs isolated from rats treated 14 days earlier with MCTP MCTP toxicity at day 7 in rats used in isolated, blood- perfused lung studies Perfusate platelet number and inflow perfusion pressure in isolated lungs from rats treated 7 days earlier with MCTP MCTP toxicity at day 14 in rats used in isolated, blood- perfused lung studies Perfusate platelet number and inflow perfusion pressure in isolated lungs from rats treated 14 days earlier with MCTP Effect of MCTP on body weight, lung weight, lavage fluid protein concentration and right ventricular enlargement Effect of MCTP treatment i_n v_i__vo on arachidonic acid- induced aggregation in platelet-rich plasma xiv 117 118 122 123 132 138 141 145 149 152 156 157 161 165 166 LIST OF TABLES (Continued) Table 29 30 31 32 33 34 35 36 Effect of MCTP :13 vitro on arachidonic acid-induced plate- let aggregation and release of Tsz Effect of treatment with ibuprofen i_n vivo on platelet aggregation i_n vitro Lack of effect of ibuprofen on the cardiopulmonary toxicity of MCTP Plasma TxB and 6-Keto PGF 0. in MCTP-treated rats following co-ztreatment with Dazmegrel Lack of effect of Dazmegrel on the toxicity of MCTP 7 days after treatment Lack of effect of Dazmegrel on the toxicity of MCTP 14 days after treatment Right ventricular pressure response to U46619 in MCTP- treated rats following co-treatment with L-640,035 Lack of effect of co-treatment with L-640,035 on MCTP- induced toxicity XV 171 173 177 178 180 186 187 AA ACE BUN CS DMF EC 5HT IBN KET LDH LT MCT MCTP PAF PAP PAS PDGF PG PGIZ PPP PRP RVE SGOT Tx LIST OF ABBREVIATIONS arachidonic acid angiotensin converting enzyme angiotensin II blood urea nitrogen control, or pre-immune, serum N,N-dimethylformamide endothelial cell 5-hydroxytryptamine ibuprofen ketanserin lactate dehydrogenase leukotriene monocrotaline monocrotaline pyrrole platelet-activating factor pulmonary arterial pressure anti-platelet serum platelet-derived growth factor prostaglandin prostacyclin platelet-poor plasma platelet-rich plasma pyrrolizidine alkaloid radioimmunoassay right ventricular enlargement serum glutamic oxalacetic transaminase thromboxane INTRODUCTION L Ifirrolizidine Alkaloids A. General Monocrotaline (MCT) is a member of a class of compounds referred to as the pyrrolizidine alkaloids (PZAs). The common chemical structure for this class of compounds is the pyrrolizidine ring, consisting of two fused S-membered rings with a nitrogen atom at the center (Figure l) (McLean, 1970). Many of the PZAs are toxic, and an unsaturated pyrrolizidine ring structure is an essential requirement for toxicity (Huxtable, 1979). To date, more than 150 PZAs have been identified and isolated, and the structures have been determined (Huxtable, 1979). The plants that contain PZAs comprise 8 families and numerous genera that are widely distributed geographically (Bull _e_t_ £11., 1968; Huxtable, 1979). Some of the more common PZA-containing genera include Crotalaria (from which MCT is extracted), Senecio, and HeliotroPium. Toxicity due to exposure to some PZAs is responsible for significant loss of livestock in the United States (Snyder, 1972) and other areas of the world, and for loss of human life as well. The occurrence and toxicity of PZAs has been reviewed (Bull g g” 1968; McLean, 1970; Huxtable, 1979). B. Toxicity to Humans Because symptoms of toxicity are often delayed following exposure and because detection of low concentrations of PZAs or their metabolites is mnOmm>a mZ_._<._.O~_UOZO<< .952 an... .52 no 85885 A 2&5 m2_._<.—O~_UOZO<< Z difficult, diagnosis of PZA poisoning is based on circumstantial evidence. Still, it h well established that humans exposed to PZAs develop veno—occlusive disease of the liver. Symptoms of this hepatic vascular disease include massive centrilobular congestion and necrosis, collagenous occlusion of small branches of the hepatic venous tree, development of collateral venous channels over the abdomen, and dilated sinusoids (Hill e_t 11., 1951). Many PZA-intoxicated people die. Exposure can occur by a number of mechanisms. Veno-occlusive disease, endemic in Jamaica, is thought to arise from the use of "bush" teas prepared from plants, some of which contain PZAs (Hill gt _at., 1951; Bras gt g}, 1954; Stuart and Bras, 1957). Grains have become contaminated with PZAs when PZA-containing plants grow as weeds in the same fields. Use of these grains to prepare bread results in low grade exposure and may be responsible for large outbreaks of liver disease such as was seen in 1975 in Afghanistan (Mohabbat gt 9, 1976) and India (Tandon gt a_.l_., 1976). In the United States, use of the commercial preparation of gordoloba yerba caused PZA poisoning in small children in Arizona (Stillman gt 11., 1977; Fox gt gt, 1978). In addition, contamination of honey used for human consumption (Dienzer gt 9, 1977) or of the milk of cows which graze on the plants (Dickinson gt 511., 1976) can result in human exposure to PZAs. C. Toxicity to Animals PZA poisoning in animals has been recognized for quite some time. As long ago as 1787 British farmers suspected PZA-containing plants were harmful to livestock (Bull gt g, 1968). The PZAs are toxic to a wide variety of species, and the symptoms associated with PZA toxicity have been given many different descriptive names. Chronic liver disease commonly occurs in pigs, dogs, and cattle, although the gastrointestinal system of cattle is also affected. Horses develop mainly a neurological syndrome, sheep exhibit hemolytic disease (Hux- table, 1979), and chickens and turkeys develop lesions of the liver and lungs (Allen gt gL, 1960, 1963). PZAs are also carcinogenic and mutagenic in animals (Cook gt gl_., 1950; Schoental gt gl_., 1954). PZA poisoning represents a great economic loss, costing millions of dollars annually in the United States alone (Snyder, 1972). As with humans, the diagnosis of PZA poisoning in grazing animals is largely based on circumstantial evidence, but the toxicity can be reproduced in laboratory animals given PZAs (Huxtable, 1979). The PZA which has attracted the most attention and which has been most extensively studied is MCT, and the remainder of this dissertation will be confined to a discussion of MCT. II. Monocrotaline A. General The PZA monocrotaline (MCT) is found in the seeds and leaves of plants of the genus Crotalaria (Heath, 1969), and in the United States, largely in the seeds of Crotalaria spectabilis (IARC). Q. sgctabilis grows in other areas of the world, but was introduced in the United States in 1921 by the Florida Agricultural Experimental Station as a leguminous cover crop. Q. spectabilis now grows wild in many southern states (Kay and Heath, 1969). MCT is the monocrotalic acid ester of retronecine (Adams and Rodgers, 1939). Its structure, illustrated in Figure 1, has been identified using nuclear magnetic resonance and infrared and mass spectrometry (Culvenor and DalBon, 1964; Bull gt _a_l., 1968). It is a colorless, crystalline powder with a melting point of 202-203°C. B. Pathophysiolgy Numerous studies have been conducted examining the pathophysiology of PZAs or MCT. Various methods, as well as routes, of administration have been employed, making comparison of these studies difficult. For example, many investigators have ground the seeds of _(_3_. spectabilis and have fed these to animals in the diet. Others have dissolved MCT in the drinking water. Both of these methods of chronic administration produce similar effects, but there may be subtle differences in the time course of those effects. Also, results from studies using these methods, in which the actual dose of MCT each animal received is merely estimated, are difficult to compare with results obtained from studies in which animals received a single administration of a known amount of MCT. The discussion below will concentrate on those studies in which MCT was admini- stered, although studies using C. spectabilis seeds will be discussed when they add new information or clarify an issue. The effects of MCT on organs other than the lung will be briefly described, while pulmonary effects will be described in detail. 1. Species affected MCT produces toxic effects in a wide variety of experimental animals including rats (Roth, 1981; Molteni gt gt” 1985), rabbits (Gardiner gt a_l., 1965), mice (Miranda gt _a_l., 1981), dogs (Miller _e_t_ _at., 1981), and non-human primates (Allen gt gt” 1965; Raczniak e_t _a_l_., 1978). Guinea pigs (Chesney and Allen, 1973b), gerbils, and hamsters (Cheeke and Pierson-Goeger, 1983) are not susceptible to MCT toxicity. MCT intoxication has been reported in other species such as cattle (Becker gt gl_., 1935), horses (Rose gt a_l., 1957), hogs (Emmel gt gt, 1935), poultry (Thomas, 1934; Allen gt _a_l., 1960, 1963; Simpson gt 9, 1963), and goats (Dickinson, 1980). Human intoxication by MCT has also been reported (Kasturi gt 9.1., 1979). 2. Pharmacokinetics Detailed pharmacokinetic analysis has not been performed for MCT because radiolabeled MCT with high specific activity is not available. Hayashi (1966) reported that 50’70% 0f 3H-MCT is recovered unchanged in the urine within 3 hours, and 30% is recovered in the bile as a metabolite. Pyrrole derivatives of MCT (Figure 1), assayed as Ehrlich—positive materials, are detected in tissues within minutes after administration of MCT (Mattocks and White, 1970), reach a peak at about 90 minutes, and then decrease to low concentrations by 48 hr (Allen gt gl_., 1972; Mattocks, 1972). 3. Hepatotoxicity Veno-occlusive disease of the liver, described above, has been the most frequently observed effect of PZA poisoning. Animals treated with MCT develop centrilobular focal cell necrosis, and portal hepatic veins become dilated and congested (Schoental and Head, 1955; Turner and Lalich, 1965; Merkow and Kleinerman, 1966). Enlarged parenchymal cells have also been observed (Turner and Lalich, 1965). Prothrombin time increases (Rose 3 gl_., 1945) and plasma glutamic pyruvic transaminase activity increases (Roth gt 9, 1981). Biliary excretion of indocyanine green is depressed although bile production is not altered (Roth gtgl_., 1981). 4. Renal toxicity The majority of renal changes caused by MCT occur in the glomeruli, glomerular capillaries, and afferent arterioles (Hayashi and Lalich, 1967). Glomeruli become swollen, and thrombosis of glomerular capillaries and afferent arterioles has been reported. Interlobular arteries thicken and the media hypertrOphies. In rats fed ground 9. spectabilis seeds for up to 8 months (Masugi gt g, 1965; Carstens and Allen, 1970), the most severe case of intoxication caused replacement of 75% of the glomeruli with a homogenous periodic acid Schiff-positive material, and glomerular capillaries were only occasionally dis- cernible (Carstens and Allen, 1970). Alterations in renal function have been reported in animals which received MCT in the drinking water for 4 weeks (Roth _et 31., 1981). Consistent with the histopathologic picture drawn from the above studies, blood urea nitrogen (BUN) increased, indicating impaired glomerular filtration. Necro- sis of renal tubules has been reported (Ratnoff and Mirick, 1949), and the accumulation of organic ions, an index of proximal tubular secretory functions, was altered by MCT treatment (Roth gt 9, 1981). Thus, the toxic effects of MCT on the kidney include vascular damage, especially at the level of the glomerular capillaries and afferent arterioles, and impaired glomerular and tubular function. 5. Cardiac effects Chronic administration of _C_. spectabilis seeds in the diet (Kay gt gl_., 1967b; Hislop and Reid, 1974; Meyrick gt g1” 1980) or MCT in the drinking water (Roth _e_t_ a_l., 1981; Molteni gt gl_., 1985) produces right ventricular enlargement (RVE). This effect is also produced by a single administration of relatively low doses of MCT (Kameji gt Q” 1980; Ghodsi and Will, 1981; Hilliker e_t _a_l., 1982; Gillespie e_t a_l., 1985). RVE follows the development of pulmonary hypertension and is believed to be a compensatory response to the increased pulmonary vascular resistance. It has also been reported that when animals previously fed 2. spgctabilis seeds are allowed to eat unadulterated feed, they recover from pulmonary hypertension, and RVE is no longer evident (Hislop and Reid, 1974). The heart has not received much attention in histopathological studies of MCT toxicity. One detailed investigation demonstrated no alterations in the left ventricle, but the number and size of mitochondria in the right ventricular myocardium increased 14 days after treatment with MCT (Kajihara, 1970). Irregularities of the right ventricle progressed such that by 25 days after treatment a majority of the right ventricular muscle cells were hypertrophied and contained enlarged, bizarre-shaped nuclei. Changes in myofibrils suggested that the heart was responding to a pressure overload. Golgi vesicles were electron- dense, cisternae of the endoplasmic reticulum were enlarged, and there were a larger number of free ribosomes in the cytoplasm, indicative of increased RNA synthesis. Others have demonstrated that protein and collagen content increase in the right but not left ventricles of MCT-treated rats (Lafranconi gt gt, 1984), and that protein synthesis increases in parallel with right ventricular weight (Huxtable g a_l., 1977). The DNA/RNA ratio decreased in the right ventricles of MCT-treated rats, largely due to an increase in RNA content (Lafranconi g gt, 1984). Although an increase in RNA would suggest a hypertrophic response, DNA synthesis was not measured in this study so that a hyperplastic response could not be ruled out. All of these changes suggested that the right ventricle was undergoing increased energy production to compensate for the excess work it had to perform. However, most myofilaments were normally arranged, and the mean diameter of thick filaments was not different from that of controls, as might have been the case if these rats were experiencing heart failure (Kajihara, 1970). Werchan and coworkers (1986) agree that right ventricular function is not impaired in MCT-treated rats. Right ventricular systolic pressure and the maximum rise and fall in pressure development (+dp/dt), measured using an isolated Langendorff preparation, were elevated at several preloads in hearts from MCT-treated rats relative to controls. In summary, RVE develops in response to MCT-induced pulmo- nary hypertension. There appears to be a hypertrophic element to the enlarge- ment although protein and collagen content increase as well. Right ventricular function does not seem to be impaired by MCT treatment. 6. Pulmonary toxicity The pathophysiology of MCT intoxication is progressive. Whether administered chronically or as a single injection, lesions and alterations observed worsen with time. An effort will be made to address this issue in the discussion of pulmonary pathology. It should also be mentioned that lesions observed microscopically are generally multifocal in nature rather than diffuse, and the severity of lesions varies within a lung (Turner and Lalich, 1965; Merkow and Kleinerman, 1966; Valdivia gt gt, 1967). a. Gross changes Following treatment with doses of MCT which produce pulmonary toxicity, rats become dyspnic. Their fur takes on a ruffled appearance and they become listless. Food consumption as well as the rate of body growth decreases. Severe respiratory distress and cyanosis after about 2 or more weeks from the onset of treatment generally indicate impending death of the animal. Grossly, regions of the lung are dark brown and atelectic, and the lungs are heavy and fluid-filled (Schoental and Head, 1955; Turner and Lalich, 1965; Merkow and Kleinerman, 1966; Gillis gtgt, 1981). b. Microscopic changes 1) Changes in endothelial cells. The earliest changes noted in the lung following treament of rats with MCT are in endothelial cells (ECs), suggesting that this cell is a target of MCT toxicity. In one report, cytoplasmic alterations were seen within 24 hours after a single injection and progressed throughout the course of the disease (Valdivia gt g" 1967). At the capillary level, alterations include alternate thinning and thickening of EC cytoplasm (Valdivia gt _a_L, 1967). Later in the development of toxicity, swollen ECs, occasionally occluding the vessel lumen, are seen in capillaries (Turner and Lalich, 10 1965; Valdivia _e_t g, 1967; Molteni _e_t gt, 1984), pulmonary arterioles (Turner and Lalich, 1965), and small muscular pulmonary arteries (Turner and Lalich, 1965; Merkow and Kleinerman, 1966). ECs of pulmonary veins are not affected (Turner and Lalich, 1965). Associated with swollen ECs are acellular (Merkow and Kleinerman, 1966) or fibrin thrombi (Turner and Lalich, 1966). Injured ECs have prominent, enlarged nuclei and an increased number of cytoplasmic organelles. Similar changes in ECs are seen in MCT-treated monkeys (Chesney and Allen, 1973a) and rats fed _C_. gpectabilis seeds (Kay e_t gt, 1969; Hislop and Reid, 1974). In rats fed _C_. spectabilis seeds, Meyrick and Reid (1982) demonstrated a proliferation of ECs in the alveolar wall and in intra—acinar arteries and veins. 3H-Thymidine uptake peaked at 7 and again at 21 days after the animals had started receiving the seeds. This suggested an early and late hyperplastic response of ECs. Unfortunately, the investigators did not look earlier than day 7, which would have been of interest in light of the report of EC changes within one day after treatment with MCT (Valdivia e_t gt, 1967). In fact, given that the EC is thought to be a target of MCT toxicity, the study of very early changes in ECs has been neglected, and further investigation and more detailed description is warranted. 2) Changes in pulmonary vessels. The vascular changes that occur following MCT (treatment, including the EC changes described above, are numerous and progressive. The most consistent finding in MCT-treated rats is an increased thickness of the medial layer of pulmonary vessels (Turner and Lalich, 1965; Hayashi and Lalich, 1967; Ghodsi and Will, 1981; Kay e_t_ gt, 1982a; Hayashi gt gt, 1984; Molteni gt gt, 1984). This is observed as soon as 8 days after MCT treatment, and appears earlier and to a greater degree in smaller pulmonary blood vessels (Turner and Lalich, 1965; Hayashi and Lalich, 1967). The increased medial size has been attributed to a proliferation of circularly-oriented smooth 11 muscle within the internal and external elastic laminae (Ghodsi and Will, 1981), although smooth muscle has also been observed outside the elastic laminae (Turner and Lalich, 1965). Increased numbers of elastic laminae have been reported as well (Molteni gt gt, 1984). Smooth muscle extends to smaller, normally nonmus- cular vessels as well (Langleben and Reid, 1985). This occurs by day 21 following a single injection of MCT, although earlier times have not been investigated. In rats fed _Ct gpectabilis seeds, this extension of vascular smooth muscle is observed by day 7 in pulmonary vessels at the level of respiratory bronchioles, and later in pulmonary vessels at the level of smaller airways (Meyrick and Reid, 197 9). Meyrick and Reid (1982) reported an increased 3H- thymidine uptake in smooth muscle cells of pulmonary arteries of _Ct spectabilis- fed rats within 3 days. However, the increase was extremely small and occurred in only one of three treated rats. In MCT-treated rats, smooth muscle cells have an increased number of mitochondria, increased rough endoplasmic reticulum, and prominent Golgi, suggesting a hypertrophic response. An electron-dense, amor- phous material that is not collagen has been described surrounding the elastic laminae and existing between and around the smooth muscle cells extending into the adventitia (Merkow and Kleinerman, 1966; Heath and Smith, 1978). Some other adventitial changes have been noted, including increased collagen and proliferation of fibroblasts, but only in rats receiving MCT chronically (Merkow and Kleinerman, 1966). A diffuse, necrotizing vasculitis has been described by some investigators (Merkow and Kleinerman, 1966) but not others (Hayashi and Lalich, 1967; Kay gt _at, 1982a). This was characterized by a periodic acid Schiff- positive material within the walls and lumina, was confined to small pulmonary arteries and arterioles, and was associated with focal hemorrhage, edema and 12 congestion. No leukocyte infiltration or inflammation was noted in smaller vessels, although in larger vessels the pseudopodia of polymorphonuclear cells (PMNs) and macrophages were seen to extend through fenestrations of the internal elastic laminae (Merkow and Kleinerman, 1966). Capillary thrombosis, associated with edema, hemorrhage, and necrosis, was evident before arterial thrombosis was observed (Hayashi and Lalich, 1967). Thrombi have been reported to contain fibrin, platelets, and red blood cells (Merkow and Kleinerman, 1966; Turner and Lalich, 1967; Heath and Smith, 1978; Kay gt gt, 1982a; Hayashi gt gt, 1984). Changes in pulmonary vessels of MCT-treated rats are consistent with those seen in rats fed _C_. spectabilis seeds (Masugi gt a_l., 1965; Kay and Heath, 1966; Kay gt gt, 1967b; HisIOp and Reid, 1974; Meyrick and Reid, 1979), although a few differences bear mentioning. No changes have been reported for the pulmonary veins of MCT-treated rats, but smooth muscle cells in the pulmonary veins of rats fed seeds were swollen and were evaginating toward the EC (Heath and Smith, 1978). Another difference is that a decrease in the number of peripheral arteries has been observed qualitatively (a decrease in background haze in contrast radiography) and quantitatively (a decrease in the number of arteries relative to the number of alveoli) (Hislop and Reid, 1974; Meyrick and Reid, 1979) in rats fed 9. spectabilis seeds, whereas there was no decrease in the total number of small pulmonary blood vessels in MCT-treated rats (Kay gt gt, 1982b). Ghost arteries, thought to be remnants of obliterated vessels, have also been observed in rats fed seeds (Hilep and Reid, 1974). It has been suggested that certain fixation procedures, such as the vascular perfusion technique employed by Meyrick and Reid (1979), artifactually decrease the number of pulmonary blood vessels observed histologically (Mooi and Wagenvoort, 1983), so that this difference between MCT treatment and Q. spectabilis feeding 13 may be explained on the basis of fixation procedures. Or, these differences may be due to the difference in chronic administration of seeds in the diet and acute administration of MCT by injection. Alternatively, the differences may be due to other substances (e.g., perhaps other PZAs) in the seeds, or to variations in the develoPment of toxicity by these two methods of exposure. 3) Parenchymal changes. In one report, interstitial alveolar edema was observed as early as 4 hours after treatment with a single injection of MCT (Valdivia g gt, 1967). This progressed such that by one week after treatment perivascular edema was noted around small pulmonary vessels (Valdivia g g, 1967 ; Molteni gt gt, 1984). Hyaline membranes, mainly fibrin and cellular debris, were associated with areas of severe alveolar edema. Simultaneous with the early alveolar edema, intersti- tial cells of the alveolar wall become swollen. Between 2 days and 2 weeks these cells migrate into the alveolar spaces (Valdivia gt gt, 1967; Kay g Q” 1982a). Focal swelling of elastic membranes has been reported within 24 hours, and by 2 weeks elastic tissue is decreased and collagen bundles appear in the alveolar walls (Valdivia gt gt, 1967). Hemorrhage and fibrosis are late changes (Turner and Lalich, 1965; Merkow and Kleinerman, 1966; Kay gt gt, 1969). Proliferation of epithelial cells has also been ob- served. Type II pneumocytes become enlarged, with increased numbers of cytoplasmic organelles (Masugi gt a_l., 1965; Valdivia gt gt, 1967; Kay gt gt, 1982a). In _C_. sEctabilis-fed rats, bronchiolar epithelium extends to alveolar ducts and alveolar spaces, replacing the normal alveolar epithelium (Kay and Heath, 1966). Peribronchiolar lymphatics become dilated and con- tain protein and red blood cells (Hayashi and Lalich, 1967; Kay gt gt, 1969). As early as 4 hours after treatment, lymphocytes and mast cells are observed in the 14 swollen alveolar walls (Takeoka gt gt, 1962; Hayashi and Lalich, 1967; Valdivia gt fl” 1967; Kay gt gt, 1967a, 1969; Sugita _et a_l., 1983b). Abnormal alveolar macrophages are observed, large and foamy in appearance and containing numer- ous electron-dense granules (Kay and Heath, 1966; Sugita gt gt, 1983b). The number of abnormal macrophages increases with increasing doses of MCT (Sugita gt g” 1983b). 4) Summary. MCT causes a progressive disease in which the number and degree of changes increases with time after treatment. The EC appears to be a primary target in MCT toxicity, and is affected early in treatment. The ECs of smaller vessels are most severely affected. Changes in vascular smooth muscle lag behind alterations in ECs, generally taking more than a week to deveIOp. Some smooth muscle extension is observed, but medial thickening is the most prominent alteration. Like the EC changes, the latter effect is observed earliest and to the greatest degree in smaller vessels. Alterations in the airway include epithelial proliferation and infiltration of mast cells, lymphocytes and macrophages. How these changes relate to each other, or to the overall response to MCT, is not well imderstood. c. Hemodynamic changes Pulmonary arterial pressure (PAP) increases in animals which are fed 9. spectabilis seeds (Kay gt gt, 1967b; Meyrick e_t a_l., 1980; McNabb and Baldwin, 1984) or which receive a single injection of MCT (Chesney and Allen, 1973a; Ghodsi and Will, 1981; Olson gt gt, 1984a). The increase in PAP is protracted, and is associated with the eventual development of RVE. Pulmonary hypertension is a secondary event to EC injury. It is unclear whether the elevation in PAP can be attributed solely to the observed increase in vascular smooth muscle or whether there is a vasoconstrictive component as well. In chronic human primary pulmonary hypertension it is 15 believed that vasoconstriction precedes and is the cause of vascular remodelling (Voelkel and Reeves, 1979). However, eight days after administration of MCT, significant medial thickening of small pulmonary vessels was reported while only a small tendency toward an increase in PAP was observed (Ghodsi and Will, 1981). This is consistent with the finding of Kay and coworkers (1982a) that medial thickening preceded increases in right ventricular systolic pressure (PRVS). However, in this latter study medial thickening was first elevated on day 7, and although the increase in PRVS in MCT-treated rats did not reach statistical significance imtil day 10, the magnitude of the increase in MCT-treated rats was the same on days 7 and 10. Therefore, this particular study did not resolve whether increases in pulmonary vascular pressure coincide with or follow vascular remodelling. The results of these two studies might suggest that some vascular remodelling precedes the development of pulmonary hypertension, but do not rule out the possibility that vasoconstriction contributes to increases in PAP. In support of the latter hypothesis, Watanabe and Ogata (1976) observed increases in right ventricular pressure prior to medial thickening of vessels in MCT-treated rats. Meyrick and coworkers (1980) suggested that vasoconstric- tion was not involved in the early development of increased PAP in g. gpectabi- Et-fed rats. This conclusion was based on the assumption that, if vasoconstriction were involved, cardiac index would be depressed and pulmonary vascular resis- tance would be elevated. When these investigators first detected pulmonary hypertension at day 14, cardiac index was elevated in treated rats and pulmonary vascular resistance was not different from control rats. There may be a problem with this conclusion that vasoconstriction does not contribute to PAP based on a comparison of cardiac index and pulmonary vascular resistance, however, because by day 14 body weight was significantly reduced in treated rats. It is not certain 16 whether reduced body weight in MCT-treated rats is due to loss of body fat or to failure to grow, but this may be significant when comparing cardiac index (which is normalized to body weight) and pulmonary vascular resistance (which is normalized to cardiac index) in MCT-treated and control rats. For example, if reduction of body weight in treated rats is due to a loss of body fat, cardiac output might not be expected to decrease commensurately with body weight. The result would be an elevation in cardiac index in treated rats relative to control rats. In the same manner, pulmonary vascular resistance would be underestimated in treated rats. Therefore, to conclude that vasoconstriction is not contributing to increased PAP on the basis of these results may be a misinterpretation. Later in the development of pulmonary hypertension (21 days) due to _C_. spectabilis seeds, pulmonary vascular resistance was elevated and cardiac index was depressed (Meyrick gt gt, 1980; McNabb and Baldwin, 1984). This suggests that vasoconstriction may play a role. later in the development of pulmonary hypertension. By 21 days after the start of seed feeding, arterial and venous oxygen contents were depressed, suggesting impaired gas exchange (Mey- rick _e_t gt, 1980; McNabb and Baldwin, 1984). Blood pH and hematocrit were not altered by feeding _Ct spectabilis seeds. Platelet number was affected by MCT treatment: the number of circulating blood platelets was decreased during days 2-10 following a single injection of MCT, then was increased above baseline by day 14 (Hilliker gt gt, 1982). In summary, although EC injury has been reported within one day of treatment with MCT, PAP does not increase until day 7 or later. The relationship between changes in ECs and vascular remodelling or pulmonary hypertension, or between vascular remodelling and increases in PAP have not been completely delineated. More detailed studies of alterations between day 1 and 17 day 7 may improve our imderstanding of MCT pneumotoxicity. Examination at intervals more frequent than one week may also enhance our knowledge of mechanisms of MCT‘s toxicity. (1. Changes in vascular smooth muscle responsiveness Alterations in responsiveness to vasoactive agents have been observed in isolated vessel segments and isolated, perfused lungs from MCT- treated rats. Vasoconstriction in response to angiotensin II, norepinephrine, or KCl was elevated in segments of pulmonary arteries isolated from rats treated 4 days earlier with a single injection of MCT compared to control rats (Altiere gt gt, 1986b). By 7 days post-treatment, the response had returned to control, and by day 14 after administration of MCT, the response in vessels from treated rats was depressed compared to controls. Response to vasodilation induced by isoproterenol or acetylcholine was not altered at day 4 or 7, but was reduced in vessels isolated from MCT-treated rats at day 14 (Altiere gt gt, 1986b). By 14 (Altiere gt _a_t, 1986b) or 21 (Coflesky gt gt, 1985) days after a single injection of MCT, vessels from treated rats demonstrated greater compliance and a reduced ability to generate force. This was associated with increased medial and adventitial areas and decreased lumen size (Coflesky gt 9, 1985). At this time the resting membrane potential of small pulmonary arteries was hyperpolarized and the resting membrane potential of main pulmonary arteries was depolarized (Suzuki and Twarog, 1982). Altered responsiveness has also been observed in isolated lungs of MCT-treated rats (Gillespie gt 9, 1986). Similar to obervations in isolated pulmonary arterial segments (Altiere g _at, 1986b), the vasoconstrictive response to angiotensin H in isolated, perfused lungs was elevated in MCT-treated rats at days 4 and 7, but not at day 14. Vasoconstriction induced by alveolar hypoxia was also greater in lungs of treated rats at day 4, but was not different 18 from control at day 7 or 14. The response to KCl in isolated, perfused lungs was not altered by treatment with MCT. In summary, these results indicate that in preparations of isolated pulmonary arteries and isolated lungs, the pulmonary vasculature of MCT-treated rats is increased in responsiveness to certain vasoactive agents shortly after treatment, and hypo-responsive at later times. This might suggest that early in the development of MCT-induced pulmonary hypertension enhanced vasoconstriction to mediators present in the pulmonary circulation could cause elevations of PAP. This might also suggest that later in the deveIOpment of pulmonary hypertension, when vascular remodelling and increased vascular smooth muscle have been reported, excessive vasoconstriction is not required to maintain elevated PAP. e. Changes in endothelial cell function In addition to providing a semipermeable barrier separating blood from parenchyma, the pulmonary endothelium serves a variety of metabolic functions. The luminal surface of these cells is endowed with transport structures, enzymes, and receptors accessible to blood constituents, and these permit the ECs to perform biosynthetic and metabolic activities. EC injury due to MCT is associated with alterations in some of these functions, which will be described here briefly and in more detail in a later section. Angiotensin-converting enzyme (ACE), located in calveolae on the luminal side of pulmonary ECs (Ryan and Ryan, 1984), converts angiotensin I to angiotensin 11 (AH) and inactivates bradykinin. Molteni and coworkers (1984) reported that, when rats were given MCT in the drinking water, ACE activity in lung homogenates was increased after one week, but returned to normal and then was decreased by 6-12 weeks. Decreased ACE activity was observed whether the activity was expressed on the basis of the whole lung, wet weight, or protein 19 content (Molteni gt gt, 1985). Using a similar MCT treatment regimen, Lafranconi and Huxtable (1983) observed a decrease in lung ACE activity when normalized to protein content, however, since MCT treatment elevated protein concentration, whole lung ACE activity was not decreased. After a single injection of MCT, lung ACE activity was decreased by 7-10 days whether expressed on the basis of wet weight, protein. content (Keane g_t_gl_., 1982; Hayashi gt gt, 1984) or whole lung (Keane and Kay, 1984). Serum ACE was not altered by MCT treatment. Removal of biogenic amines, such as 5-hydroxytryptamine (5HT) and norepinephrine (NE), is another function of pulmonary endothelium. This involves carrier-mediated uptake into the ECs and subsequent inactivation by monoamine oxidases (MAO) (Roth, 1985). Treatment with MCT in the drinking water is associated with impaired removal of perfused 5HT in isolated lungs by day 14 and impaired metabolism by day 21 (Gillis e_t gt, 1978; Huxtable g gt, 1978; Roth gt gt, 1981), although MAO activity is not altered (Gillis _e_t gt, 1978). SET removal is impaired as early as 5 days after a single injection of MCT (Hilliker gt gt, 1982). It has been reported that NE removal is also depressed in isolated ltmgs from MCT-treated rats (Gillis gt a_l., 1978; Hilliker g a_l., 1984b), although Huxtable and coworkers (1978) reported that NE removal is not altered. This discrepancy may be due to the fact that Huxtable and coworkers (1978) perfused limgs at room temperature while other reports have been from isolated lungs perfused at physiological temperature. f. Biochemical changgg 1) Lame fluid protein concentration and lactate de- hydrogenase activity. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme, and its release into the airways is a nonspecific, yet sensitive indicator of cell injury. Elevation of protein concentration in the cell-free lavage fluid recovered 20 from airways is also an index of cell injury. Protein concentration and LDH activity are increased in the lavage fluid of rats treated with MCT (Roth, 1981; Roth gt _a_l., 1981). Serum LDH activity and protein are not altered. 2) Polyamines. The polyamines (spermidine and sper- mine) and their precursor putrescine are believed to have an essential role in cellular growth and proliferation (Heby, 1981). Treatment with MCT is associated with an early increase in the lung of activities of essential enzymes in the polyamine biosynthetic pathway (Olson e_t g" 1984a,b). Polyamine concentrations are also increased in the lungs of rats one week after MCT treatment. g. Chafles in regpiratory mechanics Although MCT is recognized as a pulmonary toxicant, alterations in respiratory mechanics due to MCT have been largely ignored as an area of investigation. However, Gillespie and coworkers (1985) demonstrated recently that a single injection of MCT reduces total lung capacity, tidal volume, respiratory frequency, and dynamic or quasi-static compliance by day 20. Lung resistance was elevated in MCTdtreated rats, and the coefficient of diffusion, an index of gas exchange, was reduced. These results are consistent with the parenchymal changes described, and indicate that, at least late in the develop- ment of pneumotoxicity, MCT-treated rats are in respiratory distress and have a reduced capacity for gas exchange. This is consistent with the report that arterial and venous oxygen contents are reduced by day 21 after the introduction of Q. sgctabilis seeds into the diet of rats (Meyrick _e_t gt, 1980). C. Metabolism and Bioactivation 1. General Although little is known about the mechanism by which MCT causes toxicity, it is generally accepted that injury is not due to MCT itself, which is relatively stable and non-toxic; rather, MCT (as well as other PZAs) 21 requires bioactivation. This is mediated in the liver by cytochrome P450- containing mixed function oxidase enzymes. This contention is supported by a number of observations. Microsomes prepared from liver, but not lung, metabo- lize PZAs to N-oxide and pyrrole derivatives, a reaction which is dependent on the presence of reduced NADP and oxygen (Mattocks and White, 1971). The conversion is inhibited by carbon monoxide or SKF-525A, and is increased in liver microsomes of phenobarbital-treated rats (Mattocks and White, 1971; White and Mattocks, 1971; Chesney gt gt, 1974b). Liver slices produce pyrroles when exposed to MCT or PZAs _i_g y_it_r_g, however lung slices do not (Mattocks, 1968; Hilliker gt gt, 1983c). Pretreatment with inducers or inhibitors of cytochrome P450 increase or decrease, respectively, the severity of hepatic, renal, and pulmonary lesions and the lethality of MCT jg 311:2 (Tuchweber gt gt, 1974). So it appears that one or more toxic metabolites are produced in the liver, and then leave the liver and travel via the circulation to other tissues to produce injury (Mattocks, 1968). Some PZA metabolites, such as N-oxide derivatives, are rela- tively non-toxic (Lalich and Ehrhart, 1962; Mattocks, 1971; Culvenor gt gt, 1976). Many investigators believe that the toxic metabolites are pyrrole derivatives (Figure 1). Following administration of PZAs, pyrroles are covalently bound to injured tissues (Mattocks, 1968; Allen gt g, 1972) and are excreted in the urine (Mattocks, 1968; Hsu _e_t a_l., 1973). The amount of pyrroles produced in vitro or i_n_ ytyg correlates with the relative toxicity of PZAs (Mattocks and White, 1971; Mattocks, 1972), and the amount of pyrrole found in tissue correlates with the degree of tissue injury (Mattocks, 1972). Pretreatment with phenobarbital increased the amount of pyrrole found in liver homogenates and increased the severity of liver and lung lesions and the lethality of MCT, whereas pretreatment with chloramphenicol had the opposite effect (Allen gt gt, 1972). In addition, 22 perfusion of isolated livers with MCT caused the appearance of pyrroles in the effluent, and when isolated lungs were perfused with this effluent, pulmonary injury was observed (Lafranconi and Huxtable, 1984). In summary, MCT is bioactivated in the liver to one or more pyrrole metabolites which then pass to the lung and covalently bind to macro- molecules. It 'm not known to which macromolecules pyrroles bind, however it is likely that this binding is involved in producing the injury that ultimately deve10ps. 2. Monocrotaline pyrrole a. Similarities in pathophysiologtc effects of MCT and MCTP One pyrrole derivative of MCT is monocrotaline pyrrole (MCTP) (Figure 1). When administered to animals, MCTP reacts with the first capillary bed it encounters to produce injury. When injected subcutaneously, MCTP causes a transient necrosis at the site of injection, and ultimately EC damage (Hooson and Grasso, 1975). When MCTP is given in a mesenteric vein, liver injury ensues (Butler, 1970). Administration of low doses of MCTP in a tail vein results in pulmonary vascular injury very similar to that seen following treatment with MCT (Butler, 1970; Bruner gt 2., 1986). Approximately two days after a single administration of a low dose (2-5 mg/kg) of MCTP, alveolar edema, congestion, and hemorrhage were observed (Butler gt g” 1970). By 1 week, the alveolar wall was thickened, and fibrin and numerous macrophages were present (Butler, 1970; Butler gt gt, 1970; Chesney e_t gt, 1974a; Raczniak gt gt, 1979). The nuclei of capillary ECs were enlarged and the capillary lumen was often occluded. The severity of these lesions increased with time, and vessel leak and abnormalities of epithelial cells were observed after 2-3 weeks (Butler, 1970; Plestina and Stoner, 1972). 23 Smooth muscle cell proliferation has been observed in the media of pulmonary arteries of rats treated with a low dose of MCTP, although the time course of this change has not been delineated (Chesney gt g, 1974a; Lalich gt gt, 1977). Interstitial fibrosis, dilated lymphatics, and platelet- containing thrombi have also been reported (Butler gt gt, 1970; Chesney gt gt, 1974a; Lalich e_t gt, 1977; Raczniak e_t gt, 1979). Increases in lavage fluid protein concentration and LDH activity were observed after treatment with low doses of MCTP (Bruner gt gt, 1983; Hilliker gt gt, 1984a). Cardiac index decreased simultaneous with an increase in pulmonary vascular resistance (Raczniak gt _a_t, 1979). Pulmonary hypertension developed (Chesney gt gt, 1974a; Bruner gt gt, 1983) and eventually led to RVE (Chesney gt gt, 1974a; Lalich _e_t gt, 1977; Bruner gt gt, 1983; Hilliker gt gt, 1984a). Removal of biogenic amines was also impaired in isolated lungs and lungs slices from MCTP-treated rats (Hilliker gt fl” 1983c, 1984a). Higher doses (10-30 mg/kg) of MCTP accelerated the development of pulmonary injury, and most treated rats died within 48 hours (Butler gt gt, 1970; Plestina and Stoner, 1972; Hurley and Jago, 1975). Death was preceded by pleural effusion and accumulation of edema fluid in the perivascular, peribronchiolar, and interstitial tissue, as well as in the alveolar lumens (Plestina and Stoner, 1972; Hurley and Jago, 1975). This was accompanied by increased vascular leak, largely from capillaries in the alveolar wall, which was not evident until 15 hours after treatment (Plestina and Stoner, 1972; Hurley and Jago, 1975). Gaps were observed between EC, many of which were swollen and contained prominent nuclei, and platelet aggregates were commonly noted in vessel lumens (Plestina and Stoner, 1972; Hurley and Jago, 1975). Alveolar walls appeared thickened and Type I cells were hypertrophic (Hurley and Jago, 1975). Thus, 24 higher doses of MCTP produce vascular leak and pronounced fluid accumulation in the lungs which result in death of the animal within 48 hours. When MCTP was injected into the pulmonary artery of isolated limgs most pyrrolic material was retained by the lungs within the first 30 seconds (Plestina and Stoner, 1972). When the lungs were stained with Ehrlich reagent to detect pyrrole moieties, staining was widely distributed through the lung tissue, but was most intense in the walls of larger blood vessels (Plestina and Stoner, 1972). Lung injury was also produced in isolated lungs by MCTP (Hilliker and Roth, 1985b). Wet lung weight, lavage fluid protein concentration, and perfusion pressure increased, and metabolism of perfused 5HT decreased after injection of a moderate dose of MCTP directly into the pulmonary artery. These results suggest that most of the MCTP administered probably reacts with the pulmonary vasculature on first pass through the pulmonary circulation, and that direct exposure to MCTP can produce pulmonary injury in isolated lungs. In summary, as with MCT, treatment with MCTP causes early changes in EC. Changes in pulmonary epithelium and vascular remodelling similar to those seen with MCT also follow treatment with MCTP. And, as with MCT, there are deficiencies in our knowlege of the development of MCTP pneumotoxicity. Alterations in ECs and pulmonary vasculature shortly after treatment (i.e., before day 2) have not been examined after giving low doses that result in the eventual development of pulmonary hypertension. The progression of changes in the pulmonary vasculature has also not been detailed. Finally, how the lesions that occur at a cellular level correlate with, or are responsible for, biochemical and functional changes has not been satisfactorily addressed. Investi- gation in each of these areas could enhance our knowledge of the mechanism by which MCT and MCTP produce pneumotoxicity and pulmonary hypertension. 25 b. Differences in lathophysiologic effects of MCT and MCTP Although the similarities in the pathophysiology of MCT and MCTP are numerous, a few (minor) differences exist. The decrease in circulating platelet number observed following treatment with MCT (Hilliker gt gt, 1982) was not seen with MCTP treatment: platelet numbers were not different from control at any time through 14 days after treatment (Bruner gt gt, 1983). Alterations in Type I pneumocytes have not been reported for MCT- treated rats, but following treatment with MCTP the nuclei of Type I cells become enlarged and the cytoplasm swells and contains increased cytoplasmic organelles (Butler, 1970; Hurley and Jago, 1975). Observations of vascular responsiveness in isolated, perfused lungs also differ slightly following treatment with MCT and MCTP. While the pulmonary vasculature is hyperresponsive to vasoconstrictors early (day 4) but not later (day 7 or 14) after treatment with MCT (Gillespie gt gt, 1986), following treatment with MCTP vasoconstriction is enhanced in isolated lungs at days 7 and 14 (Hilliker and Roth, 1985a). Finally, while relatively low doses of either MCT or MCTP produce chronic pulmonary vascular injury and pulmonary hypertension, the effect of administration of larger doses which cause death within 2 days differs between MCT and MCTP. MCTP causes frank pulmonary edema and animals die from pulmonary complications (Hurley and Jago, 1975) while death shortly after MCT treatment is associated primarily with liver injury (Schoental and Head, 1955; Tuchweber gt gt, 1974). C. Bioactivation of MCTP? It does not appear that MCTP requires further bioactiva- tion. Treatment with an inducer or an inhibitor of cytochrome P450 which does alter MCT toxicity did not alter the pneumotoxicity or RVE caused by MCTP (Bruner gt 9, 1986). Although this result does not rule out the possibility that 26 P450 isoenzymes or other enzyme systems unaffected by these agents may bioactivate MCTP, no evidence exists to suggest a role of such enzymes. (1. Development of MCTP toxicity Bruner and coworkers (1983) outlined the time-course of biochemical and hemodynamic changes following a single intraveous injection of MCTP (5 mg/kg). There was a delay in the development or expression of injury such that 3 days after treatment no signs of major injury were apparent. By day 5 lavage fluid protein concentration and LDH activity were elevated in treated rats relative to controls, and these indices remained elevated through day 14. Pulmonary artery pressure did not increase until day 7, and at this time elevated wet 11mg weight and vascular leak were also evident (Bruner 3 Q” 1983, 1986). Pulmonary artery pressure remained elevated through day 14, and RVE developed between days 10 and 14. This suggested that the right ventricle enlarges in response to sustained pulmonary hypertension. D. Interest in MCT/MCTP Toxicity 1. As an environmental toxicant PZA poisoning caused by animals grazing on PZA-containing plants is responsible for considerable livestock and, therefore, economic losses (Snyder, 1972; Huxtable, 1979). Human exposure is often fatal as well (Hill gt gt, 1951; McLean, 1970), therefore PZAs represent an environmental health problem. 2. As a pneumotoxic metabolite moduced in the liver MCT also represents a model compound for the study of toxi- cants which are bioactivated in the liver to metabolites which produce pulmonary toxicity. 3. As a model for human pulmonaryvascular disease Much of the pathophysiology described in MCT-treated rats is similar to that seen in humans with chronic pulmonary vascular diseases such as 27 primary pulmonary hypertension (PPH) and adult respiratory distress syndrome (ARDS). For this reason, the MCT-treated rats presents a good animal model for the study of these human diseases. a. Primary pulmonary hypertension PPH is pulmonary hypertension of unexplained etiology. A diagnosis of PPH is reached if three criteria are met: 1) right ventricular hypertrophy in the absence of other cardiac abnormalities; 2) elevated pulmonary arterial pressure and normal pulmonary wedge pressure; and 3) the absence at autopsy of evidence for my other etiology of pulmonary hypertension (Voelkel and Reeves, 1979). Pulmonary pathology very similar to that reported in MCT-treated rats has been described for patients with PPH. Intimal fibrosis, pulmonary arteritis, swollen ECs, platelet thrombi, and vessel occlusion have been observed (Walcott gt a_l., 1970; Watanabe and Ogata, 1976; Voelkel and Reeves, 1979; Palevsky and Fishman, 1985; Reid, 1986). The pulmonary arterial media hypertro- phies, the number of small vessels decreases, and ghost arteries appear in lungs of these patients. Plexiform lesions are considered a hallmark of PPH in man, and have also been observed in MCT-treated rats (Watanabe and Ogata, 1976). In addition, as in MCT-treated rats, alterations in respiratory mechanics are observed (Fernandez-Bonetti gt gt, 1983), and pulmonary extraction of biogenic amines is impaired (Sole _e_t gt, 1979). Clinically, PPH presents a problem because signs and symptoms are non-specific and appear only after marked vascular alterations have develOped. Therapy is largely ineffective, and the prognosis of these patients is quite poor (McLeod and Jewitt, 1986). Thus, monocrotaline provides a useful animal model to study a problematic human disease. 28 b. Adult regpiratory distress syndrome ARDS is a complication in people who suffer physical trauma, sepsis, or pneumonia (Bernard and Brigham, 1985). Early stages of ARDS are associated with increased vascular permeability due to leakage from capilla- ries and microvascular disruption, and this results in interstitial edema and alveolar flooding (Bernard and Brigham, 1985). EC injury is observed accompanied by compromised pulmonary extraction of 5HT (Morel gt gt, 1985), and pulmonary arterial pressures are elevated (Snow gt gt, 1982). Airway abnormalities are also evident, including destruction of Type 1 cells and decreased compliance (Bernard and Brigham, 1985). These changes are similar to those observed following administration of high doses of MCTP to rats (Plestina and Stoner, 1972; Hurley and Jago, 1975). As ARDS progresses, changes seen are similar to those observed following treatment with relatively low doses of MCTP. Later stages of ARDS are associated with pulmonary hypertension and vascular remodelling (Snow gt gt, 1982). Medial thickness of small pulmonary arteries increases, and there is a trend toward a decrease in the external diameter of partially muscular arteries, suggesting that small, nonmuscular arteries are developing into partially muscular arteries (Snow gt gt, 1982). Thus, the alterations associated with the early stage of ARDS are similar to those seen following high doses of MCTP, and alterations associated with later stages of ARDS are similar to those seen following modest doses of MCTP. Therefore, MCTP may be useful as a model of this human pulmonary vascular disease. E. Problems Associated with Studying this Model As with many animal models, there are a number of difficulties inherent in the study of MCT-induced pneumotoxicity. A few of them bear 29 mention here, in that they relate to experimental design and interpretation of results. For example, the requirement for bioactivation of MCT introduces difficulties in interpreting the results of some studies in which "specific" drugs are used in the hope of uncovering a mechanism of action of MCT. If such a drug attenuates the toxicity of MCT, the question must be raised as to whether this protective effect can be attributed to the specific action of the drug or whether it is a consequence of inhibition of bioactivation of MCT to a toxic metabolite. To circumvent this problem, drug treatment studies have been performed in rats treated with the pyrrole metabolite, MCTP, which apparently does not require bioactivation. Another difficulty arises from the complex nature of the vascular alterations caused by MCT or MCTP. Because these effects are numerous, and span several cell types and various cell functions, the mechanism of action likely involves the interaction of many cells and mediators. Therefore, studies °_1g m or in intact organs where the pulmonary architecture is maintained will likely provide the most useful information. Hayashi and coworkers (1979) demonstrated that retardation of growth by restriction of food intake attenuated MCT-induced pneumotoxicity and RVE. This phenomenon also presents a problem in studying the mechanisms of MCT's actions. Because of the subacute nature of the toxicity, any attempt to ameliorate the effects of MCT by co-treatment with a specific drug necessitates treatment with that drug and/or observation of animals for one week or longer. Many drugs, when given at effective doses for this length of time, reduce body growth by themselves, again raising the possibility that a protective effect may not be due to the specific action of the drug, but may be due instead to a non- specific effect of retarded growth. 30 Yet another problem derives from the observation that daily intraperi- toneal injections of water decrease the right ventricular hypertrophic response to MCT (Langleben and Reid, 1985). This protective effect was attributed to a response to the stress of handling and injection, and emphasizes the need for appropriate controls in drug treatment studies. Despite the problems associated with studying MCT's mechanism of action, it is a useful animal model for examining pulmonary vascular disease, and it can provide important information in this area. III. Mechanisms of Action of MCT[MCTP A. General Although early studies of MCT or MCTP were aimed largely at describing the associated pathophysiology, more recent investigations have been designed to determine the mechanism by which MCT causes pulmonary vascular damage. Several avenues of investigation have arisen, and the evidence for or against these potential mechanisms will be described in this section. A number of fundamental questions remain to be answered, however. For example, does vasoconstriction contribute to increased pulmonary vascular resistance at any time during the develOpment of pulmonary hypertension? Evidence suggests that in human pulmonary hypertension smooth muscle proliferation and vascular remodelling are the result of vasoconstriction (Voelkel and Reeves, 1979). In MCT-induced pulmonary hypertension, increases in pulmonary artery pressure have been reported to precede (Watanabe and Ogata, 1976) or to follow (Ghodsi and Will, 1981) the appearance of medial hypertrophy. Thus, this issue has not been clearly resolved. Another question pertains to what role, if any, vessel leak and the resulting pulmonary edema play in the development of pulmonary hypertension in this model. Increases in vessel leak are observed prior to RVE 31 (Valdivia g gt, 1967; Sugita gt _a_t, 1983a), suggesting that increased vascular permeability could contribute to pulmonary hypertension. Yet another curious issue arises from the observation of a 3-day delay following treatment with MCTP prior to the onset of major lung injury (Bruner gt g” 1983). This is probably 24-48 hours after the disappearance of the pyrrole (Allen gt gt, 1972; Mattocks, 1972), and suggests that toxicity may somehow be mediated indirectly. These and other imanswered issues serve to emphasize that the vascular changes following administration of MCT or MCTP are varied and complex. To search for a single answer or mechanism may be imrealistic. Rather, a less naive approach may be to attempt to uncover factors which contribute to the response, and this is the approach most investigators are now taking. B. The Role of Angiotensin Converting Enzyme An increase in lung ACE activity was observed one week after administration of MCT (Molteni gt gt, 1984). Although later in the development of toxicity lung ACE activity was depressed (Keane gt a_l., 1982; Molteni gt _a_l., 1984; 1985), it was conceivable that the early increase in ACE activity would contribute to the development of pulmonary hypertension. ACE inactivates the vasodilator bradykinin and converts inactive angiotensin I to the vasoconstrictor angiotensin II, and an increase in these activities could produce enhanced vasoconstriction in the pulmonary vascular bed. It has been reported that co-treatment with the ACE inhibitor Captopril attenuated the development of RVE and the ultrastructural changes in MCT-treated rats (Molteni _et a_l., 1985). However, treatment with Captopril alone reduced the weight of the right ventricle, which could have contributed to 32 the decrease in RV/(LV+S) observed in MCT-treated rats co-treated with Capto- pril. In addition, body weight was significantly lower in Capt0pril-treated rats than in controls, and this non-specific effect to retard body growth may have contributed to protection against RVE. More importantly, however, Captopril did not decrease lung ACE activity at the dosing regimen used in this study, and did cause an increase in serum ACE activity. This finding suggests that inhibition of ACE was not responsible for the observed decrease in RVE. Similar protection has been obtained with nonsulfhydryl-containing ACE inhibitors at doses which did or did not reduce lung ACE activity (Molteni _e_t gt, 1986). Therefore, the results of these studies do not present a convincing case that ACE activity is involved in the pathogenesis of MCT-induced pulmonary hypertension. Interestingly, Captopril did reduce the amount of hydroxyproline in limgs of MCT-treated rats, indicating a reduction in collagen content in these ltmgs and suggesting that an anti-fibrotic activity of Captopril may be responsible for its protective effect. C. The Role of Collagen Synthesis Molteni and coworkers (1985; 1986) also investigated the role of collagen synthesis in MCT-induced pulmonary injury by co-treating rats with penicillamine, an inhibitor of collagen maturation. Using hydroxyproline content as an index of fibrosis, these investigators demonstrated that penicillamine, at a dose which prevented the MCT-induced increase in lung hydroxyproline content, decreased the pulmonary ultrastructural changes caused by MCT treatment. The increase in wet lung weight and RVE due to MCT were not affected by co- treatment with penicillamine. These results suggest that collagen synthesis may contribute to MCT-induced ultrastructural alterations. However, penicillamine also functions as a copper chelator, and serum copper levels are elevated in patients with primary pulmonary hypertension (Ahmed and Sackner, 1985) and in 33 rats with pulmonary hypertension induced by a metabolite of MCT (our own observations). Thus, if c0pper is involved in the ultrastructural changes induced by MCT, penicillamine may alter this response through chelation of copper. D. The Role of the Immune Effectors Because the delay in toxicity and the histopathologic profile seen following treatment with MCTP were consistent with an immune response, it was hypothesized that immtme effectors may be involved in MCTP-induced pneumo- toxicity (Bruner, 1985). A possible role for both a cell-mediated and a humoral- mediated immtme response was examined. The effect of co-treatment with immtmosuppressant agents was deter- mined in MCTP-treated rats. Immunosuppression induced with an antiserum directed against rat lymphocytes did not alter the toxicity of MCTP. Co- treatment with the immunosuppressant drug cyclosporin A attenuated the lung injury and RVE caused by MCTP, however, rats co-treated with cyclosporin A lost weight relative to controls, so that the possibility that this partial protection was due to a retardation in growth cannot be ruled out. When lymphocytes from MCTP-treated rats were transferred into MCTP-treated rats, pneumotoxicity was not altered. The results of these experiments suggest that a cell-mediated immune response is not involved in MCTP toxicity (Bruner, 1985). MCTP also did not activate complement _ig gtt_rg or jg v_iyg, and depletion of complement did not attenuate MCTP-induced lung injury. Thus, it appears that immune effectors are not involved in the response to MCTP. E. The Role of Phggocytic Cells Following a single injection of MCT, large numbers of cells were observed in the alveolar space which, on the basis of functional and ultrastruc- tural studies, were determined to be abnormal alveolar macrophages (Sugita e_t _a_l., 1983b). The number of these cells increased with increasing doses of MCT and 34 with the degree of elevation of right ventricular systolic pressure and RVE. It was suggested that these abnormal alveolar macrophages may represent a marker of MCT-induced pulmonary hypertension (Sugita _e_t gt, 1983b). One mechanism by which macrophages or other phagocytic cells could contribute to the response to MCT or MCTP is through generation and release of reactive oxygen species which could cause cell injury. The ability of cells recovered in the bronchoalveolar lavage fluid to generate the superoxide anion was examined in MCTP-treated rats (Dahm g a_l., 1986). The total number of macrophages was lower in the lavage fluid of treated rats compared to controls 5 days after treatment, but not at 7, 10, or 14 days after treatment. In the lavage of treated rats compared to controls, the total number of neutrOphils was elevated at days 7, 10, and 14, the total number of eosinophils was increased at day 10 and the total number of lymphocytes was increased at day 14. Superoxide production by cells in the lavage fluid was decreased in MCTP-treated rats compared to controls at days 7, 10, or 14 (Dahm gt gt, 1986). This could mean that these cells had released all superoxide before or during the lavage procedure and were not capable of releasing any more superoxide after recovery. If superoxide were released i_n_ v_iy_9_ (before collection of lavage fluid), this reactive oxygen species could contribute to the tissue injury observed in lungs of MCTP- treated rats. F. The Role of Reactive Oxygen The role of reactive oxygen in MCTP-induced pneumotoxicity was further examined by Bruner (1985). MCTP-treated animals were co-treated with drugs which either prevent the formation of reactive oxygen species or degrade or scavenge them once produced. Desferroxamine chelates iron, which is necessary for the formation of hydroxyl radicals through the Fenton reaction. Catalase 35 causes the breakdown of hydrogen peroxide, and dimethysulfoxide (DMSO) sca- venges hydroxyl radicals. Toxicity due to MCTP was unaffected by co-treatment with either desferroxamine, catalase, or DMSO (Bruner, 1985). These results suggest that oxygen radicals are not involved in MCTP toxicity. G. The Role of Leukotrienes Leukotrienes (LTs) arise from the membrane phospholipid arachidonic acid by action of the enzyme 5'-lipoxygenase (Figure 2). LTs produce a variety of biological effects. LTC4, D 4 and E 4 are now known to be what was once called slow reacting substance of anaphylaxis (SRS-A). tn; _vjtgg these three LTs produce smooth muscle contraction in a variety of tissues (Creese gt gt, 1984; Sirois gt gt, 1985), reduce coronary blood flow (Hedqvist gt gt, 1983), and may stimulate release of cyclooxygenase metabolites of arachidonic acid (Creese gt a_l., 1984; Sirois e_t g” 1985). _Ig giyg LTC4 and D4 permeability, and pulmonary vascular resistance (Feddersen gt Q” 1983; Leffler alter pulmonary compliance, vascular e_t gt, 1984). LTB4 causes chemotaxis, aggregation, and adherence of granula- cytes (Hoover gt _a_l_., 1984). It is a less potent stimulator of smooth muscle than SRS—A LTs, but also causes release of cyclooxygenase products (Sirois gt gt, 1985). Due to the inflammatory response produced by MCT treatment, and the biological effects of LTs, LTs have been proposed to be mediators of the pneumotoxic response to MCT. There is some evidence that LTs are elevated in lungs due to MCT treatment. SRS-A-like activity was observed in lavage fluid of lungs from rats one week after a single injection of MCT (Stenmark gt gt, 1985). SRS—A-like activity was not observed in lavage fluid of control rats. Lavage fluid of treated rats also contained greater concentrations of LTC4 than controls, and the presence of LTB . Two or 3 weeks after a single injection of MCT, no SRS—A—like 4 activity was observed in the lavage fluid of treated or control rats. At 3 weeks, 36 cm“ ARAC HIDO NIC ACID CYClooxygeV Nowgenase P662 12HPET‘E SHPET‘E-fl SHETE Hydrop eroxidase l LTA I ”WW 4 W / \GSH / “3‘32 ms4 .L'I‘C4 \ 0W.“ i G-Keto PGFm Figure 2. Pathways of arachidonic acid metabolism. 37 lavage fluid from treated rats inhibited contractile activity induced in isolated, guinea pig ileum by addition of exogenous LTC4 or LTD 4. Homogenates of lungs from rats treated 3 weeks earlier showed SRS-A-like activity (Stenmark gt Q" 1985). These results suggested that LT synthesis may be activated by MCT treatment. In rats treated 3 weeks earlier with MCT, co-treatment with diethyl- carbamazine (DEC), which inhibits LT production (Mathews and Murphy, 1982), attenuated pulmonary hypertension and RVE (Stenmark gt a_l., 1985). This is in contrast to the effect of co-treatment with DEC on the toxicity of MCTP (Bruner, 1985). DEC attenuated MCTP-induced lung injury at day 7, but did not alter pneumotoxicity or RVE at day 14. There are a number of possible explanations for the disparity in the results of the two studies. In the study with MCT, co-treatment with DEC began two days prior to treatment with MCT and continued through the end of the study, whereas co-treatment with DEC began 3 days after treatment with MCTP in the study of Bruner (1985). It is possible that the difference in dosing regimens could explain the different results obtained in these two studies. Another explanation could be that DEC was acting to protect against MCT toxicity in some manner Lmrelated to LT inhibition. For example, body weights were not reported in the study of DEC co-treatment of MCT-treated rats, and it is possible that DEC suppressed body weight gain. Alternatively, DEC could have inhibited biotransformation of MCT to its toxic metabolite. DEC is not a specific inhibitor of LT synthesis, and more specific drugs that inhibit LT biosynthesis or act as LT receptor antagonists have not been used in this model. Therefore, additional experiments to determine if a role for LTs exists in MCT-induced pulmonary hypertension need to be performed. 38 H. The Role of Polyamines Increases in lung polyamine concentrations are observed one week after treatment with a single injection of MCT (Olson _e_t gt, 1984b). Activities of the enzymes associated with polyamine biosynthesis are elevated in lungs early (day 1), and late (days 14-21) in the development of MCT toxicity (Olson gt gt, 1984a,b) and in ECs in culture exposed to MCT metabolites (Altiere gt a_l., 1986a). Because polyamines may be essential for cell growth, it was hypothesized that they may be involved in the proliferation and hypertrophy of vascular smooth muscle observed following treatment with MCT. If the increase in vascular smooth muscle contributes to MCT-induced elevation of pulmonary arterial pressure, then polyamines may play a role in MCT-induced pulmonary hyperten- sion. Ornithine decarboxylase (CDC) is the rate-limiting enzyme in the biosynthesis of polyamines. Co-treatment with the CDC inhibitor, a-difluoro- methylornithine (DFMO) attenuated the development of RVE and the increase in pulmonary artery pressure caused by MCT (Olson g gt, 1984b). However, in this particular study, drug effectiveness was not confirmed, body weight data was not presented, and the effect of DFMO on bioactivation of MCT was not addressed. A subsequent study using the same protocol dealt with some of these issues (Olson gt gt, 1985). In this second study, DFMO prevented MCT-induced medial thickening, perivascular edema, and increased lung weight. DFMO partially suppressed the elevation of lung polyamine levels caused by MCT, and did not affect body weight gain. The production of Ehrlich-reactive products (pyrroles) from MCT in isolated livers or in liver slices was not altered by DFMO, suggesting that DMFO did not prevent the bioactivation of MCT. However, Ehrlich reactivity is positive for all pyrroles, and DFMO could have suppressed synthesis of a pyrrole metabolite responsible for lung injury without affecting total pyrrole production. 39 The results of these studies suggest that polyamines may somehow be involved in MCT-induced pneumotoxicity. However, as mentioned above, the issue of alteration of MCT bioactivation has not been sufficiently addressed. Furthermore, this conclusion is drawn from studies using only one inhibitor of polyamine biosynthesis. It is possible that DFMO may have other actions in addition to inhibition of ODC. Along this line, it is of interest that DFMO prevented all manifestations of MCT toxicity examined. This perhaps should not be surprising considering the multiplicity of actions that polyamines have in biological systems. Clearly, more studies should be done in this interesting and promising area. The case for polyamines would be strengthened if other drugs which inhibit polyamine synthesis or activity also protected against MCT-induced lung injury and pulmonary hypertension. L The Role of Blood Platelets 1. Evidence that platelets may be involved A couple of observations initiated the thinking that blood plate- lets may be involved in the response to MCT. The first was that platelet- containing thrombi were seen in the pulmonary vessels of rats treated with MCT or MCTP (Merkow and Kleinerman, 1966; Chesney gt gt, 1974a; Hurley and Jago, 1975; Heath and Smith, 1978). The second was that, following treatment with MCT, a mild thrombocytopenia was observed (Hilliker gt gt, 1982). These two observations were consistent with the damaged ECs observed by electron micro- scopy (Turner and Lalich, 1965; Merkow and Kleinerman, 1966; Butler, 1970; Chesney gt gt, 1974a), and it was hypothesized that EC injury promoted plate- let/vessel wall interaction. Activated or modified platelets could then in some way contribute to pulmonary hypertension. This hypothesis was supported by the finding that antibody- induced thrombocytopenia attenuated the development of RVE in MCTP-treated 40 rats (Hilliker gt gt, 1984a). Rats made moderately thrombocytopenic (circulating platelet number approximately 15% of normal) from days 3-5 or from days 6-8 following a single treatment with MCTP developed less severe RVE at day 14 than rats with normal platelet numbers. Since RVE is thought to be a response to sustained increases in pulmonary artery pressure, this suggested that platelet depletion reduced MCTP-induced pulmonary hypertension. Lung injury assessed at day 14 was unaffected by platelet depletion. There are several possible explanations for this last result. One interpretation is that the platelet does not play a role in MCTP-induced pulmonary injury but rather is involved in the response to MCTP-induced injury (i.e., pulmonary hypertension). Alternatively, reducing the number of circulating platelets could delay MCTP-induced toxicity. Normally, major lung injury appears between days 4 and 8 whereas RVE is not evident until day 14 after MCTP treatment (Bruner gt gt, 1983). If thrombocyto- penia were to delay the onset of toxicity by a few days, then at day 14 lung injury may still be apparent but RVE would not be. At any rate, there is some evidence to suggest that blood platelets may be involved in the pulmonary hypertensive response to MCTP. No protective effect was observed if the period of platelet depletion was from days 0-2 following administration of MCTP. This suggests that platelets may not be involved in the early injury caused by MCTP, and that platelet involvement may not be a factor until several days after MCTP treatment. 2. Mechanisms by which platelets may be involved There are a number of mechanisms by which platelets could be involved in MCTP-induced pulmonary hypertension. For example, platelets function by some ill-defined mechanisms to maintain normal, uninjured endothe- lium (Roy and Djerassi, 1972; Kitchens and Weiss, 1975), and an alteration of this function could conceivably contribute to increased pulmonary vascular pressures. 41 Alternatively, platelet-containing thrombi could occlude pulmonary vessels and increase pulmonary vascular resistance. Another possible mechanism is that platelets encountering damaged endothelial cells in the pulmonary vascular bed could become activated and release mediators which alter vascular tone or permeability or which stimulate smooth muscle cell proliferation. This last potential mechanism will now be further discussed. 3. Platelet mediators that may be involved When platelets interact with an injured vessel wall they can become activated, meaning that they can adhere to the surface of the vessel, aggregate, and undergo a release reaction (Ratliff e_t gt, 1979; Weiss, 1982). Intravascular platelet aggregation causes increased pulmonary vascular resis- tance, increased pulmonary vascular permeability, and alterations in respiratory function (Bd and Hognestad, 1972; White gt gt, 1973; Vaage gt gt, 1974, 1976). These responses are believed to be mediated by products of the release reaction. Mediators released by stimulated platelets include 5HT, arachidonic acid metabo- lites (such as 12HPETE, PGH TxAZ), platelet-derived growth factor (PDGF), and 2, platelet-activating factor (PAF). a. S-Hydroxytryptamine Platelets accumulate 5HT by an active uptake process (Born gt a_l., 1972; Talvenheimo _et fl” 1979) and most of this 5HT is stored in dense granules, although some extravesicular storage pools also exist (Tranzer gt g_1., 1966; Holmsen gt g, 1969; Costa e_t g, 1982; Given and Longenecker, 1985). 5HT released during platelet aggregation can cause vasoconstriction in a number of vascular beds (DeClerck and Van Nueten, 1982; Mullane fl gt, 1982; McGoon and Vanhoutte, 1984), including the lung (Rickaby gt Q” 1980; Tucker and Rodeghero, 1981). 5HT can also potentiate the response to other vasoconstricting agents (DeClerck and Van Nueten, 1982) and can increase vascular permeability 42 (DeClerck gt gt, 1984, 1985). Thus, 5HT could contribute to MCTP-induced pulmonary hypertension through vasoconstriction, potentiation of the action of other vasoconstrictors, or alteration of vascular permeability. b. Arachidonic acid metabolites Arachidonic acid (AA) is a 20-carbon fatty acid found most frequently esterified at the sn-2 position of membrane phosphoglycerides. AA is liberated from phospholipids by the action of phospholipase A2 or phospholipase C. Free AA is metabolized by two major pathways: conversion by the enzyme cyclooxygenase produces prostaglandins (PGs) and thromboxanes (TXs); conversion by lipoxygenase enzymes produces hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), and leukotrienes (LTs) (Figure 2). Al- though small amounts of 12—HPETE are produced in platelets, the major pathway of AA metabolism in platelets is via the cyclooxygenase enzyme to produce the endoperoxides PGG and PGH 2 2’ TxA2 (Needleman gt gt, 1976; Longenecker, 1985). which in turn degrade to form PGE PGFZO.’ and 2’ TxA is the major AA metabolite synthesized by platelets. 2 By inhibiting adenyl cyclase, and thereby decreasing platelet cyclic AMP, TxAz promotes platelet aggregation (Hamberg gt 9, 1975b; Meyers _e_t gt, 1979; Parise gt fl” 1984; Longenecker, 1985). h addition, TxA2 causes vasoconstriction (Hamberg gt gt, 1975b; Svensson gt gt, 1977; Farrukh gt g, 1985). Therefore, by virtue of its functions to promote platelet aggregation and to stimulate vascular smooth muscle to contract, TxA2 may contribute to MCTP-induced pulmonary hypertension. The pro-aggregatory and vasoconstrictive activities of TxAz are opposed i_n_ m by the actions of prostacyclin (PGLz), synthesized largely by vascular endothelial cells (Moncada gt a_l., 1976; Weksler gt gt, 1977; Tateson e_t gt, 1977; MacIntyre gt gt, 1978). It has been proposed that the 43 balance of TxAz and PGI2 is an important factor in vascular homeostasis (Gryglewski gt gt, 1976; Bourgain gt a_l., 1982; Flynn and Demling, 1982; Saldeen and Saldeen, 1983). Endothelial cell injury associated with MCTP may alter PGI2 synthesis, thereby allowing greater expression of the vasoconstrictive activity of TxAz. In addition, normal endothelial cells may "steal" platelet-derived PGH2 as a substrate for PGIZ synthesis (Gorman, 1979), and if this function is suppressed due to EC injury following treatment with MCTP, increased TxAZ synthesis by the platelet might result. This presents another mechanism by which TxAZ could contribute to MCTP-induced pulmonary hypertension. c. Platelet-derived growth factor Platelet-derived growth factor (PDGF) is a polypeptide contained within platelet alpha granules and released during platelet activation (Deuel and Huang, 1984). PDGF stimulates DNA synthesis and proliferation of a number of cells including fibroblasts and smooth muscle cells. Since vascular smooth muscle proliferation and extension into normally nonmuscular vessels is a feature of MCTP-induced pulmonary hypertension, PDGF may contribute to the response to MCTP by stimulating smooth muscle cell proliferation. d. Platelet-activating factor Platelet-activating factor (PAF; acetylglycerylether phos- phorylcholine) is a lipid mediator that aggregates platelets (Chignard gt gt, 1979; Macconi g gt, 1985) and polymorphonuclear neutrophils (Camussi gt gt, 1980). PAF also causes smooth muscle contraction, pulmonary edema, and alterations in pulmonary vascular permeability (Findlay gt a_l., 1981; Mojarad g gt, 1983; Lichey gt gt, 1984; Benveniste and Chignard, 1985). These actions of PAF might contribute to MCTP-induced pulmonary hypertension. PAF caused pulmonary hypertension and edema in isolated rabbit lungs perfused with human platelets, 44 and this response was mediated by PAF-induced release of TxAz (Heffner _e_t gt, 1983). J. Summary In summary, it appears that MCT or MCTP produce pneumotoxicity and pulmonary hypertension by a mechanism(s) which likely involves multiple mediators. One promising area of research is that of the contribution of polyamines to MCT-induced toxicity, and this area warrants further investigation. The remainder of this thesis will focus on studies which examine a possible role for the platelet in MCTP-induced pulmonary hypertension. IV. Specific Aims Platelets appear to be involved in the pulmonary hypertensive response to MCTP by an undetermined mechanism. It is apparent that a number of platelet mediators could contribute to pulmonary hypertension due to MCTP. The hypothesis that platelet-derived mediators contribute to MCTP-induced pulmo- nary hypertension was tested by performing experiments to: A. Determine the dose/response relation for MCTP. B. Describe the histopathology of the deve10pment of MCTP-induced pneumotoxicity. C. Examine the influence of diet restriction on MCTP-induced cardiopul- monary toxicity. D. Determine the effect of thrombocytopenia on MCTP-induced pulmo- nary hypertension. E. Examine the role of 5HT in MCTP-induced cardiopulmonary toxicity. F. Examine the role of TxAz in MCTP-induced cardiopulmonary toxicity. MATERIALS AND METHODS L Animals Respiratory disease-free, male, Sprague-Dawley rats (CF:CD(SD)BR) (Charles River Laboratories, Portage, MI) were used in these studies. They were housed on corn cob bedding in plastic cages under conditions of controlled temperature and humidity. An alternating 12-hour light/dark cycle was main- tained. Whenever possible, cages were kept in an animal isolator (Contamination Control, Inc., Lansdale, PA) so that the rats breathed only HEPA-filtered air. With the exception of rats used in the diet restriction study, all animals were allowed free access to food (Wayne Rodent BloxR, Continental Grain Company, Chicago, IL) and water. An adult, female Nubian goat was used for the preparation of anti~rat platelet antibody. The goat was kept outdoors in a small fenced-in pasture on which there was also provided a wooden shelter. Grass and water were freely available. H. Diet Restriction Rats were housed individually (one per cage) for this study. Standard rat chow was ground to a powder and was provided in small glass jars placed inside the cages. Rats were allowed to eat gt m (average daily food consumption was 22 g/rat) or were restricted to 9 g food/rat/day. Water was provided fl libitum for all rats. 45 46 III. Monocrotaline Pyrrole (MCTP) A. Synthesis of MCTP MCTP was synthesized from monocrotaline (MCT) (TransWorld Chemi- cals, Washington, D.C.) via an N-oxide intermediate as described by Mattocks (1969). The MCTP isolated from the synthesis procedure has Ehrlich activity (an indication of pyrrole moieties) (Mattocks and White, 1971), and a structure consistent with MCTP as determined by mass spectrometry (Mattocks, 1969; Culvenor e_t gt, 1970; Bruner e_t a_l., 1986) and nuclear magnetic resonance (Bruner fl gt, 1986). MCTP was stored imder nitrogen in the dark at -20°C. B. Treatment with MCTP MCTP was dissolved in N,N-dimethylformamide (DMF) at appropriate concentrations so that the volume injected was 0.5 ml/kg to achieve the desired dose. Rats were treated via the tail vein with DMF or with MCTP at doses ranging from 2-5 mg/kg. IV. Assessment of Cardiopulmonary Injury A. Bronchoalveolar lavagg The trachea of rats anesthetized with sodium pentobarbital (50 mg/kg, i.p.) was cannulated with a blunted 18 gauge hypodermic needle. The abdomen and thorax were then opened and the lungs were carefully removed. A known volume of room temperature saline (0.9%) was instilled into the airway and then removed. This procedure was repeated once, and the recovered lavages were combined. The volume of saline instilled was determined by multiplying the mean body weight (in kg) of all rats to be killed on a given day (control and treated) by 23 ml/kg (Mauderly, 1977). The lavage fluid was spun in a centrifuge (600 x g, 10 minutes), and the activity of lactate dehydrogenase (LDH) was determined in the C811~free supernatant on the day the lungs were lavaged. LDH activity was 47 measured spectr0photometrically according to the method of Bergmeyer and Bernt (1974) and was quantified as the conversion of the cofactor NADH to NAD+ as pyruvate substrate was converted to lactate. Protein concentration was determined in the cell—free lavage fluid using the method of Lowry and coworkers (1951). Bovine serum albumin was used as the protein standard. B. Pulmonary sequestration of radiolabelled protein as a marker of pplmonary vascular leak Pulmonary vascular leak was assessed as the accumulation of 125I in the ltmgs following an intravenous injection of 12'SI-labelled bovine serum albumin (RSI-BSA), using a modification of the method of Johnson and Ward (1974). Rats were given an intravenous injection of 125I-BSA (0.2 ml, 1.0 mg BSA/ml) containing approximately 400,000 cpm. Four hours later, rats were anesthetized with sodium pentobarbital, and 500 units of heparin (in 0.5 m1 saline) were injected into the inferior vena cava. One minute later, one ml of blood was 12'SI-blood) withdrawn and placed in a tube for determination of radioactivity ( (Tracor 1185 series Gamma Counter, Chicago, IL, or Micromedic Automatic Gamma Counter, Horsham, PA). The trachea was cannulated as described above for bronchoalveolar lavage, and the pulmonary artery was cannulated with a saline-filled catheter (PE 190). The lungs and heart were removed from the thorax and the left atrial appendage was cut. The lungs were periodically ventilated with a small volume of room air while 10 ml of saline were gently perfused through the pulmonary arterial cannula to clear blood from the vascula- ture. If lavage fluid LDH activity or protein concentration was to be determined, the ltmgs were lavaged at this point as described above. The lungs were trimmed from the trachea and connective tissue, rinsed with saline, blotted dry and placed in tubes for determination of radioactivity. If the lungs had been lavaged, one m1 of lavage fluid was also placed in a tube for determination of radioactivity. The 48 total radioactivity in the lung (RSI-lung) was the radioactivity in the lung tissue plus, where applicable, the radioactivity removed in the lavage fluid (cpm/ml lavage fluid x ml lavage fluid recovered). An increase in the ratio 12'SI-lung/ 1251- blood was considered to indicate vascular leak. C. Ltmg weight Wet lung weight was determined as the difference in the weight of the 11mg plus connective tissue prior to bronchoalveolar lavage and vascular perfusion and the weight of the connective tissue after the lung was trimmed away. Dry hmg weight was determined after lungs, placed in pre-weighed vials and kept in a drying oven (92°C), reached a constant weight. D. Pulmonary artery pressure Pulmonary artery pressure (PAP) was measured in anesthetized rats. The distal end of a 3.5 French umbilical vessel catheter was warmed and bent to approximately a 45° angle. The catheter was introduced through the right jugular vein, carefully advanced into the right ventricle, and then gently manipulated into the pulmonary artery (Stinger gt gt, 1981). Pressure was measured with a Statham P23ID pressure transducer and was recorded on a Grass Model 7 polygraph. Location of the catheter was determined by characteristic pressure tracings. E. Right ventricular enlargement Right ventricular enlargement (RVE) was assessed as an increase in the ratio of the weight of the right ventricle to the weight of the left ventricle plus septum (RV/(LV+S)) (Fulton gt gt, 1952). The heart was removed, and the atria were carefully trimmed away. The wall of the right ventricle was then trimmed free of the left ventricle plus septum, and each tissue was weighed separately. 49 F. Other indices of injury Blood urea nitrogen (BUN) was determined using a standard diagnostic kit (Urea Nitrogen No. 535; Sigma Chemicals, St. Louis, MO). Serum glutamic oxalacetic transaminase (SGOT) was also determined using a standard diagnostic kit (aspartate aminotransferase No. 505; Sigma Chemical, St. Louis, MO). V. HistOpatholOJy A. Fixation procedure Following measurement of PAP in anesthetized rats, an abdominal incision was made and 500 Units of heparin (in 0.5 ml saline) were injected into the inferior cava. The trachea was cannulated (PE 210 tubing) and the thorax was opened. The pulmonary artery was cannulated (PE 190 tubing), the heart was removed, and the lungs were carefully excised from the thorax. The lungs were gently inflated with room air several times to distend atelectic portions. A modified Karnovsky's fixative (1% glutaraldehyde; 2% paraformaldehyde; 0.1 M cacodylate buffer; pH=7.4) was infused at constant pressure into the pulmonary artery from a height of 32 cm H20, and into the trachea from a height of 26 cm HZO’ for a minimum of 15 minutes. Sections of lung tissue (2-3 mm) were then cut and fixed in Karnovsky's fixative for an additional 4 hours at 4°C. Lung sections were rinsed twice in cold cacodylate buffer (0.2 M, pH=7.4) and were stored for future processing. B. Tissue processng and stainirg A longitudinal section (2-3 mm thick) of the left lung lobe, as well as sections from areas with obvious lesions or from areas randomly selected, were embedded in paraffin. Sections (4 um) of paraffin embedded tissues were stained with hematoxylin and eosin stain (H and E). 50 Sections from areas of the lungs with gross lesions or from the right caudal lobe were selected for glycol methacrylate (plastic) embedding. These tissues were cut (1-2 um) and were stained with toluidine blue and modified Gill's H and E stains. VL Preparation of Goat Anti-Rat Platelet Antibody A. Preparation of a pre-immune (control) serum Prior to exposure to the platelet membrane antigen, the goat was bled on several occasions to obtain control serum (CS). Blood (approximately 300 ml) was collected from the external jugular vein using a 16 gauge needle attached to PE260 tubing. The blood was allowed to clot at 37°C for 2 hours, after which it was spun (1000 x g) in a centrifuge for 10 minutes. The supernatant was incubated at 56°C for 45 mintues to deactivate complement, then cooled in an ice bath to approximately room temperature before being spun again as described above. The supernatant (CS) was then kept frozen (-2°C) until use. B. Preparation of antigen: Rat platelet membranes Retired breeder donor rats were anesthetized lightly with ether, and blood (10-20 cc) was withdrawn from the abdominal aorta into syringes containing 3.8% sodium citrate. The citrate volume was then adjusted so that the final concentration in the blood was 0.38%. The pooled blood was spun (150 x g, 10 minutes) and the platelet-rich plasma (PRP) was removed with a pipette, being careful to avoid red blood cells and the 'buffy coat" in the process. This step was repeated until no more PRP could be collected. The PRP was then sptm (600 x g) for 10 minutes to obtain a platelet pellet. This procedure was repeated until the supernatant was exhausted of platelets as determined by the lack of appearance of a pellet. The platelet pellets were resuspended and washed 3 times in 0.01% EDTA in saline (1 ml), and were 51 pooled. The pooled platelet pellet, suspended in a minimum of 0.01% EDTA, contained 1.0x107 pt/ul. No red or white blood cells were seen when the suspension was examined microsc0pically. This suspension was frozen and thawed twice to facilitate lysing the platelet membranes. After the second thawing, the suspension was sonicated using a Sonicator Cell Disruptor (Heat Systems-Ultra- sonics, Inc., Plainview, NY) to further disrupt the platelets. This suspension had less than 200 pt/ul. The protein concentration was determined, and the remaining suspension was diluted with 0.01% EDTA to adjust the protein concentration to 20 mg/ml (corresponding to approximately 6x106 pt/ul). Before use, an equal volume of Freund's complete adjuvant was emulsified with the platelet membrane solution using a homogenizer. Antigen was prepared on a number of occasions by the above proce- dure, each time attempting to achieve approximately the same final concentra- tion of protein. C. Preparation ofjoat anti-rat platelet serum The goat's back was shaven, and a total of 1 ml of the platelet membrane antigen emulsification was injected intradermally into 15-20 spots along her back (approximately 0.05 ml/injection). One week and two weeks later she received another 1 ml of antigen in an identical fashion ("booster"). Ten days later, blood was withdrawn from the external jugular vein and anti-platelet serum (PAS) was prepared from the blood as described above for CS. The goat was bled 5 more times, each time preceded by a booster injection of antigen. PAS collected on different days was stored separately, and the potency of each was determined g 1133 as described below. D. Absorption of sera with red blood cells To reduce any toxic effects of the sera, both the CS and the PAS were absorbed to washed, rat red blood cells (RBC's) before use. Blood was collected 52 from the abdominal aorta of sodium pentobarbital-anesthetized donor rats into syringes containing 1000 Units of heparin (in 1 ml saline). The blood was centrifuged (600 x g for 10 min) and the plasma discarded. The RBC's were then washed with saline 3 times. Washed RBC's were added to the serum (1 ml RBC's/ 5 ml serum) and this was kept at room temperature for one hour. After removing the RBC's by centrifugation (600 x g for 10 min), the absorption procedure was repeated once. Absorbed antisera was kept frozen until immediately before use. E. Efficacy of PAS in vivo The efficacy of the PAS was determined E m. Rats were treated with one of several doses of PAS intraperitoneally to establish a dose which produced the degree of thrombocytopenia desired. Once established, this dose was given (i.p.) to several rats immediately after taking blood from the tail for a baseline platelet count. Blood was then taken at various times after administra- tion of the PAS, and platelet count and hematocrit were determined. Total white blood cell counts and differential white cell counts were also determined 12 hours after treatmentnwith the PAS. VII. Cell Countirg Platelet number was determined in platelet-rich plasma or in whole blood collected from the abdominal aorta or from the tail. Blood collected from the abdominal aorta was diluted with sodium citrate in a plastic syringe during collection so that the final concentration of citrate in blood was 0.38%. When blood was sampled from the tail, approximately 0.45 ml was allowed to drip into a polypropylene tube containing 50 ul of 3% trisodium EDTA in saline. Aliquots of blood or platelet-rich plasma were diluted with ammonium oxalate buffer using a UnopetteR diluting system (Becton Dickinson, Rutherford, NJ). Platelets were 53 counted in a Neubauer hemacytometer using phase contrast microscopy (Brecher and Cronkite, 1950). White blood cells were counted in whole blood as described above for platelets. Differential cell counts were determined from Wright's stained-smears by counting and identifying 100-200 cells. VIII. Platelet Aggregation A. Blood collection Rats were lightly anesthetized with ether, and blood was collected from the abdominal aorta. Plastic syringes containing 3.8% sodium citrate were used, and the volume of citrate was adjusted to a final blood:citrate ratio of 9:1. B. Preparation of platelet-rich plasma and platelet poor plasma Platelet-rich plasma (PRP) was prepared by one of two methods. In the first method, blood samples were spun 3 times at 150 x g for 5 minutes in an IEC HN SH centrifuge. After each centrifugation, the PRP was transferred to a clean polypropylene tube and the PRP fractions for each blood sample were pooled. In the second method, PRP was collected after each of two l-minute centrifugations of whole blood at 600 x g in an IEC HN SH centrifuge. Platelet number in the PRP collected by either manner was similar. Platelet-poor plasma (PPP) was collected after a 20-minute centrifu- gation (600 x g) of the remaining blood. Autologous PPP was used to adjust the 3 platelet count of the PRP to approximately 106/mm for platelet aggregation studies. C. Platelet gggregation PRP (0.5 ml) was transferred to silanized glass cuvettes, and was allowed to settle for 30-60 minutes at room temperature before aggregation. Platelet aggregation was measured as an increase in light transmittance through a 54 cuvette of PRP (Born and Cross, 1963) using a dual channel platelet aggregometer (Payton Assoc., Buffalo, NY) and was recorded on an Omniscribe Chart recorder. Unstimulated PRP was used to set 0% transmission, and autologous PPP was used to set 100% transmission. Cuvettes containing PRP were inserted into the aggregometer and were allowed to equilibrate for 2 minutes (37.5°C, stirred at 900 rpm) before addition of the aggregating agents. Arachidonic acid (1.5 mM; sodium arachidonate, BioData Corp., Hatsboro, PA) was used as the stimulus for aggregation. Three aspects of platelet aggregation were recorded: the maximum aggregation (as a percent of the difference in light transmittance through PPP and unstimulated PRP), the rate of aggregation (slope = % aggregation/min, taken during the linear portion of the aggregation tracing); and the delay to aggregation (the time required for the shape change). These are illustrated in Figure 3. D. Determination of platelet 5-hydroxytryptamine Blood was collected from ether-anesthetized rats into syringes con- taining sodium citrate (0.38% final concentration) and pargyline HCl (0.1 mM final concentration; Abbott Labs, Abbott Park, IL). Platelet-rich plasma (PRP) was prepared according to the second method described above. An aliquot of the PRP was taken for determination of platelet number. Another aliquot (100 111) was transferred to a microcentrifuge tube and was spun in an EppendorfR microcentri- fuge for 5 minutes. HCl (2.5 N) was added to an aliquot of the supernatant to adjust the pH to 2.5, and this platelet-poor plasma was reserved for determination of 5-hydroxytryptamine (5HT). The pellet was washed with 1 ml of 0.01% NazEDTA containing 0.1 mM pargyline HCl, and then was resuspended in 1 ml of a phosphate buffer containing 0.1 mM pargyline HCl (0.05M phosphate, 0.03 M citrate, 15% methanol, pH=2.7). The platelets were disrupted by persistent sonication (Sonicator Model W-220F, Ultrasonics, Inc., Plainview, NY). The platelet membrane solution was then spun in a microcentrifuge for 5 minutes, and 55 .nomumwoumws "Manama u o .nomumwoumwm uwfimxdaufisa u n A<< mo nominee 832: gm poumgamua u m 63:58 E pommounxo fl Egon on“ can 6338\n05mwouwwm 3 as pommounko mm nomummoumwm mo was." 9E. Aoeov mmm pouflsafimg pad Agcoc mam E nonsuumamamh 3w: 5 098.8336 o5 mo mmmunooumm m an emergence mm somuwmoummm we assume 23. .fime maflmmouwwm u << .pofiauouop as.» meH can commons msB nofluwou 93 £038 «a nomumwouwmm marge 330m pad poSmmoE 9858.922“ somummoummm «333% mamaommop sense nomummawww 133.5 .4. .m magma 56 <—-n < 4 SDNVIIIWSNVHI lHOl'l TIME é—O Figure 3 57 the supernatant was frozen until determination of 5HT by high performance liquid chromatography (HPLC). The protein concentration of the platelet pellet membrane was determined by the method of Lowry and coworkers (1951). 5HT was measured with an HPLC-electrochemical detection (HPLC- EC) system (Shannon g Q” 1986). Fifty microliters of the sample supernatant were injected onto a C18 reverse phase column (4.6 mm i.d. x 25 cm; 5 11m spheres, Biophase ODS, Bioanalytical Systems, Inc., West Lafayette, IN) which was protected by a precolumn cartridge filter (10 um spheres, 4.6 mm i.d. x 3 cm, Bioanalytical Systems, Inc.). The HPLC column was coupled to an electrochemi- cal detector (LC4A, Bioanalytical Systems, Inc.) equipped with a TL-5 glassy carbon electrode set at a potential of +0.75 V relative to a Ag/AgCl reference electrode. The HPLC mobile phase (pH=2.7) consisted of 0.1 M citrate phosphate buffer, 0.1 mM EDTA, 1.5 mM sodium octyl sulfate and 25% methanol. Separa- tions were performed with a flow rate of 0.8 ml/min and a pressure of approximately 2400 psi. The amount of 5HT in a sample was determined by comparing sample peak heights measured by a Hewlett-Packard 3390A Integrator (Hewlett-Packard, Avondale, PA) with standards that were run each day. The limit of sensitivity was 20 pg. E. Determination of platelet-derived thromboxane B, Blood was collected from rats treated 1, 4, 7, or 14 days earlier with MCTP. PRP was collected according to the first method described above, and aggregation was induced with arachidonic acid (1.5 mM). The aggregation response was terminated by addition of 100 1.11 of 2 N HCl to the PRP. The PRP was then transferred to a 3 ml conical microcentrifuge tube and was spun in an microcentrifuge for 5 minutes. The supernatant was transferred to a clean tube and was frozen until determination of Tsz by radioimmunoassay (RIA). 58 Tsz generation was determined in unstimulated PRP, and in PRP which was at half-maximal or maximal aggregation following stimulation with AA. This is illustrated in Figure 3. F. Effect of MCTP in vitro oglatelet aggtggation and Tszgeneration The effect of MCTP added it y_i_tr_g to PRP was determined. Blood was collected from untreated rats and PRP was prepared as described above. DMF (1 111) or MCTP (31-500 11g) in 1 111 of DMF was added to the PRP 1 minute prior to addition of AA. The concentration of Tsz in the PRP was determined at maximal aggregation. IX. Isolated, Perfused Lungs A. Surgery Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the trachea was cannulated with PE240 tubing. The abdomen was opened, and 500 U of heparin in 0.5 ml saline were injected into the inferior vena cava and allowed to circulate for approximately 1 minute. The diaphragm was cut along the abdominal wall, and then the ribcage was cut while carefully avoiding the lung tissue. The thymus was discarded, and the pulmonary artery was cannulated just above the right atrial appendage. The cannula used was made of PE190 tubing that was filled with saline and was then plugged. The heart was removed by cutting just above the ventricles, and was set aside for determination of RVE (RV/(LV+S)). The lungs were carefully removed from the thorax, and were inflated and deflated several times to prevent atelactasis. They were then transferred to the perfusion apparatus. B. General perfusion procedure Two different perfusion apparatuses were used. The one depicted in Figure 4 was used when the lungs were perfused with a buffer. The chamber in 59 L Ventilator {(1 95% 02 lsz COJ Recorder i : ‘khnnudfiier’ Pressure Transducer Peristoltic ;o_'.::.'.'.::=' Pump I ‘ 37° Water Bath FEE Perfusate Reservoir Figure 4. Diagram of isolated perfused lung preparation. 60 which the lung was suspended by the tracheal and pulmonary arterial cannulae was surrounded by water at 37°C. The perfusion medium, kept cold in an ice bath outside of the apparatus, was warmed as it circulated through the tubing in the water jacket. When blood was used as the perfusion medium, the perfusion apparatus consisted of a chamber maintained at 37°C, and the blood perfusate was stored in the chamber throughout the perfusion. The lungs were prevented from drying in the heated chamber by surrounding them with a "curtain" of moistened gauze pads. Perfusion medium was pumped at a constant flow through the pulmo- nary artery and, after exiting the vasculature through the pulmonary vein, was either allowed to drip back into the perfusion medium reservoir (recirculating system) or was collected or discarded (single-pass system). When blood was used as the perfusion medium, a silk filter was inserted between the pump and the lung to prevent thrombi from reaching the lung. For some experiments, the lungs were statically inflated with 2-3 ml of room air. For other experiments, the lungs were ventilated with warmed, humidifed 95% 02/5% COz using a small animal respirator (Mallard Medical Co., Irvine, CA). Inspiratory pressure was 13-16 cm H O, and a positive end- 2 expiratory pressure of 2-3 cm H20 was maintained. Inflow perfusion pressure was monitored with a Statham P23ID pres- sure transducer and was recorded on a Grass Model 7 polygraph. X. Effect of Ketanserin on the Vascular Response to 5HT in Isolatedi’erfused hues: Rats to be used as blood donors or lung donors were treated with MCTP (4 mg/kg) and were co-treated with ketanserin or its vehicle, distilled water, as described below. Fourteen days after treatment with MCTP, legs from rats treated with ketanserin or water were perfused with blood from rats treated with 61 ketanserin or water, respectively. Perfusions were performed 10-12 hours after the final dose of ketanserin or vehicle. Blood donors were lightly anesthetized with ether, and blood was withdrawn from the abdominal aorta into syringes containing heparin (final concentration = l U/ml). Blood was stored in the perfusion chamber until the perfusion. A 30 ml reservoir and a recirculating system was used. A gentle stream of humidifed 95% 02/5% C02 was passed over the blood to maintain pH. In addition, during the perfusion pH was monitored, and Na CO3 (0.5M) or NH 2 4 maintain pH between 7.35 and 7.45. CI (0.01 M) was added to the blood as appropriate to A 22 gauge needle, inserted through the stopper in the bubble trap close to the pulmonary arterial cannula, was attached to a 3-way stopcock and was used for delivery of drugs. Lungs were perfused for 10 minutes at 10 ml/min, then for an additional 10 minutes at 20 ml/min. After this equilibration period, saline (0.2 ml), angiotensin H (AH; Ciba-Geigy Corp., Summit, NJ, 0.5 11g) and 5HT (creatinine sulfate complex, 50 11 g) were infused into the pulmonary artery in that order. After injection of each drug, an additional 0.2 ml saline was injected. At no time did saline produce a change in perfusion pressure, and, following infusion of AH, pressure was allowed to return to baseline before infusion of 5HT. The perfusions were terminated 5 minutes after injection of 5HT. XL Prostanoid Release in Isolated, Buffer-Perfused Lungg A. Day 7 after MCTP treatment Ltmgs from rats treated 7 days earlier with MCTP (4 mg/kg) were perfused at 10 ml/min in a single pass system using Krebs-bicarbonate buffer containing 4% bovine serum albumin (BSA) (Fraction V, Miles Biochemicals, Inc., Elkhart, IN). The limgs were perfused for 30 minutes and ventilated as described above. Effluent samples were collected periodically into polypropylene tubes 62 containing indomethacin (final concentration = 100 11M) and samples were kept frozen (-70°C) until determination of prostanoid concentrations. In one experiment, lungs from a separate group of rats not treated with MCTP, but treated 1-2 hours earlier with indomethacin (10 mg/kg, i.p.) were perfused in a similar manner with perfusion medium containing indomethacin (0.5 mM). B. Day 14 afer MCTP treatment Lungs from rats treated 14 days earlier with MCTP, and lungs (from untreated rats) used to determine the concentration-response relation to arachi- donic acid, were perfused at a flow of 8 ml/min in a non-recirculating system. These lungs were pre-perfused for 15 minutes with a Krebs-bicarbonate buffer containing 4% BSA. During this period the lungs were ventilated as described above. At time 0 (t = 0), the perfusate was switched to a BSA-free, Krebs- bicarbonate buffer, and the lungs were statically inflated with 2-3 cc room air. After a 10-minute stabilization period to wash out any remaining BSA, effluent samples were collected every minute as described above. After an additional 3 minutes (at t = 13 minutes), arachidonic acid (AA), prepared as described below and at the concentration indicated, was infused directly into the pulmonary artery at 0.1 m1/min. The AA was delivered from a 5 cc syringe using a syringe pump (Model 341, Orion Research Inc., Cambridge, MA). PE20 tubing connected the syringe to a T-connection at the bubble trap and was also fitted from the T- connection through the st0pper in the bubble trap to the pulmonary arterial cannula. The effect of edema formation on the release of prostanoids was determined in isolated, perfused lungs from untreated rats. The left atrium was cannulated and outflow pressure was elevated to approximately 8 cm H20. The lungs were perfused with a BSA-free buffer at a flow rate of 40 ml/min until they 63 appeared to have taken on fluid. At that time, outflow pressure was returned to 0 cm H20’ and the flow was decreased to 8 ml/min. After 10 minutes, effluent samples were collected every other minute for 12 minutes. C. Preparation of arachidonic acid for infusion into isolated, perfused lungs Arachidonic acid was obtained from Sigma Chemical Company (St. Louis, MO) as the free acid. It was dissolved in absolute ethanol and stored frozen under nitrogen. Just prior to use, NaOH was added to convert it to the sodium salt, and appropriate dilutions were made with saline. Purity of the arachidonic acid solution used in the perfusions was checked periodically by thin-layer chromatography (TLC). High performance silica gel plates (Whatman LHP-K) and a solvent system of ethyl acetate/acetic acid (99/1, v/v) were used. The plates were developed with iodine vapors and only one spot (average retention factor of 0.62:0.3) was observed. XH. Prostanoid Release in Blood-Perfused Lungg Arterial blood was collected from untreated, ether-anesthetized rats into syringes containing sodium citrate (0.38% final concentration). Approximately 50 ml of blood were collected for each perfusion. This was kept at 37°C in the perfusion apparatus, and a gentle stream of humidifed 95% 02/5% C02 was passed over the blood to maintain pH. Lungs were ventilated as described above. The perfusate flow was 8 ml/min, and a single-pass system was used. The lungs were pre-perfused with a Krebs-bicarbonate buffer containing 4% BSA for 30 minutes to clear the vasculature. After this stabilization period, the lungs were perfused for 4 minutes with blood. Platelet number, hematocrit and pH of the inflow and effluent blood perfusate were measured for each perfusion. Values for each of these were not different in the effluent blood perfusate of lungs from treated and control rats. 64 A sample of the blood was taken from the reservoir just prior to the perfusion, and samples of the effluent were collected every minute after switching to blood perfusate. Blood was collected into polypropylene tubes containing indomethacin (final concentration, 100 11M), and these were immedi- ately spun in a centrifuge to obtain plasma. The plasma was then frozen at -70°C until analysis of prostanoids by radioimmunoassay. The concentration of Tsz was determined in unextracted plasma, and the concentration of 6-Keto PGFla was determined in plasma extracted as described below. In addition, for lungs from rats treated 14 days earlier with MCTP, samples of buffer from the last minute of the pre-perfusion period were collected as described above for determination of prostanoids. Occasionally, platelet activation occurred during the process of drawing blood for the perfusion. This was indicated by a relatively high concentration of Tsz in the blood prior to perfusion. Therefore, any lung for which the preperfusion concentration of TxBZ was greater than 0.2 ng/ml plasma was omitted from the study. XIII. Determination of Prostanoids by Radioimmunoassay A. General Procedure The concentrations of thromboxane B2 (Tsz) and 6-keto prostaglandin F1 (1 (6-keto PGF1 a)’ which are stable metabolites of thromboxane AZ and prostacyclin (PGIz), respectively, were determined in biological fluids by radio- immunoassay (RIA). Specific antibodies and antigens were purchased from Seragen Inc. (Boston, MA), and radioactive antigens (3H) were purchased from Amersham (Arlington Heights, IL). The cross-reactivites of the antibodies as reported by Seragen, Inc., are shown in Table 1. 65 TABLE 1 Cross Reactivities (at 50% B/Bo) of Specific Antisera Used in Radioimmunoassays A Anti-6-Keto PGFla Anti-Tsz E Tsz < 0.1 100 6-Keto-PGF1a 100 < 0.1 PGFm 7.8 < 0.1 6-Keto-PGE1 6.8 NR PGFZQ 2.2 < 0.1 PGEl 0.7 < 0.1 PGEZ 0.6 < 0.1 PGDz < 0.1 < 0.1 PGA1 < 0.1 < 0.1 PGAz < 0.1 < 0.1 PGB1 < 0.1 < 0.1 PGBZ < 0.1 < 0.1 1 5-Keto-PGF2a < 0.1 NR 1 S-Keto-PGEz < 0.1 NR Dihydroxy keto E2 < 0.1 NR Dihydroxy Keto F2“ < 0.1 NR Dihydroxy Keto E1 NR < 0.1 Dihydroxy Keto Fla NR < 0.1 S-HETE NR < 0.1 12-HETE NR < 0.1 15-HETE NR < 0.1 As reported by Seragen, Inc. Cross reactivity = amount A bound; 100 HETE = hydroxyeicosatetraenoic acid NR = not reported. amount B bound 66 The antibodies were reconstituted in phosphate buffered-saline con- taining 0.5% gelatin (Difco Laboratories, Detroit, MI) (PBSG) at a dilution that would bind 40% of the labelled antigen ("trace") in the absence of unlabelled antigen. The trace was also prepared in PBSG. A stock standard solution of unlabelled antigen was prepared in PBSG, and this was diluted with the appropri- ate medium for working standards used in the assay. The appropriate medium for standards used in an RIA was the same biological fluid as the samples to be analyzed. Charcoal-stripped plasma, prepared as described below, was used to construct standard curves for determination of prostanoid concentration in plasma. Equal volumes (100 111 each) of trace, antibody, and standard or sample were mixed in an assay tube. A complete standard curve was run with each assay. In addition, tubes were prepared for determination of non-specific binding of the trace to the antibody and for determination of total radioacivity bound in the absence of unlabeled antigen (B0). The tubes were refrigerated for 12-24 hours, then decolorizing carbon (NoritA, J.T. Baker Chemical Co., Phillipsburg, NJ) coated with dextran (T70, Pharmacia Fine Chemicals, Uppsala, Sweden) (dextran- coated charcoal) was used to separate the trace that was bound to antibody from the tmbound trace. After exposure to the charcoal solution for 12 minutes, the tubes were spun in a centrifuge at 0°C (300 x g, 12 minutes). The supernatant, containing the antibody bound to the trace and to the unlabelled antigen, was decanted into scintillation vials and was mixed with 15 ml of scintillation cocktail (Safety Solve, Research Products International Corp., Mount Prospect, E). The vials were placed in a liquid scintillation counter (Beckman LS-3150P) and were counted for 5 mimites for determination of radioactivity. Non-specific binding was subtracted from all standards and samples, and the ratio 0‘ the amount 0‘ 3H bound to the total possible 3H bound (Bo) was 67 calculated. Standard curves were constructed as the logit of this ratio yg. the log of the amount of unlabelled antigen in the tube. The amount of antigen in the sample was then determined from this standard curve. B. Preparation of charcoal-stripped plasma Charcoal-stripped plasma was used as the medium in which standards were made for determination of prostanoids in plasma. Rats were treated with indomethacin (10 mg/kg, i.p.) one hour before blood was drawn from the abdominal aorta. Blood was collected into syringes containing sodium citrate (0.38% final concentration), and was spun for 10 minutes (600 x g). The plasma was then transferred to a clean tube, and a concentrated solution of dextran- coated charcoal was added (100 111 charcoal solution/1 ml plasma). This was vortexed, kept on ice for 15 min, then spun in a centrifuge at 0°C (300 x g, 12 min). The supernatant was then decanted and frozen until use. C. Extraction of prostanoids In some experiments, prostanoids were extracted with ethyl acetate before analysis by RIA (Jaffe and Behrman, 1974). Acidified samples were vortexed with ethyl acetate (3 x volume of sample), then were spun in a centrifuge (600 x g, 10 min). The supernatant was transferred to a clean tube and evaporated to dryness under nitrogen in an ice bath. The residue was dissolved in PBSG, and frozen until analysis by RIA. To determine extraction efficiency, a minimal amount of radioactive antigen was added to a sample of the biological fluid before extraction. The radioactivity in the sample before and after extraction was determined. The extraction efficiency was the radioactivity in the extracted sample as a percent- age of the radioactivity in the sample before extraction. Values for the concentrations of prostanoid in all extracted samples were corrected for extrac- tion efficiency. 68 XIV. Drug Treatments A. Ketanserin Ketanserin was supplied as a gift from Janssen Pharmaceutica, Beerse, Belgium. Ketanserin (2.5 mg/kg) or its vehicle, distilled water, was administered by gavage twice daily. Treatment started three days after administration of MCTP and continued through day 14. The effectiveness of the dosing regimen was verified in two studies. In the first study, the end-point examined was the change in the transmittance of light (shape change) observed in PRP in response to 5HT (Drummond and Gordon, 1975; Laubscher and Pletscher, 1979). At various times following administration of ketanserin, rats were anesthetized with ether, and blood was collected from the abdominal aorta for preparation of PRP by the first method described above. The shape change of the platelets in response to 5HT (creatinine sulfate complex, 1 11g), measured as the maximal decrease in light transmittance (Born, 1970), was observed using a Payton dual channel platelet aggregometer. In the second study, the effect of co-treatment with ketanserin on the vascular response to 5HT was examined in isolated, perfused lungs of MCTP-treated rats as described above. B. Ibuprofen Ibuprofen was supplied as a gift from The Upjohn Company (Kalama- zoo, MI) by Mr. Peter Chelune. Sodium ibuprofenate (10 or 17.5 mg/kg ibuprofen) or its vehicle, saline, was administered to rats three times daily by gavage. Treatment began at the time of administration of MCTP and continued through the end of the study. Drug effectiveness was determined as inhibition of the platelet aggregation response to sodium arachidonate, and as a decrease in the concentra- tion of thromboxane B2 in the plasma of treated rats. 69 C. Dazmegrel UK38485 (Dazmegrel, 3-(1H-imidazol-l-yl-methyl)-2-methyl-lH-indo- le-l-propanoic acid) was a gift from Pfizer Central Research (Sandwich, Kent, England). Dazmegrel (50 mg/kg; dissolved in alkaline saline, pH=8.5) or its vehicle was administered twice daily by gavage starting at the time of administration of MCTP and continuing through the end of the study. Drug effectiveness was determined as the decrease in the concentra- tion of thromboxane B2 in the plasma, platelet-rich plasma, or platelet-poor plasma of treated rats. D. L-640,035 L-640,035 ((3-hydroxymethyl)dibenzo [b,f] thiepin-5,5-dioxide) was sup- plied as a gift from Merck Frosst Canada, Inc. Polyethylene glycol (approximate molecular weight = 200, diluted with an equal volume of distilled water) was used as the vehicle. L-640,035 (50 mg/kg) or its vehicle was administered by gavage three times daily starting on the day of administration of MCTP and continuing through the end of the study. The effectiveness of this dosing regimen was confirmed as a decrease in the right ventricular pressor response to intravenous administration of a stable endoperoxide analogue and thromboxane mimic, U46619 (5z,9 (1,11 a,13e,153)-11,9- (epoxymethano)prosta-5,13-dien-1-oic acid (supplied by Mr. Peter Chelune of The Upjohn Company, Kalamazoo, MI). U46619, dissolved in ethanol and diluted with saline, was introduced through a catheter (PE10) positioned in the left femoral vein. Right ventricular pressure was measured as described above. XV. Statistical Analysis Data are expressed as mean i S.E.M. In experiments having only two groups, the Student's t-test was used to compare means (Steel and Torrie, 1980). 70 The t-test for percentages was used to compare the survival of MCTP-treated and control rats. A completely random design one-way analysis of variance (ANOVA) was used to make comparisons among three or more groups. Prostanoid release from isolated, perfused lungs was analyzed using a mixed-design ANOVA, and individual comparisons were made with the least significance difference (lsd) test for between groups comparisons and Dunnett's test for within group comparisons. The effect of drug treatments on MCTP toxicity was evaluated using a two-way factorial ANOVA, and Tukey's w-procedure was used to make individual comparisons. Homogeneity of variance was tested using the Fmax procedure, and, when data violated the assumption of homogeneity, logarithmic transformation of the data was performed. If the data remained non-homogeneous, pre-planned comparisons were made using the Wilcoxon-Mann-Whitney two-sample test (rank sum test). In all cases, a 95% confidence level was used as the criterion for significance. RESULTS L Dose/Response Relation for MCTP A single intravenous injection of 5 mg MCTP/kg body weight produces pulmonary injury and pulmonary hypertension in rats, and by 14 days after treatment, right ventricular hypertrOphy has developed (Bruner gt gt, 1983). However, using this dose of MCTP, in several studies we had experienced high mortality (approximately 50%). The objective of this study was to determine a dose of MCTP which would produce right ventricular enlargement (RVE) by day 14 after treatment but would result in fewer deaths among the treated rats. On day 0, rats received an intravenous injection of DMF or one of 4 doses of MCTP. The doses used were 2, 3, 4, and 5 mg/kg. The concentration of MCTP in DMF was adjusted so that each dose was delivered in a volume of 0.5 ml DMF/kg, and controls received an equivalent volume of DMF. Fourteen days later, the rats were killed and lung injury and RVE were assessed. By day 14, 4 of the 9 rats treated with the highest dose of MCTP (5 mg/kg) had died (mortality = 44%). No other animals in this study died over the 14 day period (n=7 for all other groups). Table 2 shows the effect of the different doses of MCTP on body weight. Body weight gain was suppressed by MCTP treatment at doses greater than 2 mg/kg. RVE developed by day 14 at each dose of MCTP tested (Figure 5A). Wet lung weight/body weight was also elevated in all groups of MCTP-treated rats (Figure SB), as were lavage protein concentration and LDH activity (Figures 5C and 5D). 71 72 TABLE 2 Effect of Treatment with Various Doses of MCTP on Body Weight Gain Treatmenta 3W0 13wF A BW DMF 261: 6 338: 9 +77_+_ 8 2 mg MCTP/kg 256i 7 330110 +74: 5 3 mg MCTP/kg 259: 6 279:19b +2oil9b 4 mg MCTP/kg 26z_+_ 6 270:131’ +9121b 5 mg MCTP/kg 261113 217_+_16b -45: 9b a‘Rats received either i.v. MCTP or DMF on day 0 and were killed 14 days later. BW = initial body weight; BW = final body . o F weight. N = 5-7. bSignificantly different from DMF. 73 O 5 N (D ‘ o s o 0.330 5 15" A O O D ‘9 o 2 12" O :10 an 35 9‘ a \ > 3 1 ° 05 0.301 e 5 i E 5’ 3.. O 25 0 - O 2 3 4 S 0 2 3 4 S MCTP (mg/kg) MCTP (mg/kg) 5.0% 2‘T0 ‘3 4 0r 20» o \ a A o m o "' 16‘- 5 3.01» g v 121 C .2. 2 Cl 111 :0 I 8.. 1- D O 0 1.00 -‘ ' E a 4 o o m 0 son 0 2 3 4 S 0 2 3 4 S MCTP (mg/kg) MCTP (mg/kg) Figure 5. Dose/response relation for MCTP. Rats received either i.v. MCTP or DMF on day 0 and were killed on day 14. (A) RV/(LV+S) = right ventricular enlargement; (B) WL/BW: WL = wet lung weight; BW = body weight; (C) Lavage fluid protein concentration; (D) Lavage fluid LDH activity. N = 5-7. a = significantly different from control (0 mg/kg = DMF). 74 On the basis of these results, doses of 3.5, 4, and 5 mg MCTP/kg were chosen for subsequent studies. H. HistOpathglggy (Development of MCTP-induced Toxicity) The purpose of this study was to correlate the biochemical and physiological alterations caused by MCTP with changes identified histologically. To this end, rats received either MCTP (3.5 mg/kg), DMF, or saline intravenously on Day 0, and were killed 3, 5, 8, or 14 days later. One group of animals was used for the determination of lung injury (lavage protein concentration, LDH activity, and lung weight) and vascular leak, while in a second group pulmonary arterial pressure was measured and the lungs were subsequently fixed and processed for histological examination. All groups of animals were used for the determination of RVE. Animals from all groups were killed on the same day. Lungs were fixed via the vasculature and the airways as described in Methods. Tissues were examined by light microscopy by Dr. James Reindel without prior knowledge of treatment group. Alterations were graded on a 0 (no change) to ++++ (most severe change) scale. There were no differences at any time observed histologically in the lungs from rats which received saline and DMF, therefore only DMF data are presented. Three days after treatment with MCTP, lavage LDH activity was elevated, vascular leak was evident, and the wet/dry lung weight ratio was decreased (Table 3). Lavage protein concentration and the wet lung/body weight ratio were not affected by treatment at this time, and PAP and RV/(LV+S) were not different from control. Changes at the light microsc0pe level were minimal in rats treated 3 days earlier with MCTP (Table 4). 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Lymphatics in this interstitial space were distended with edema fluid. A mild perivascular cellular infiltrate was observed, consisting largely of lymphomononuclear cells with fewer polymorphonuclear cells (PMNs). Less frequently, and in more severely affected tissue, an increased margination of white blood cells (WBCs) was noted in muscular and partially muscular vessels, large foamy macrophages were noted in alveolar spaces, and slightly hypertrophic Type II pneumocytes were observed in alveoli. By day 5 after treatment, wet lung weight, wet/dry lung weight ratio, and lavage LDH activity and protein concentration were elevated in treated rats (Table 3). Vascular leak was also evident although pulmonary hypertension and RVE were not manifest at this time. Perivascular and peribronchiolar edema was more pronounced than at day 3 (Table 4), and was associated with smaller-sized vessels and airways. Lymphatics in the interstitial space were dilated. Peri- vascular cell infiltrates were also more prominent than at day 3, again consisting primarily of large lymphomononuclear cells, some of which were hypertrophic, and occasional mast cells. Marginated WBCs were frequently seen. In patchy areas, alveolar septal walls were thickened with edema fluid. In addition, there was a mild cellular infiltrate in the septal wall, mainly lympomononuclear cells. HypertrOphied alveolar Type II cells were observed, and enlarged macrophages were noted in the alveolar walls and lumen. In areas of marked perivascular edema, separation of vascular smooth muscle cells and hypertrophy of individual cells were observed. Lavage protein concentration, lavage LDH activity, wet lung weight, and vascular leak were increased at day 8, but RVE was not evident (Table 3). Two of the five rats for which PAP measurements were obtained had pressures in the normal range so that the mean of all treated animals at this time was not significantly different from control. However, the PAPs of the other 3 treated 78 rats were greater than the PAP of any control rat. When the histopathology of these 5 treated rats was compared, one treated rat with normal PAP showed only mild changes, not very different from control lungs. Changes in the other rat with normal PAP were not unlike changes in treated rats with elevated PAP. At day 8, variability in the severity and extent of lesions observed histologically was noted among the eight treated rats evaluated, and also among and within the lung lobes of each rat. Perivascular edema was associated with large, medium, and small muscular vessels, and perivascular cellular infiltrates were mostly lympho- mononuclear cells with fewer PMNs (usually eosinophils). WBC margination was prominent and platelet thrombi were also occasionally observed in vessels of various sizes. Accumulation of edema fluid in the alveolar septae and alveolar lumen was more pronounced at day 8 than at earlier times examined (Table 4). Hypercellularity of the alveolar wall was observed, due to infiltration of lymphomononuclear cells and hypertrOphy of interstitial cells. Large foamy macrophages, containing phagocytized cellular debris, were commonly seen in the alveolar spaces. In the bronchiolar epithelium, surface blebbing of Clara cells was less pronounced. A general proliferative response was evident: hypertrophy of interstitial cells and alveolar Type 11 cells, and increased numbers of mitotic figures in the perivascular and alveolar septal wall interstitium, alveolar sacs, and bronchiole epithelium were observed. Nuclear dust from degenerate cells was frequently associated with mitotic figures. In areas of severe perivascular edema and marked cellularity endothelial cell hypertrophy and separation of medial smooth muscle cells were observed, as well as hypertrophic smooth muscle cell nuclei. By day 14, wet lung weight, the wet/dry lung weight ratio, lavage protein concentration, lavage LDH activity, and vascular leak were increased in MCTP- treated rats (Table 3). Pulmonary hypertension and RVE were also evident. 79 Alterations observed by light microsc0pe were similar to those seen at day 8, only increased in severity and extent (Table 4). In addition, marked changes were noted in the bronchiolar epithelium, and the bronchiolar epithelium had extended and lined alveolar ducts and sacs, replacing the normally thinner epithelium. Alveolar lumens often contained fibrin strands and macrophages with phago- cytized cellular debris. Although vascular alterations were, for the most part, mild, endothelial cell blebbing and smooth muscle cell separation and hypertrophy were frequent observations. Although morphometric analysis was not performed, in areas of severe injury some vessel walls appeared slightly thickened. In summary, the first alterations observed, at day 3 after MCTP treatment, were vascular leak and perivascular edema. By day 5 lavage protein concentra- tion was elevated, and infiltration of lymphomononuclear cells was observed in perivascular connective tissue. Lung lesions progressed as indices of lung injury worsened. By day 8 there was marked cell proliferation in the airways, and edema fluid and increased numbers of macrophage were noted in the alveoli. These alterations were more extensive and severe at day 14, and there was some indication of smooth muscle hypertrophy in pulmonary vessels at this time. For the most part, vascular alterations were unimpressive, and the most marked alterations were observed in the airways. III. Diet Restriction and MCTP-induced Cardigmilmonary Toxicity Hayashi and coworkers (1979) demonstrated that reduction of food intake that was associated with retarded growth inhibited the cardiopulmonary changes induced by MCT and prolonged survival in MCT-treated rats. Because diet and nutritional status can alter metabolism of xenobiotic agents (Kato and Gillette, 1965; Mgbodile _e_t 31., 1973), it was unknown whether this protective effect was due to decreased bioactivation of MCT to MCTP. The purpose of this study was 80 to determine whether diet restriction had a similar protective effect against the cardiopulmonary response to MCTP, which apparently does not require bioactiva- tion to exert its toxicity (Bruner gt 31,, 1986). A 2x2 factorial design was used. Rats received either MCTP (5 mg/kg, i.v.) or DMF, and were either allowed to eat a_d_ libitum or were restricted to 9 g food/rat/day. All rats were killed 14 days after treatment. A. Effect of dietary restriction on MCTP-induced cardiopulmonary toxi- city Body weights for the 4 groups of animals are shown in Figure 6. MCTP-treated rats that were allowed free access to food gained less weight than control rats which ate a_d_ li_b. The body weights of MCTP-treated and control rats restricted to 9 g food/rat/day declined at a similar rate. Only two animals died during the course of the study; both were MCTP-treated, diet-restricted rats, and both died on day 12 after administration of MCTP. Diet-restricted animals in both the MCTP-treated and control groups had lower liver weights than the respective ad lib groups (Table 5). Kidney weight and SGOT were not affected by MCTP treatment or by diet restriction. BUN was elevated in DMF diet-restricted rats compared to the ad _l_i_l_)_ controls, perhaps reflecting protein catabolism in diet-restricted rats. MCTP treatment produced an increase in lavage protein concentration (Figure 7A) which was significantly lower in MCTP-treated, diet-restricted rats than in MCTP-treated rats which ate a_d_ Q. The LDH activity was elevated in the lavage fluid of MCTP-treated rats from both the diet-restricted and _a_d_ Lib groups, and there was no significant difference between these two groups (Figure 7B). The lung/body weight ratio was elevated by MCTP treatment and diet restriction attenuated this increase (Figure 7C). MCTP-treated rats allowed to eat £1 li_b had RVE, as indicated by an increased RV/(LV+S) ratio (Figure 7D). However, in MCTP-treated rats fed a 81 O DMF 52 . D DMF RESTRICTED DIET O MCTP AD LLB. I MCTP RESTRICTED DIET 300 - I O 250- - BODY WEIGHT (9) V 200 1504 O 2 4 6 8 10 l2 14 DAYS AFTER TREATMENT Figure 6. Effect of diet restriction on body weight of MCTP-treated rats. Rats were treated with either MCTP (5 mg/kg) or DMF and were allowed to eat ad libitum or were restricted to 9 g food/rat/day. Open circles = DMF ad libitum; open squares = DMF-restricted diet; closed circles = MCTP 1‘1 libitum; closed squares = MCT-restricted diet. N = 7-10. 82 TABLE 5 Liver Weight, Kidney Weight, Blood Urea Nitrogen (BUN) and Serum Glutamatic Oxalacetic Transaminase (SGOT) Activity in Diet~restricted, MCTP-treated Rats Treatmenta DMF MCTP . Restricted . Restricted £9 £12 Diet fl L41) Diet Liver Wt/BW (x100) 4.19:0.12 3.3 810.101) 3.98:0.13 3.43._+_o.11C Kidney Wt/BW (x100) 8.0103 9.43303 9.2.10.8 9.6103. BUN (mg %) 17: 2 29: 4" 22:3 30: 4 SGOT (SF Units/ml) 101:10 123:20 91:7 99:11 3‘Rats were treated with MCTP (5 mg/kg) or DMF and were allowed to eat fl libitum or were restricted to 9 g food/rat/day. Animals were killed 14 days after treatment. BUN = blood urea nitrogen; SGOT = serum glutamic oxalacetic transaminase. N = 7-10. bSignificantly different from DMF a_(l libitum group. c:Significantly different from MCTP £1 libitum group. 83 Figure 7. Effect of diet restriction on (A) lavage protein concentration, (B) lavage LDH activity, (C) lung weight/body weight, and (D) RV/(LV+S) in MCTP-treated rats. Rats were treated with a single i.v. injection of either MCTP (5 mg/kg) or DMF and were allowed to eat 1"; libitum or were restricted to 9 g food/rat/day. Animals were killed 14 days after treatment. Open bars = a_d_ libitum; solid bars = restricted diet. N = 7-10. a = significantly different from DMF a_d_ libitum group. b = significantly different from DMF-restricted diet group. c = significantly different from MCTP ad libitum group. - RESTRICTED DIET Emma 84 D mm, - RESTRICTED DIET Jul __m Ll__m . T..— w .|_ m .I .H. D C D mmm .89... Eon! 50.26.25 oz... 3.2.3. Q25‘ 1 d d u _d d I 141 l21 0A5« 11 Lm Ila—l Jul J _m w Dw A B a! w. w w m. a a . u u e e .. .. at)... 2:02 .93: 322:3 .92: Figure 7 85 restricted diet, this ratio was significantly less than in MCTP-treated rats fed g1 lib and was not different from diet-restricted controls. B. Effect of Diet Restriction on Survival of MCTP-treated Rats Because it appeared that diet restriction afforded some protection from the cardiopulmonary toxicity of MCTP, it was of interest to determine whether a reduction in food intake allowed MCTP-treated animals to live longer. As shown in Figure 8, between 10 and 2.8 days following treatment with MCTP, the fraction of rats surviving was greater in the food-restricted group. However, after day 29, there was no significant difference in the number of rats surviving, suggesting that the effect of diet restriction to prolong survival time of MCTP- treated rats is temporary. Animals surviving through day 41 were killed, and lavage fluid LDH activity and RVE were determined. The lavage fluid LDH activity was not significantly different between the two groups (Table 6) and was close to that of rats not treated with MCTP (compare Figure 7B). The RV/(LV+S) was significant- ly lower in the diet-restricted group, suggesting that the effect of diet restriction against MCTP-induced RVE is sustained through 40 days following treatment. The results of these studies indicate that reduction of food intake or body weight gain attenuates the cardiopulmonary effects of MCTP. Therefore it is important in drug treatment studies to choose a dose of the drug which does not by itself retard weight gain. IV. Effect of Thrombocytopenia on MCTP-induced Pulmonary Hypertension One of the first experiments performed to test whether the platelet might be involved in the response to MCTP was to examine the toxicity of MCTP in rats with decreased numbers of circulating platelets (Hilliker gt a_l., 1984a). In 86 $38 madam on» no 95% 8333 Ivlm. 80$ moonouowfiw «QUEEN? oumomvfi mxmmuoum...‘ .3 n 9,833.2 no .8982” finfimfi 42.5 550.25 thumquom m 0 on noaomuumou who? .8 3:3 @303 noon on mucous ovum coupons who? was Amfimfl 3 @902 5MB @3865 our? 3mm .mfifiefism 3.2 noumobIgUE mo gonads 65 no noflombmou “036 no «oommm .w 6.3th 87 mm on ma w 2&3 02555.5 «>43 cu m— o— ..mg ‘0“ ol'l' mmmqllll N— m. ONIAIABflS 51W :10 HBBWflN 88 TABLE 6 Lavage LDH Activity and Right Ventricular Enlargement in Rats Surviving 41 Days After MCTP Treatment Treatmenta Ad _L_i_b Restricted Dietb Lavage LDH (U/dl) 4.5:0.8 4.910.? RV/(LV+S) 0.434:0.063 029410.020‘: N 4 7 aRats were treated with i.v. MCTP (5 mg/kg). Animals surviving 41 days were killed and their lungs were lavaged as described in METHODS. bRestricted diet = 9 g food/rat/day. CSignificantly different from g libitum group. 89 preliminary experiments a goat anti-rat platelet antiserum (PAS) was admini- stered to rats, and the number of circulating platelets could be decreased to 10- 20% of control for a period of 2 days. Treatment with the PAS for a longer period of time caused the animals to become ill as evidenced by ruffled appearance, inactivity and loss of weight, and some of the animals died. When rats treated with MCTP on day 0 were made thrombocytopenic with this PAS from days 0-2, lung injury and RVE assessed at day 14 were not different from MCTP-treated rats with a normal platelet count (Hilliker g1_: g1.” 1984a). However, when rats were made thrombocytopenic from days 3-5 or days 6-8, RVE was not as severe at day 14 as in rats treated with control serum (CS). This protective effect was greater when the period of thrombocytopenia was from days 6-8 than days 3-5. MCTP-induced lung injury was not altered in rats killed at day 14 by co-treatment with the PAS. This attenuation of RVE suggested that the platelet may be involved in MCTP-induced pulmonary hypertension since RVE is thought to be a consequence of the sustained increase in pulmonary arterial pressure in this model. To determine whether decreasing the number of circulating platelets attenuated the increase in pulmonary arterial pressure as well as RVE, MCTP- treated rats were co-treated with PAS, and pulmonary artery pressure was measured directly. It was first necessary to develop another antiserum to rat platelets, and this was performed in a goat as described in METHODS. The potency and specificity of the PAS was determined jg fig. The PAS was then used to examine the effect of severe thrombocytopenia (< 5%) and moderate thrombocytopenia (IO-2.5%) on MCTP-induced cardiopulmonary toxicity. A. Antiserum Characterization Preliminary experiments were performed in untreated rats to deter- mine a non-toxic dose of the PAS which would deplete platelets. Treatment with 90 up to 2.0 ml (i.p., a single dose) of the new PAS produced no overt signs of toxicity such as loss of weight or ruffled appearance, and platelet number was depressed by approximately 95%. In a more extensive study, rats were bled from the tail for an initial platelet count, and they were then treated with 1.5 m1 (i.p.) of CS or PAS at time 0. These rats received a "booster" of 0.5 m1 (i.p.) CS or PAS (respectively) at 36 hours. Blood samples were also taken from the tail at various times between 6 hours and 6 days after treatment with CS or PAS, and platelet count and hematocrit were determined. Total white blood cell counts and differential white cell counts were determined at 12 hours. There was no difference in body weight of rats treated with CS or PAS through 6 days after treatment (Table 7). The fact that these rats did not gain weight over this 6-day period could be due to treatment with the sera or to the trauma of being bled so frequently. Platelet number was decreased by 6 hours in rats treated with PAS relative to rats receiving the CS (Figure 9). Platelet number remained low through 48 hours, and it had returned to normal by 144 hours after treatment. Using this dosing regimen, platelet number in PAS-treated rats was 5-10% of the control value (which was approximately 106/111). Hematocrit tended to decrease (non-significant) over time after treatment in both groups (Figure 10), probably due to frequent blood sampling from these rats. At 96 hours after treatment, hematocrit was significantly lower in PAS-treated rats than controls. Total white blood cell number was not different in rats treated with PAS and CS 12 hours earlier (Table 8). The distribution of white cells was largely unaffected by treatment with PAS, except for a greater percentage of basophils in PAS-treated rats. 91 TABLE 7 Effect of Treatment with PAS on Body Weight (g) Time After Treatmenta Treatment (Days) CS PAS 0 35918 3631'; 3 1 346:7 339110 2 338:6 333110 3 342:6 334:10 4 341 :5 33232.10 6 343:8 337110 aRats received CS or PAS at time 0 (1.5 ml) and again 36 hours later (0.5 ml). N = 4-5. 92. 683 953 «5 «m m0 89G 333va haugommmnmmm n i .m Iv u 2 .898—5 «2033 m0 :oflwfifiumumv new :3 93 Beam noxmu mm? @005 .98 m4: muaon om am Emma .98 :8 m.3 o QES um mm PAS MCTP CS PAS CS DMF lfignell 101 TABLE 10 Effect of Severe Thrombocytopenia (Days 6-8) on Toxicity 14 Days after Treatment with MCTP Treatmenta DMF MCTP cs PAS cs PAS Bwiniti a1 (g) 210:4 213:4 210:4 215:4 Bwfinal (g) 318:6 315:9 304:8 291:7 WL/BW (x1000) 4.0:o.1 4.1:o.1 6.7:o.4b 7.5_+_o.5c WL/DL 5.3:o.1 5.3:o.1 60:02 62:03 Lavage LDH Activity 2.8:o.2 2.9:o.3 6.5:1.2b 8.1:1.6C (u/dl) Lavage Protein 0.08:0.02 0.17:0.08 1.12:0.29ID 1.31:0.34c (mg/ml) Platglet Number ND 0.48:0.14 ND 0.37:0.07 (1:10 All) 3‘Rats were treated as described in Table 9, and were killed on day 14. BW = body weight; WL = wet lung weight; DL = dry lung weight; Platelet number was determined 24 hours after the first treatment with PAS. ND = not determined. N = 6-110 bSignificantly different from DMF/CS. cSignificantly different from DMF/PAS. 102 numbers (Figure 12). The same was true of right ventricular pressure. MCTP- treated rats had developed RVE by this time, and this was not different in MCTP- treated rats co-treated with CS or PAS (Figure 13). The results of these studies indicated that this degree of thrombocytopenia neither protected against lung injury nor attenuated the pulmo nary hypertensive response to MCTP. Although both at day 8 and at day 14 there was a slight decrease in pulmonary artery pressure in MCTP-treated rats made thrombocytopenic relative to MCTP-treated rats receiving the control serum, these differences did not reach statistical significance. This was also true of RVE at day 14. Z. Rats made thrombocytOpenic from days 8-10 or 10-12 In a previous study, less severe RVE had been observed when the period of thrombocytopenia was from days 6-8 than when it was from days 3-5 (Hilliker _e_t_ _a_l., 1984a). This observation raised the possibility that when platelets were depleted later in the development of MCTP toxicity, more protection was afforded. To test this, rats were treated with MCTP (3.5 mg/kg) or DMF on day 0, and were co-treated with CS or PAS either at days 8 and 9 or at days 10 and 11 to induce thrombocytopenia. Rats were killed at day 14, and lung weight and right ventricular enlargement were assessed. In rats treated with PAS from days 8-10, the platelet number was less than 5% of the platelet number in rats treated with CS (Table 11). However, co-treatment with PAS did not attenuate the MCTP-induced increase in wet lung weight or RVE. When the window of thrombocytopenia was moved to days 10-12, the increase in lung weight and RVE caused by MCTP were not altered by co- treatment with PAS (Table 12). Although platelet number was less than 5% of 103 Figure 12. Effect of severe thrombocytopenia on (A) mean pulmonary artery pressure (PAP) and (B) right ventricular pressure (RVP) 14 days after treat- ment with MCTP (3.5 mg/kg) or DMF. Rats were treated as described in Figure 11 and PAP and RVP were measured on day 14. N = 6-11. a = significantly different from DMF/CS. b = significantly different from DMF/PAS. 104 I b / //////////A 40-- 36-- h b b b a d 1 q 2 8 3 P 1 20-- 16 u 2 2 Amxesv dm PAS MCTP CS PAS CS DMF Figure 12 105 .mfiazo an: 8235 €862:an u n .moEeS 862a 8283 3283?? n n .23 n z .: 28E an 8888 an cannons 68? 3mm .maqd Ax? cmumobuoo mum." vmumobthUE E 3 how «a 80803.2an adusomuaaoar 39% .MH magma 106 . mH 2de deoz dzo m< mu mu 0. «was —-\\\\\\\\\\\\ -rmN. -tmm. mm. (S+A7)/AH 107 TABLE 11 Effect of Severe Thrombocytopenia (Days 8-10) on the Toxicity of MCTP Treatmenta cs PAS cs PAS Platglet number 11.0:o.2 0.24:0.1b 11.4:o.3 0.13:0.1d (:10 / 111) WL/BW (x1000) 3.8:o.1 4.0:o.1 6.7:o.4b 8.2:1.2C RV/(LV+S) o.2so:o.011 0.258:0.013 o.415:o.osszb o.420:0.023C aRats received MCTP (3.5 mg/kg) or DMF on day 0 and were then co- treated with either CS or PAS on days 8 and 9. Lung weight and right ventricular enlargement were assessed on day 14. Platelet number was determined 24 hours after the first dose of CS or PAS. WL = wet lung weight; BW = body weight. N = 3-4. bSignificantly different from DMF/CS. cSignificantly different from DMF/PAS. dSignificantly different from MCTP/PAS. 108 TABLE 12. Effect of Severe ThrombocytoPenia (Days 10-12) on the Toxicity of MCTP Treatmenta cs PAS cs PAS Platglet number ND 2.1:Z.l ND 0.10:0.02 (110 / 111) WL/BW (x1000) 3.7:o.1 3.3:o.1 7.0:o.sb 3.6:1.1C RV/(LV+S) 0.263:0.007 0.281:0.006 o.373:o.013b o.396:o.021c 3‘Rats were treated with MCTP (3.5 mg/kg) or DMF on day 0, and were then co-treated with either CS or PAS on days 10 and 11. Ltmg weight and right ventricular enlargement were assessed on day 14. Platelet number was deter- mined 24 hours after the first dose of PAS. ND = not determined. WL = wet lung weight; BW = body weight. N = 3-5. bSignificantly different from DMF/CS. cSignificantly different from DMF/PAS. 109 normal in MCTP rats co-treated with PAS, DMF rats co-treated with PAS had a circulating platelet number approximately 20% of normal. The results of these studies indicate that moving the window of thrombocytopenia to times later in the development of toxicity does not alter the response to MCTP. C. Effect of Moderate Thromboqtopenia The studies in which the circulating platelet number was depressed to less than 5% of normal failed to confirm the finding of a protective effect due to co-treatment with PAS. It was hypothesized that there may be a critical level for platelets, and that dr0pping the circulating platelet number below that level may of itself induce injury. Accordingly, a lower dose of the same PAS was given to rats to decrease platelet number to approximately 20% of normal. This was the degree of thrombocytopenia that occurred in the earlier study (Hilliker e_t 11., 1984a) in which attenuated RVE was found. Rats were given MCTP (3.5 mg/kg) or DMF on day 0, and then were co-treated with CS or PAS on days 6 (0.75 ml, i.p.) and 7 (0.5 ml, i.p.). All rats were killed on day 14, and lung injury and pulmonary hypertension were assessed. In a separate experiment using this same protocol, rats treated with PAS were bled from the tail daily through day 14, and platelet number was determined. 1. Effect on pulmonary hypertension The platelet number in rats which received PAS was 23-24% of the platelet number in rats which received CS (Table 13). At this degree of thrombocytopenia, the increases in wet lung weight, lavage LDH activity and lavage protein concentration due to MCTP treatment at day 14 were not affected by co-treatment with PAS. Body weight gain was suppressed in MCTP-treated rats receiving the CS, but not in those receiving the PAS. The wet/dry lung 110 TABLE 13 Effect of Moderate Thrombocytopenia on MCTP-induced Toxicity Treatmenta DMF MCTP cs PAS cs PAS 13winiti a1 (g) 247:5 249:4 250:4 247:4 b 13wfinal (g) 347:6 333:9 323:7 323:5 WL/BW (x1000) 3.5:o.1 3.4:o.1 ss.9:o.4b 5.0:o.2c WL/DL 5.1:o.1 s.o:o.1 5.13:0.2b 5.4:o.1 Lavage LDH Activity 1.7:o.2 2.0:o.3 7.9:1.3b 6.3:o.7c (U/dD Lavage Protein o.os3:o.004 o.1oo:o.009 1.39:0.28b 1.14:0.23C (mg/ml) Plagelet Number 10.1:o.6 2.3:o.4b 9.7:0.6 2.3:o.3d (10 / 111) aRats were given MCTP (3.5 mg/kg) or DMF on day 0. They were co- treated with CS or PAS from days 6-8, and were killed on day 14. WL = wet lung weight; BW = body weight; DL = dry lung weight; Platelet number was determined 24 hours after the first CS or PAS treatment. N = 7-14. 1)Significantly different from DMF/CS. “Significantly different from DMF/PAS. dSignificantly different from MCTP/CS. lll weight ratio was also elevated only in MCTP-treated rats with normal platelet numbers. RVE, present in MCTP-treated rats with a normal platelet count, was abolished in MCTP-treated rats with a decreased platelet number (Figure 14). Pulmonary hypertension developed in MCTP-treated rats co-treated with CS, but in MCTP-treated rats made moderately thrombocytopenic, pulmonary artery pressure was not different from control (Figure 15). This was true of the increase in right ventricular pressure was well. These results indicated that at a relatively modest degree of thrombocytopenia, MCTP—treated rats do not develop pulmonary hypertension and RVE. Lung injury is not attenuated by decreasing the circulating platelet number when examined at day 14. This confirms the previous finding that thrombocyto- penia affords a protective effect against RVE, and extends that finding to protection against elevation of pulmonary artery pressure. 2. Platelet rebound The purpose of this experiment was to examine the effect of co- treatment with PAS on platelet number through day 14 following MCTP treat- ment. Rats were given i.v. MCTP (3.5 mg/kg) or DMF on day 0. On days 6 and 7 they received i.p. either CS or PAS. One group of DMF- and MCTP-treated rats co-treated with PAS (”bled") was bled from the tail immediately prior to the first injection of PAS and every 24 hours thereafter through day 14. Blood samples were taken from the tails of another group of DMF-and MCTP-treated rats co- treated with PAS ("not bled") only 24 hours after the first injection of PAS and again at day 14. Blood samples from rats co-treated with CS were only taken at day 14. Rats were killed on day 14, and lung injury and RVE were assessed. As in previous studies, the increases in wet lung weight and lavage protein concentration due to MCTP were not affected at day 14 by 112 .858: 89G 80.8330 Euamoamawmm n n fining/HQ 80am “.8833 3280389.." n .m .31» u z .3 how no vommommm mm? Aw+>q<>m .wto 93v 80$ wand 8 mu 5?» woumohtou m8? was as how no REG 8 AwVQmE m.mv mBUE $4 83m 083 End .33 woumouuthUE E unofiomuflno 8302827 «am? no «mammoaoonaoufi 32388 no 803m .3 0.39m 113 T —k\\\\\\w_e__ ”\\\\af ._._ l l r—t m (S+/\‘l) //\&j 37» . 34+ .28+ 25 114 Figure 15. Effect of moderate thrombocytopenia on (A) mean pulmonary artery pressure (PAP) and (B) right ventricular pressure (RVP) in MCTP- treated rats. Rats were given i.v. MCTP (3.5 mg/kg) or DMF on day 0, and were co—treated with CS or PAS from days 6-8. PAP and RVP were measured on day 14. N = 7-14. a = significantly different from DMF/CS. b = significantly different from MCTP/CS. 115 32-- 20 4.. 17 PAS MCTP CS PAS DMF CS rI/wflMMMMm O " u u u u u 9. nu n. R. 4 2 2 2 1 1 1 1 Amzssv m>m 10 PAS CS PAS CS MCTP DMF lfiguels 116 decreasing platelet number (Table 14). Lavage LDH activity was significantly higher in MCTP-treated rats co-treated with PAS than in those co-treated with CS. Body weight at day 14 was decreased in MCTP/PAS rats compared to DMF/PAS or MCTP/CS rats. RVE developed in MCTP-treated rats with a normal platelet number, but not in MCTP-treated rats made thrombocytopenic. The platelet number in PAS-treated rats 24 hours after the first dose of PAS was approximately 12% of normal (Table 15). The platelet number in the bled group of rats prior to injection of PAS was slightly but significantly different in DMF (11.610.4x105/ul) and MCTP-treated (10.0:0.5x105/ul) rats (p <0.05). Platelet number in the bled rats remained below 20% of the pre-PAS value through day 8 for both DMF- and MCTP-treated rats (Figure 16). On days 9 and 10 the platelet number was significantly higher in MCTP-treated rats, but there was no difference between these two groups at any other time. From day 11 through day 14, platelet number was above the pre—PAS value for both groups of rats, in MCTP-treated rats reaching approximately twice the normal number of circulating platelets (Figure 16). The magnitude of the overshoot tended to be greater in MCTP-treated rats than controls, but this difference was not statisti- cally significant. On day 14 after MCTP (day 8 after PAS), platelet number was significantly higher in both DMF— and MCTP-treated rats co-treated with PAS relative to their respective CS controls (Table 15). There was no difference in platelet number at day 14 in the bled and not bled groups receiving either DMF or MCTP. These results indicate that not only does treatment with PAS decrease platelet number during the window of thrombocytopenia, but it also causes an overshoot such that platelet numbers are greater than normal later in the progression of MCTP toxicity. This raises a question as to whether the protective effect of treatment with PAS on MCTP toxicity is due to the early 117 TABLE 14 Effect of Thrombocytopenia on Body Weight, Lung Injury, and Right Ventricular Enlargement in MCTP-treated Rats Treatmenta DMF MCTP CS PAS CS PAS Bwinitial (g) 220: 5 21232 214: 5 2.09: 2. c,d 13wfinal (g) 320:13 296:7 288110 246:10 WL/BW (x1000) 3.6:o.z 3.8:0.1 6.410.5b 8.311.6c Lavage LDH Activity 2.2.10.1 Z.2_+_0.1 9.9:2.2b 13.8:O.9¢’d (U/dl) Lavage Protein 0.14:0.02 0.13:0.02 1.84:0.4Zb 2.23:0.71C (mg/ml) RV/(LV+S) 0.24910010 0.245:0.009 o.394_.;o.033b o.313:o.014‘1 aRats were treated on day 0 with MCTP (3.5 mg/kg) or DMF, were co- treated with CS or PAS on days 6 and 7, and were killed on day 14. BW = body weight; WL = wet lung weight. bSignificantly different from DMF/CS. cSignificantly different from DMF/PAS. dSignificantly different from MCTP/CS. 118 TABLE 15 Platelet Number in MCTP-treated Rats Receiving PAS Treatmenta Days After Treatment with DMF MCTP MCTP PAS cs PAS cs PAS 7 1 ND 1.23:0.1 ND 1210.1 14 8 10.5:o.4 14.4:03b 10:110.? 16.7:1.0° aRats received MCTP (3.5 mg/kg) or DMF on day 0, and resceived either CS or PAS on days 6 and 7. Values are platelet number x 10 I111. ND = not determined. N = 7. bSignificantly different from DMF/CS. (:Significantly different from MCTP/CS. 119 :36 08.8 65 do 302»: 80.5 «dogma? Eugoflmammm n * .ficooH 339013 on com: mm? weed mo ”8306.9: “mum may 0... nomum 30336688“ um.— nomo mo vooS may 3 .8983“ «33.2% 38382“ «£3.39 .«0 nofimfiaumuou 9: now 2.2335 .50: «AN am :3 may 80pm “8&3 was» @005 .98 m.ov h pad :8 2.6V 0 Show um m to eeoz eased exec 'ON netezotd 121 event (decreased platelet numbers) or the later event (increased platelet num- bers). V. Examination of the Role of 5HT in MCTP-induced Cardiopulmonary Toxicity Two experiments were undertaken to examine the possible role of 5HT in MCTP-induced cardiopulmonary toxicity. Platelets compete with lung endothe- lium for uptake of 5HT (Steinberg and Das, 1980), and removal of 5HT is depressed in isolated lungs from MCTP-treated rats (Hilliker gt a_l., 1983a). As a result of this, platelets from MCTP-treated rats may store more 5HT than platelets from control rats. Accordingly, the concentration of 5HT in platelets from MCTP-treated rats was compared to that in platelets from control rats. In addition, the effect of co-treatment with a 5HT receptor antagonist on MCTP toxicity was determined. A. Determination of Platelet 5HT Rats were treated with MCTP (3.5 mg/kg) or DMF on Day 0, and were killed on Day 14. Arterial blood (5 ml) was collected from ether-anesthetized rats, and PRP was prepared as described in Methods. The concentration of 5HT was determined in platelet-poor plasma and in the supernatant from sonicated, washed platelet pellets by HPLC. HPLC analysis was performed by Nancy J. Shannon. Fourteen days after treatment with MCTP, ltmg weight was elevated and lavage protein concentration was higher in treated rats compared to controls (Table 16). MCTP treatment also caused RVE. Platelet number was not different in PRP prepared from the blood of MCTP-treated rats compared to controls (Table 17). There was also no difference in the platelet concentration of protein. The 5HT concentration in PPP from 122 TABLE 16 MCTP Toxicity in Rats used to Determine Platelet 5HT Content Treatmenta DMF MCTP WL/BW (x1000) 3.93:0.3 9.110s" Lavage protein (mg/ml) 0.11:0.03 2.07:0.28b RV/(LV+S) 017210.007 0.399:0.026b aRats were treated on Day 0 with MCTP (3.5 mg/kg) or DMF and were killed 14 days later. WL = wet lung weight; BW = body weight; RV = right ventricular weight; LV+S = weight of left ventricle plus septum. N = 5-7. 1)Significantly different from DMF control. 123 TABLE 17 Effect of MCTP Treatment on Platelet Number and Platelet Protein Concentration in PRP Treatmenta DMF MCTP Platelet Number (r105) 8.53:1.1 8.4:1.0 Platelet Protein 0.45:0.10 03310.10 (mg/ml PRP) aRats were treated as described in Table 16 and PRP was collected as described in METHODS. N = 5-7. 124 treated rats was not different from controls (Figure 17B). The platelet content of 5HT was also not different in treated and control rats (Figure 17A). B. Effect of Co-treatment with Ketanserin In this study, rats were treated with MCTP (4 mg/kg) or DMF, and were co-treated with ketanserin (KET) (2.5 mg/kg) or the vehicle (VEH) (distilled water) orally twice daily. The purpose was to determine if KET, a 5HT receptor antagonist, would protect against the cardiopulmonary effects of MCTP. The effectiveness of this dosing regimen to antagonize platelet and vascular 5HT receptors was confirmed. 1. Confirmation of drug effect a. 5HT-induced platelet shape change. 5HT produces a change in the transmittance of light (shape change) in rat PRP (Drummond and Gordon, 1975; Laubscher and Pletscher, 1979), and this is inhibited i_n_ litrg by KET (Lampagnani and DeGaetano, 1982). To determine if the platelet receptors responsible for the 5HT-induced shape change were effectively blocked at this dose of KET (2.5 mg/kg orally, twice daily), blood was collected from rats treated with KET or the vehicle at various times after treatment. PRP was harvested from the blood, and the shape change in response to 5HT was determined. In PRP from control rats, addition of 5HT (0.1-2 ug) dissolved in Tris-HCl buffer consistently resulted in a change in the transmittance of light (shape change) subsequent to a shift in the baseline. A typical recorder tracing from the platelet aggregometer is shown in Figure 18A. Addition of Tris- HCl buffer alone produced only a shift in the baseline (Figure 18B). When rats were treated with KET (2.5 mg/kg, orally) twice daily for 4 days, the shape change in response to 5HT (1 ug) was abolished (Figure 18C) at both 6 and 12 hours after the final KET dose. After treatment for 14 days, the change in light 125 Figure 17. 5HT in (A) platelets and in (B) PPP from rats treated 14 days earlier with MCTP (3.5 mg/kg) or DMF. Platelets were collected and 5HT was determined by HPLC as described in METHODS. N = 5-7. T\\\\\\\\\\\\\\\\\\\\\\\\r \\\\\\\\\\\\n 2O 15" O 5 0 322a... oozes :3 3%. .635 in 127 .838“. no 93m «0 nomfipvm no “.309 6802.3 "“3084 Show w you Add aw30 U038?” was“ some was 63:38 m“ as names 255:8 astound unmaofiia .2: 9:: 323:: #9 commas“ Eon owuognomfis .mQOFHmE E ponmuomou as young. 3582.803 302% mouuuom ow omdommmu am mmS: vomsmuom .vouflomm Bonn hum 30on mo ommoaom .mN 03mg 144 2 drama COflmDmem @O mwpDCwE vm mm ON m: m: 3 NH OH . so ouoconfifiuoti .. my I... glean... t. M l \a Li.» . . n e 4 22;; \ dam a d .\ a m \ p . in . .\ r a. . :m / \.\r/./. \\ w ./1. rim ( he mph 145 TABLE 21 MCTP Toxicity at Day 14 in Rats used in Isolated, Buffer-Perfused Lung Studies Treatmenta DMF MCTP Bwinitial (g) 238111 250115 13Wfinal (g) 338:7 253:14.b WL/BW (x1000) 4.1:0.2 13.9:2.0b WL/DL 6.0:0.2 9.610.9b RV/(LV+S) 0.261:0.005 0.363:0.016b aRats received DMF or MCTP (4 mg/kg, i.v.) on day 0 and were killed on day 14. BW = body weight; WL = wet lung weight; DL = dry lung weight. RV/(LV+S) = right ventricular weight/weight of left ventricle plus septum. N=8. bSignificantly different from DMF controls. 146 Figure 24. Release of (A) 6-keto PGF1 and (B) TxB into the effluent of lungs isolated from control (solid lines) anch MCTP-treated rats (broken lines) 14 days after treatment. The lungs were perfused with Krebs bicarbonate buffer, and at 13 minutes arachidonic acid (80 uM, 0.1 ml/min) infusion into the pulmonary arterial cannula was begun. Samples were collected and prostanoid concentrations were determined by RIA. N=8. a = values significantly different from the value at 12 minutes for the same group. b = significant differences between groups at the times indicated. E-Keto PCqu (mg/m1) A TxBZ (mg/m1) 147 L‘Arochidonote T] 12 14* 16 f is ' zb I. /I / I/ b L: I // b b b O L1Arochidonota ] 12 14 1E5 18 20 Minutes 0? PerFusion Ffigue24 148 TxBZ release increased during AA infusion in lungs from both groups of animals (Figure 243). The concentration of Tsz was higher in the effluent of lungs from MCTP-treated rats than controls both before and during AA infusion. Inflow perfusion pressure was significantly higher in lungs from MCTP-treated rats initially and at the end of the perfusion (Table 22). However, the increase in pressure during the perfusion was not different in the two groups. Because isolated lungs from MCTP-treated rats released more TxB than lungs from control rats at day 14, and because these lungs were 2 also more edematous than control lungs (as evidenced by an increase in wet lung weight, Table 21), the effect of edema formation on the release of 6-keto PGF1 a and Tsz was examined. The mean lung weight/body weight (x1000) following perfusion at increased outflow pressure was 12.811.6 (n=3), similar to that seen in isolated, perfused lungs from rats treated 14 days earlier with MCTP (Table 21). The release of 6-keto PGFla from lungs made edematous by increasing outflow pressure was similar to that seen in lungs from rats treated with MCTP or DMF 14 days earlier, and it did not change during the perfusion (Figure 25A, compare to Figure 24A). The release of Tsz from edematous lungs was relatively high initially, close to the concentration seen in lungs from MCTP-treated rats, but leveled off by 16 minutes of perfusion to a concentration similar to that seen in control lungs (Figure 25B, compare to Figure 24B). The concentration of Tsz in the effluent at 10 minutes was not significantly different from the concentration at 20 minutes. This suggests that the pr0pensity for lungs from MCTP-treated rats to take on fluid may contribute to increased Tsz release. 149 TABLE 2.2 Inflow Perfusion Pressure in Lungs Isolated from Rats Treated 14 Days Earlier with MCTP Treatmenta DMF MCTP b PPiniti 31 (mmHg) 5.810.5 8.010.6 b PPfinal (mmHg) 6.510.4 11.011.4 APP (mmHg) 0.810.4 3.011.1 aRats were treated and lungs were perfused as described in the legend to Figure 24. PP = inflow perfusion pressure. N=8. bSignificantly different from DMF controls. 150 B-keto PGFIO (ng/ml) N U1 10 12 14 16 18 20 22 TxBZ (ng/ml) O 8 0.101va t : i 1 t : I 10 12 14 16 18 2O 22 Time OF Perfusion (min) Figure 25. Release of (A) 6-keto PGF and (B) TxB in isolated lungs made edematous. Lungs from untreated rats were perfused with Krebs bicarbonate buffer at a flow of 40 ml/min. The left atrial cannula was elevated so that outflow pressure was 8 cm H 0. When the lungs appeared to have taken on fluid, outflow pressure was returne to 0 cm H O and flow was decreased to 8 ml/min. After 10 min, effluent samples were collected every other minute. Prostanoid concentrations were determined by RIA. N=3. 151 2. Release in lungs perfused with blood The results of the above studies in which isolated lungs were perfused with buffer indicated that release of TxB but not 6-keto PGFla was 2 greater in lungs from MCTP-treated rats than controls later (day 14) in the development of pulmonary hypertension. Therefore, it was of interest to examine this relationship in isolated lungs perfused with a platelet containing medium to investigate whether the presence of platelets would enhance the difference in TxBZ release in lungs from treated and control rats. Although whole blood contains several cell types which can contribute to the plasma Tx concentration, whole blood was the preferred medium to maintain the platelets in the most natural environment. Blood for the perfusions was obtained from untreated rats. Lungs from rats treated 7 or 14 days earlier with MCTP (4 mg/kg) or DMF were isolated, preperfused with a buffer, then perfused for 4 minutes with blood. Samples of the blood effluent were collected every minute and immediately spun in a centrifuge, and the concentrations of 6-keto PGF1 a. and Tsz were deter- mined in the plasma effluent by RIA. The concentrations of these prostanoids were also measured in the blood prior to perfusion. Lung weight and RVE were recorded as well. a. Day 7 after treatment with MCTP. Seven days after treatment with MCTP, lung weight was significantly greater in treated animals (Table 23). Body weight gain and RV/(LV+S) were not affected by MCTP. treatment at this time. Prior to perfusion, the concentrations of TxB in the blood 2 was not significantly different in the two groups (Figure 26B). The same was true of the concentration of 6-keto PGF1 a in the blood (Figure 26A). The concentra- tion of 6-keto PGFla in the plasma effluent was not significantly different in 152 TABLE 23 MCTP Toxicity at Day 7 in Rats used in Isolated, Blood-Perfused Lung Studies Treatmenta DMF MCTP 13winiti all (g) 241:6 241:7 Bwfinal (g) 29117 2.7616 WL/BW (x1000) 4.4:0.1 5.9:0.3b RV/(LV+S) 0.2310.01 0.2410.01 aRats received MCTP (4 mg/kg) or DMF i.v. on day 0. On day 7, rats were killed and their lungs were isolated and perfused. BW = body weight; WL = wet lung weight. N = 5-6. bSignificantly different from DMF control. 153 Figure 26. (A) 6-Keto PGF dB() TxB in the plasma effluent of isolated lungs from DMF control (solifian lines) and MCTP-treated (broken lines) rats 7 days after treatment. Rats were given MCTP (4 mg/kg) or DMF on day 0 and were killed on day 7. Lungs were isolated, preperfused with buffer, then perfused for 4 minutes with blood. Prostanoid concentrations in the plasma were determined by RIA. P = pre-perfusion. N = 5-6. a = significantly different from the respective pre-perfusion value. 154 p u 5 s 4 3 2 1 O a~e\mcu canon oncx-m o a II: .. 4 / / rml/ 3 / / / ,4 .2 , n r e 1 7.03 +9: 3 TITITITITITITITITITITI. 2 6 2 8 4 O a_5\mcu mmxe Minutes 0? Perfusion' Figure 26 155 lungs from treated and control rats at any time during the perfusion, and it did not change during the period of perfusion (Figure 26A). There was no significant difference in the concentration of Tsz in the plasma effluent of lungs from control or MCTP-treated rats at any time during the perfusion (Figure 263). The concentration of Tsz in the effluent increased as the time of perfusion increased in lungs from both control and treated rats. The numbers of platelets in the blood perfusate and efflu- ent were not different for lungs of MCTP-treated and control rats (Table 24). The difference in platelet number between inflow and effluent perfusate was not different from zero for either of the two groups, indicating that large numbers of perfused platelets were not adhering to the pulmonary vasculature. Perfusion pressure was not significantly higher in lungs from MCTP—treated rats at this time. Hematocrit and pH of the blood effluent at the end of the perfusion was also not different in the two groups (data not shown). To determine any contribution to changes in prostanoid concentration by perfusing blood through the perfusion apparatus alone, similar "perfusions" were performed in the absence of a lung. The concentrations of Tsz and 6-keto PGF in the plasma effluent of these sham perfusions were not In significantly different from those in the lungs of control rats. Platelet number, hematocrit, and pH were also not different (data not shown). b. Day 14 after treatment with MCTP. Body weight gain was suppressed 14 days after treatment with MCTP (Table 25). MCTP treatment also caused an elevation in lung weight and RVE at this time. The plasma concentrations of TxBZ (Figure 273) in the blood before perfusion did not differ between lungs from control rats and those from MCTP-treated rats. The same was true for 6-keto PGFla (Figure 27A). The concentration of 6-keto PGFla was not significantly different in lungs from Perfusate Platelet Number and Inflow Perfusion Pressure 156 TABLE 24 in Isolated Lungs from Rats Treated 7 Days Earlier with MCTP Treatmenta DMF Pt (#x106/u1) 1 1+0 1 inflow ° - ' 6 Pteffluent (#xlo /ul) l.210.1 APt (#x106/ul) 0.0710.03 -0.1l10.07 PPinitial (mmHg) 12.910.5 PPfinal (mmHg) 12.8:0.5 APP (mmHg) -0.0810.3 -0.7010.2 aRats were treated and lungs were isolated and perfused as described in the legend to Figure 26. Pt = platelet; APt difference in platelet number in inflow and effluent blood; PP perfusion pressure. N = 5-6. 157 TABLE 25 MCTP Toxicity at Day 14 in Rats used in Isolated, Blood-Perfused Lung Studies Treatmenta DMF MCTP 13winitial (g) 274:13 267:12 13wfinal (g) 349: 6 265:20b WL/BW (x1000) 5.1:0.4 13.3:2.6b RV/(LV+S) 0.23:0.01 0.31 :0.02b aRats received MCTP (4 mg/kg) or DMF i.v. on day 0. On day 14, rats were killed and their lungs were isolated and perfused. BW = body weight; WL = wet lung weight. N = 7. bSignificantly different from DMF control. 158 Figure 27. (A) 6-Keto PGF and (B) TxB in the plasma effluent of isolated lungs from DMF control (so 13 lines) and NECTP-treated (broken lines) rats 14 days after treatment. Rats were treated i.v. with MCTP (4 mg/kg) or DMF on day 0 and were killed 14 days later. Lungs were isolated and perfused as described in the legend to Figure 26. N = 6-7. P = pre-perfusion. a = significantly different from the respective pre-perfusion value. b = significant difference between MCTP and DMF at the indicated time. 159 1.6" ~ p d 1 2 8 1|“ 0 ALE\mcu cunua cadx-c - p h n n - q q — c S 3 S 2 5 1 3. 2 L Aue\mcu mmxe Minutes 0? PerFusion 160 control and MCTP-treated rats at any time during the perfusion. The concentra- tion of TxB did not change significantly during perfusion of lungs from control 2 rats (Figure 27B). However, Tsz increased after 2 minutes of perfusion in lungs from MCTP-treated rats. After 4 minutes of perfusion, the concentration of Tsz was significantly greater in the effluent of lungs from treated rats than in that of lungs from control rats. In neither the inflow nor the effluent perfusion medium did platelet numbers differ between treatment groups (Table 26). Also, the differ- ence between the platelet numbers in the inflow and effluent perfusion medium was not different from zero for lungs from either treated or control rats. Inflow perfusion pressure at the end of the perfusion was higher in lungs from MCTP- treated rats than in lungs from control rats. Hematocrit and pH of the blood effluent after perfusion through the lung was not different between the two groups (data not shown). Isolated lungs remove circulating Tsz (Robinson g 1511., 1982). The ability of lungs from MCTP-treated rats to extract TxBZ from the blood was evaluated by comparing the difference between the pre-‘perfusion plasma concentration of Tsz and the plasma concentration in the effluent at 1 minute. This is depicted graphically for individual lungs in Figure 28. The extraction value ((preperfusion - l min)/pre-perfusion) was not significantly different for lungs from control (0.3710.07)and MCTP-treated (-0.0910.26) rats. B. Generation of TxB, in Platelet-Rich Plasma One mechanism by which the platelet may contribute to MCTP- induced pulmonary hypertension is through increased release of vasoactive media- tors such as TxAZ. Accordingly, the hypothesis that MCTP treatment i_n vivo alters platelet release of Tx was investigated. 161 TABLE 26 Perfusate Platelet Number and Inflow Perfusion Pressure in Isolated Lungs from Rats Treated 14 Days Earlier with MCTP Treatmenta DMF MCTP 6 Ptinflow (i/XlO lul) 1.010.04 1.010.06 6 Pteffluent (#xlO lul) 0.910.07 1.010.06 APt (#x106/ul) -0.1:0.05 -0.03:0.06 PPinitial (mmHg) 15310.9 23.7143 b PPfinal (mmHg) ll.81l.6 19.9133 APP (mmHg) -3o6ilol -3o3ilo3 aRats were treated and lungs were isolated and perfused as described in METHODS. Pt = platelet; APt = difference in platelet number in inflow and effluent blood. PP = perfusion pressure. N = 6-70 bSignificantly different from DMF by rank sums test (p<0.05). 162 .502 483389 was“ 8.9339: 8 3280292 00: £00m 43538 fi 23 m5: 0”: nmsohfi made 0350 0 8:0 @003 0:“ no 0883 05 3 meh. m0 dogma—“80000 u 80% E30389 08m0n 0003 0.: mo 080.03 0:» 5 meB «0 ”830300280 0 0pm .3 08mg 0”— E8m0~ 05 5 00920030 8 8038a 08B mwndq QED 8 PHD—2 8:8 83.80 998 3 0080.5 m8» Bony awed“ @0883 ha mmxh. mo nomuombxm .mm 0.5th 3. £ng 05.: Hie , .. n. Hmong wig 300 .E Dd / i / --m.o x m 8 1 Z :0; W E / w -m; U. 164 Rats were treated with MCTP (4 mg/kg) or DMF and were killed 1, 4, 7 or 14 days later. Blood was collected and PRP was prepared. Arachidonic acid (AA) was the stimulus for aggregation, and Tsz was measured in the PRP supernatant prior to and during the aggregation response (see Figure 3). The effect of MCTP in mg on platelet aggregation and TxBZ release was also examined. Lung weight, lavage fluid protein concentration, and RV/(LV+S) were determined at days 4, 7, and 14. Body weight gain was suppressed in MCTP-treated rats 7 or 14 days after treatment (Table 27). Lung weight was elevated in MCTP-treated rats by day 4 and remained elevated through day 14 (Table 27). This was also true for the concentration of protein in the lavage fluid. RVE was not evident until day 14. The aggregation response of PRP to AA was largely unaffected by MCTP treatment i_n_ v_iy_g (Table 2.8). The extent of aggregation (maximal aggregation) was not different in PRP from treated and control rats at any time following treatment with MCTP, although there was a tendency toward less aggregation in PRP from treated rats at days 4 and 7. The rate of aggregation (slope) was significantly lower in PRP from treated rats at day 4, but was not different from control at any other time. The delay to aggregation was also not different in PRP from MCTP-treated and control rats. The concentration of Tsz was higher in unstimulated PRP from MCTP-treated rats than control rats at day l, but it was not different from control at any other time (Figure 29A). Comparison of the units for Tsz concentration in Figures 29A and 29B demonstrates that Tsz was released during aggregation, and most of this release had occurred by the time the platelets had reached half-maximal aggregation (compare Figures 298 and 29C). There was a small but significant decrease in the TxBZ released at half-maximal aggregation in PRP from rats treated 7 days earlier with MCTP, but release was not different 165 TABLE 27 Effect of MCTP on Body Weight, Lung Weight, Lavage Fluid Protein Concentration and Right Ventricular Enlargement Days a Fin 1 Lavage Following Treatment a WL/BW (x1000) Protein RV/(LV+S) BW Treatment (mg/ml) 1 DMF 2501 3 ND ND ND MCTP 2481 4 ND ND ND 4 DMF 297115 3'5i0°3b 0.1510.03b 0.28610.028 MCTP 305115 5.410.6 0.8410.02 0.27610.010 7 DMF 2961 4b 4.9_+_0.3b 0.4210.12b 0.22110.012 MCTP 2401 8 8.11l.0 2.8210.49 0.24410.014 14 DMF 374114~b 4.110.?b 0.2410.10b 017910.012b MCTP 278128 10.111.5 2.1310.47 0.37710.016 aOn Day 0 rats received either MCTP (4 mg/kg) or DMF via the tail vein. BW = body weight, WL = wet lung weight. N = 3-8. 1’Significantly different from DMF control. ND = Not determined. 166 TABLE 28 Effect of MCTP Treatment Lg Vivo on Arachidonic Acid-induced Aggregation in Platelet-rich Plasma Days a Maximal Slo e Dela Following Treatment Aggregation 7 p . . y Treatment (%) ( o max/min) (minutes) 1 DMF 721 4 291 3 0.7710.09 MCTP 55118 35111 0.7610.22 4 DMF 60112 341 7b 0.6910.02 MCTP 401 8 181 l 0.9110.09 7 DMF 741 3 291 8 0.6410.08 MCTP 46113 231 4 0.6010.04 14 DMF 53111 261 9 0.7210.07 MCTP 53113 311 9 0.6710.08 aOn Day 0 rats received either MCTP (4 mg/kg) or DMF via the tail vein. Blood was collected, and PRP was prepared. Arachidonic acid (1.5 mM) was used to induce aggregation. For an explanation of the parameters measured, see METHODS. N = 3-8. bSignificantly different from DMF control. 167 10 I D DMF a ‘ (a RV MCYP "(52 (helm!) ‘ - W//// 2.0- 1.6" I 0.4 ‘ 2.2. _L .3— I _L 0.0 ‘ 0.4 ‘ O ‘ ‘— .— '— .— 1 4 7 34 DAYS FOLLOWING TREATMEN? magmas» _ l a. ' I 1—' W , . 1% 1X07 (palm!) Figure 29. TxB generated in (A) tmstimulated PRP and in PRP at (B) half- maximal or (C) maximal aggregation with arachidonic acid (1.5 mM). Rats were treated on day 0 with MCTP (4 mg/kg) or DMF. PRP was prepared as described in METHODS and TxB was measured by RIA. N = 3-8. a = significantly different from DMF control on the same day. 168 at any other time following treatment with MCTP (Figure 293). The concentra- tion of TxB in PRP at maximal aggregation was not affected by treatment with 2 MCTP (Figure 29C). To determine if MCTP had any direct effect on platelets, MCTP was added _ig li_tgg to PRP from untreated rats, the platelets were induced to aggregate with AA, and TxBZ generated at maximal aggregation was measured. DMF (1 111) did not affect AA-induced aggregation or release of Tsz in PRP (Table 29). MCTP, in amounts up to 125 ug in 0.5 ml PRP, also had no effect on the aggregation response to AA or on the release of TxBZ. When 500 11g of MCTP was added to the PRP, the aggregation response was abolished and Tsz release was depressed. The results of this study suggested that platelets from MCTP-treated rats do not respond to stimuli with enhanced release of TxBZ, and that, except at high concentrations, MCTP does not directly alter Tsz release or the platelet aggregation response. C. Effect on MCTP-induced Pneumotoxicity of Drugs Which Interfere with the Smthesis or Action of TxA, If TxAz contributes to MCTP-induced pulmonary hypertension, then co-treatment with drugs which interfere with the synthesis or action of TxA2 should attenuate the toxic response to MCTP. Two different drugs which interfere with the synthesis of TxA were used. Ibuprofen inhibits the enzyme 2 cyclooxygenase (Longenecker gt _a_l., 1985) which catalyzes the conversion of arachidonic acid to PGHZ, the cyclic endoperoxide precursor to TxAz, P612 and PG's of the A-F series. Dazmegrel inhibits thromboxane synthetase (Fischer 311 a_l., 1983), the enzyme responsible for conversion of PGH2 to TxAZ. In addition, a Tx receptor antagonist, L-640,035, was used (Carrier 3111., 1984). 169 TABLE 29 Effect of MCTP Q Vitro on Arachidonic Acid-induced Platelet Aggregation and Release of Tsz Addition to Maximal Slope Delay TxB PRPa Aggregation (%) (% max/min) (minutes) (ug/mzl) None 68110 4119 0.6010.10 l.810.2 DMF 55114 3217 0.6810.l9 1.810.5 31 ug MCTP 2.9115 1715 0.4410.06 1.5-10.4 62 ug MCTP 33:16 22:9 0.4210.08 1.3:o.4 125 1.1g MCTP 28112 16:5 0.38:0.09 1.3:o.2 500 ug MCTP 0:0b NA NA o.5:o.1b a‘DMF (1 111) or MCTP (in 1 ul DMF) was added to 0.5 ml PRP from untreated rats 1 minute prior to addition of Arachidonic acid (1.5 mM). The concentration of TxB 2 bSignificantly different from DMF. NA = Not applicable. was determined at maximal aggregation by RIA. N = 3-4. 170 l . Ibupro fen The purpose of this study was to determine the effectiveness of ibuprofen to alter the response to MCTP. Rats were treated wtih MCTP (4 mg/kg) on Day 0, and were then treated with ibuprofen (10 or 17.5 mg/kg) or its saline vehicle by gavage 3 times daily through the end of the study. Rats were killed 14 days later, and lung injury and RVE were assessed. A preliminary study was performed to determine a dose of ibuprofen which would inhibit Tx synthesis and yet would not retard body weight gain. The drug effect was also confirmed in MCTP-treated rats. a. Confirmation of drug effect. In a preliminary study to determine a dosing regimen which would effectively inhibit platelet aggregation and Tx synthesis, rats were treated with ibuprofen (IBN; 10 mg/kg orally, 3X daily) for 4 days. Blood was collected and PRP was prepared according to the second technique described in Methods. Platelet aggregation was induced with various concentrations of AA, and maximal aggregation was observed. The concentration of Tsz was also determined in PRP prior to aggregation. The concentration of Tsz in PRP from rats treated with ibuprofen was significantly lower (0.410.1 ng/ml) than that from controls (1.210.2 ng/ml). The platelet aggregation response to a low concentration (0.6 mM) of AA was abolished in PRP from rats treated with EN (Table 30). In contrast to the response in saline control rats, the platelet aggregation response in PRP from rats treated with IBN was dose-dependent, and the inhibition due to EN treatment was overcome at higher doses of AA. On the basis of these results, doses of 10 and 17.5 mg/kg (orally, 3 times daily) were chosen for co-treatment of MCTP-treated rats. Co-treatment of MCTP-treated rats with either dose of IBN reduced the concentration of circulating plasma TxB plasma TxB 2’ 2 171 TABLE 30 Effect of Treatment with Ibuprofen (IIBN)a g Vivo on Platelet Aggregation _IE. Vitro Concentration of % Maximal Aggregatlon Na+-Arachidonate (mM) SAL IBN 0.6 74:2 0:0b 0.9 73:2 33:15 1.5 71:2 72:7 aRats received IBN (10 mg/kg) or saline orally 3 times daily for 4 days. Platelet aggregation responses in PRP were observed as described in METHODS. N = 4-6. bSignificantly different from SAL control (p < 0.05, rank sums test). 172 concentrations were 126190 pg/ml in controls, and 30120 and 2718 pg/ml in rats treated with 10 and 17.5 mg/kg IBN, respectively (N = 4-6). These differences did not attain statistical significance due to the large variability in control values. b. Effect of ibuprofen on MCTP-induced cardiopulmonary toxicity. Co-treatment with ibuprofen did not affect body weight in MCTP- treated rats. By comparison of the values in Table 31 to DMF controls at day 14 in Table 27, it can be seen that administration of MCTP resulted in elevation of lung weight and RVE. Ibuprofen, at either of the doses employed, was ineffective at reducing the MCTP-induced toxicity: lung weight/body weight and RV/(LV+S) in either ibuprofen group was not significantly different from the saline control group. The results of this study indicate that co-treatment with the cyclooxyge- nase inhibitor ibuprofen does not attenuate the response to MCTP. 2. Dazmegrel Inhibition of cyclooxygenase can decrease synthesis of PG].Z as well as TxAz, and the vasodilatory and antiaggregatory effects of PGIz may be beneficial in MCTP-induced pulmonary hypertension. Therefore, a drug aimed more specifically at inhibiting TxAZ biosynthesis was employed. The ability of the Tx synthetase inhibitor Dazmegrel to alter the cardiopulmonary response in MCTP-treated rats was examined at the onset (day 7) of pulmonary hypertension and after pulmonary hypertension was well-established (day 14). Treatment with Dazmegrel (50 mg/kg by gavage twice daily) or its saline vehicle began at the time of administration of DMF or MCTP (day 0, 3.5 mg/kg) and continued through the end of the study. Lung injury was assessed at day 7 and day 14, and RVE was assessed at day 14. A preliminary study was performed to determine an appropriate and effective dosing regimen. 173 TABLE 3 1 Lack of Effect of Ibuprofen (IBN) on tahe Cardiopulmonary Toxicity of MCTP IBN (mg/kg) Cotreatment Saline 10 17.5 Winiti a1 (g) 229: 3 225:1 236: 3 BWfimll (g) 241:17 234:3 268110 WL/BW (x103) 8.611.1 7.3:o.9 8. 110.6 RV/(LV+S) 0.34:0.02 0.30:0.02 0.35:0.02 aRats were treated with IBN or with saline three times daily for 14 days following a single injection of MCTP (4.0 mg/kg, i.v.). BW = body weight; WL = wet lung weight. There were no significant differences among any of the means (one-way ANOVA, p < 0.05). N = 4-7. 174 a. Confirmation of drug effect In a preliminary experiment to determine a dosing regimen which would effectively inhibit Tx synthesis, a small number (n = 2-3) of rats were treated with Dazmegrel or saline and were killed 3, 8, or 24 hours later. Blood was collected and PRP was prepared by the second technique described in Methods. The concentration of Tsz was determined in PPP and in PRP stimulated with AA (0.9 mM). The concentration of 6-keto PGF1 a was also determined in PPP. Three doses of Dazmegrel were tested: 25, 50, and 100 mg/kg. The concentration of 6-keto PGF in PPP was not 101 affected by Dazmegrel treatment (Figure 30C). The TxB in PRP and PPP 3 and 2 8 hours after treatment was reduced by all three doses of Dazmegrel (Figures 30A and B). By 24 hours the inhibitory effect disappeared. Both 50 and 100 mg/kg appeared to be slightly more effective at reducing the concentration of Tsz than 25 mg/kg, and on the basis of these data a dose of 50 mg/kg given twice daily was chosen for treatment of MCTP-treated rats. Co-treatment with Dazmegrel decreased the concentration of TxB in the plasma of rats treated 14 days earlier with MCTP or DMF, but it 2 did not affect the plasma concentration of 6-keto PGFla (Table 32). b. Effect of Dazmeggel on MCTP-induced cardiopulmonary toxicity. Co-treatment with Dazmegrel did not alter any of the indices of lung injury examined 7 days after treatment with MCTP (Table 33). Wet lung weight, lavage LDH activity and lavage protein concentration were elevated in MCTP- treated rats, and this increase was not affected by co-treatment with Dazmegrel. The wet/dry lung weight ratio was increased and body weight was decreased at day 7 in MCTP-treated rats co-treated with Dazmegrel. 175 .mum N 2 Jam .3 0088.880 0.03 "830080980 08800095 .328 0.3 080 08003008 8:3 0u0w0umm0 9. 000508 008 080 00980000 00 00890.3 003 mmm .0832, 05000 080 8 <8 H0um08n0Q fits 8:000 088: 2088” 838A: 0N 8 508m 0 00m8v w .2080m no: m 00000.3 30.” mo mam 8 human 30x10 mo 8830080880 0:“ RE 080 mam Am: 080 mmm 33 8 mxh. no 838580880 09H. .om 0.3me W m .' 'o'o‘o' 43:00: 3.0138 I V V U C I! H - 6 a 6 ¢ (l‘“l°U)~'i94 "fin-9 M .\\\\\\\" 3 "DUI! é Q 'C...'.‘. 0‘. o o o 0 0:. 00009‘ 0.0%”... o‘. O fig}??? (1%) ‘au 1 O Iuoufl UNOUIS I H00" 176 W .\\\V E I t:w1°u)"ioa °"’|'9 % m 'o:¢:9:0:0:0:0: .90...o.o.o.a.o O “wfiu, ‘9'] UNI“) ‘9‘: 100 um: 50 0A2 (mglkg) 0 1|0%§ j H N 0663 .— ”I (palm!) 394 "“1-9 100 24 HOURS *9) W3" 0 900'. 0‘9 9 o 0.9 o 9 009 000 $459.”. 25 DAZ (mg .r i 0" O (ltulbu) ‘9 I1 Moots § IOO 0A2 (mg/I19) 7 .x\\\\\\\\\\\\\ 0 I Y 2 S Mums “”1“” ‘9‘] Figure 30 177 TABLE 32. Plasma Tsz and 6-Keto PGFla in MCTP-treated Rats Following Co-treatment with Dazmegrel (DAZ) Treatmenta DMF MCTP SAL DAZ SAL DAZ Tsz (pg/ml) 2901190 14: 5b 25035110 27:10c 6-Keto pGFm (pg/ml) 230: 34 150:43 203: 36 279:73 aRats received DAZ (50 mg/kg) or saline (SAL) orally, twice daily, for 14 days following a single injection of MCTP (3.5 mg/kg) or DMF, iv. '1‘sz and 6- keto PGF1 a were determined by RIA as described in METHODS. N = 6-9. bSignificantly different from DMF/SAL. c:Significantly different from MCTP/SAL. 178 TABLE 33 Lack of Effect of Dazmegrel (DAZ) on the Toxicity of MCTP 7 Days After Treatment Treatment“:l DMF MCTP SAL DAZ SAL DAZ meiti a1 (3) 281:4 279:5 27 5:3 275:3 swiml (g) 324:5 332:7 304:8 302:6° WL/BW (x1000) 3.7:0.2 3.3:0.1 7.3:O.8b 7.23:1.1C WL/DL 5.0:0.1 5.2:0.2 6.6:0.5 7.1:0.7c Lavage LDH Activity 2.1 :0.3 2.0:0.2 zz.3:3.4b 18.3:3.3c (U/dl) Lavage Protein 0.13:0.02 0.13:0.02 2.46:0.59ID 1.24:0.45C Concentration (mg/ml) aRats were treated with MCTP (3.5 mg/kg) or DMF on day 0 and were co- treated with DAZ (50 mg/kg, orally, twice daily) or SAL. Animals were killed 7 days later. BW = body weight; WL = wet lung weight; DL = dry lung weight. N = 6-100 bSignificantly different from DMF/SAL. cSignificantly different from DMF/DAZ. 179 Body weight in rats treated 14 days earlier with MCTP was not affected by co-treatment with Dazmegrel (Table 34). Administration of MCTP resulted in an increase in wet lung weight and vascular leak, and these indices of lung injury were not affected by co-treatment with Dazmegrel. Similarly, Dazmegrel did not attenuate the elevation in lavage fluid protein concentration or LDH activity caused by MCTP. The wet/dry lung weight ratio was only elevated in MCTP-treated rats co-treated with Dazmegrel. Additional- ly, at day 14, RVE was not attenuated by co-treatment with Dazmegrel (Figure 31). These results indicate that co-treatment with a thrombox- ane synthetase inhibitor does not afford protection from the toxicity of MCTP. 3. L-640,035 There is evidence that PGHZ, the precursor to TxAZ, can stimulate Tx receptors i3 gi_t_1_-g (Hamberg e_t_ 1131974; Kadowitz _e_t_ a_l., 1977). If this occurs i_n_ v_iyg, then treatment with a thromboxane synthetase inhibitor may not be sufficient to eliminate thromboxane receptor-mediated activities. There- fore, this study was undertaken to investigate whether co-treatment with a Tx receptor antagonist (L-640,035) could attenuate the pulmonary hypertensive response to MCTP. Rats were treated with MCTP (3.5 mg/kg) or DMF on day 0, and were co-treated by gavage with either L-640,035 (50 mg/kg) or its vehicle, polyethylene glycol (approximate molecular weight = 200, diluted with an equal volume of distilled water), three times daily. Treatment with L-640,035 started on day 0 and continued through day 14, when the animals were killed. Lung injury and pulmonary arterial pressure were assessed. As in previous drug treatment experiments, a preliminary study was performed to determine an effective dose. 180 TABLE 34 Lack of Effect of Dazmegrel (DAZ) on the Toxicity of MCTP 14 Days After Treatment Treatmenta DMF MCTP SAL DAZ SAL DAZ Bwiniti a1 (g) 275:4 272:6 278:3 278: 3 BW . (g) 336+5 337+7 300+8 286+12 final — - — - WL/BW (x 103) 3.6:0.1 3.6:o.1 6.710.6b 9.4:1.1c WL/DL 5.5_+_0.6 5.0:0.2 6.1:0.1 7.0:0.4.C Lavage LDH activity 2.5:0.2 2.2:0.2 13.7:2.3b 13.8:1.9° (U/dl) Lavage protein 0.16:0.02 0.20:0.03 2.89:0.28b 2.85:0.55.C concentration (mg/ml) lzsl—Lung/IZSI-blood 0.22:0.03 0.26:0.03 0.64:0.10b 0.87:0.13C aRats received DAZ (50 mg/kg, orally, twice daily) or SAL for 14 days following treatment with MCTP (3.5 mg/kg) or DMF. BW = body weight; WL = wet lung weight; DL = dry lung weight. N = 6-17. bSignificantly different from DMF/SAL. cSignificantly different from DMF/DAZ. 181 .N “3me .3 2:03 .37 182. to CD m N (S+/\—l) //\H .25 DAZ SAL DAZ SAL MCTP DMF Figure 3 1 183 a. Confirmation of druj effect In a preliminary study to determine a dosing regimen of L- 640,035 which would antagonize vascular Tx receptors, rats not treated with MCTP were given a single oral dose of L-640,035 (50 mg/kg) or its vehicle. Two or eight hours later the increase in mean right ventricular pressure induced by the thromboxane mimic U46619 was measured. Baseline right ventricular pressure was slightly reduced in L-640,035-treated rats (control, l4.2:0.8 mmHg; L-640,035-treated, 10.7:0.9 and 9.7:1.7 mmHg at Z and 8 hours, respectively; N = 3-6), and this difference was statistically significant at 8 hours. The increase in right ventricular pressure in response to U46619 was dose-dependent in treated and control rats (Figure 32). Two or eight hours after treatment, the right ventricular pressure response to the same dose of U46619 was less in L-640,035-treated animals than controls. The response to the intermediary dose of U46619 (1.5 ug/kg) was depressed by 73% two hours after treatment and by 59% eight hours after treatment. Based on these results, a dosing regimen of 50 mg/kg, 3 times daily was chosen. The effectiveness of L-640,035 in MCTP-treated rats was confirmed by its ability to antagonize the right ventricular pressure response to the thromboxane mimic. This response was depressed by 63% in MCTP-treated rats by co-treatment with L-640,035 (Table 35). b. Effect of L-640L035 on MCTP-induced cardiopulmonary toxicity. Body weight gain was less in MCTP-treated rats co-treated with L- 640,035 (Table 36). Administration of MCTP resulted in increases in lung weight and in lavage fluid protein concentration which were unaffected by co-treatment with L-640,035. Lavage fluid LDH activity was elevated in both groups of MCTP- treated rats, however, this only reached statistical significance in rats co-treated with L-640,035. 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CD (\J LO (\J O m mm (Bwa> dVd uoew DISCUSSION L The Dose/Response Relation for MCTP This study was undertaken to determine a dose of MCTP which would produce right ventricular enlargement (RVE) by day 14 after treatment and yet would not cause significant mortality. Results from previous studies indicated that RVE was apparent at day 14 following treatment with 5 mg MCTP/kg (Bruner e_t_ 2.1., 1983), however, in subsequent studies as many as 50% of animals treated with this dose of MCTP died within that time period. This raised the concern that animals which survived through the end of the study may represent a resistant population. Chesney and coworkers (1974a) demonstrated that MCTP at doses of 2 and 4 mg/kg produced lung injury and RVE by 4 weeks after treatment. Therefore, in this study, doses of MCTP ranging from Z to 5 mg/kg were employed. By day 14, lung injury and RVE were apparent at all doses of MCTP (Figure 5), while deaths were only observed at the highest dose (5 mg/kg) of MCTP. These results indicate that, at doses between 2 and 4 mg/kg, MCTP produces RVE (and presumably pulmonary hypertension) without causing substantial mortality. Based on these results, in subsequent studies MCTP was given at doses ranging from 3.5 to 5 mg/kg. IL The Development of MCTP-Induced Toxicity Although there have been numerous studies describing the pathology associ- ated with MCT treatment, fewer have been reported for MCTP, and none of these 191 192 have attempted to correlate biochemical and physiological alterations with changes observed at the light microscope level. For this purpose, the develop- ment of MCTP-induced pneumotoxicity was assessed by both biochemical methods and histological examination. The earliest time after MCTP treatment examined was three days. This time was chosen because in previous studies no biochemical or physiological indices of lung injury were observed at day 3 (Bruner _e_t_ g” 1983), and it was of interest to determine if subtle changes were occurring at this time that could only be detected by histologic examination. In contrast to previous results (Bruner gt 2.1., 1983, 1986), cell injury, assessed as an increase in lavage fluid LDH activity, and vascular leak was evident 3 days after treatment with MCTP (Table 3). The magnitudes of these increases, though, were not as great as were observed at later times after treatment. Consistent with increased vessel leak, perivascular edema was noted histologically. Although the perivascular edema was observed predominantly around medium and large muscular vessels, it was not clear in which vessels increased leak was occurring. Perivascular edema was associated with dilation of lymphatics in the interstitium so that it is possible that vessel leak was occurring in capillaries, increasing lymph drainage to the larger vessels, and causing perivascular edema in these vessels. Alternatively, fluid escaped from larger vessels could have contributed to perivascular edema noted there. The mild cellular infiltrate observed may have increased the dry weight of the hing, resulting in a decreased wet/dry lung weight ratio at this time. Although endothelial cell (EC) changes have been reported 24 hours after treatment with MCT (Valdivia e_t g” 1967), in the present study, 3 days after MCTP no intimal alterations of vessels were observed by light microscopy. This is consistent with the findings of Butler and coworkers (1970), who observed no changes in the ECs two days after administration of MCTP, but did observe 193 changes one week after treatment. Tissues in the present study were only examined at the light microscope level, and perhaps examination by electron microscopy would have revealed subtle changes in ECs not detectable by light microscopy. The pulmonary EC has been considered to be the target of MCT and MCTP toxicity, as metabolites of MCT or MCTP arrive in the lung via the blood and E03 are the first pulmonary cells encountered. However, evidence for remarkable alterations in ECs shortly after treatment with MCTP is limited. Certainly in this study pulmonary vascular alterations observed histologically at day 3 were unimpressive, even though some evidence of minor pulmonary injury was already apparent. Five days after administration of MCTP perivascular edema was more severe and was associated with smaller sized vessels. This correlated with an increase in wet lung weight, in the wet/dry lung weight ratio, and in vascular leak. Cell injury was evidenced by an increase in lavage fluid protein concentration and LDH activity, and this was accompanied by increased margination of WBCs and an infiltration into the perivascular connective tissue of hypertrophic lympho- mononuclear cells and occasional mast cells. As at day 3, vascular lesions were largely imimpressive, and the more pronounced changes occurred in the inter- stitium and the airways. Alveolar septae were thick with edema and infiltrated cells, alveolar Type 11 cells were hypertrophic, and enlarged macrophages were common in alveolar lumens. This suggested that at day 5 the major response to injury was occurring in the airways and interstitial spaces, not the vasculature. By day 8 a generalized proliferative response of many cell types was seen in limgs of treated rats, and nuclear dust and increased numbers of mitotic figures were observed. Consistent with previous reports for MCT (Kay e_t_ al, 1982a; Sugita _e_t_ 31., 1983a) and MCTP (Butler gt $1,, 1970), Type II pneumocytes and 194 alveolar interstitial cells were hypertrOphic. Clara cell blebbing was less pronounced, suggesting that these cells may be differentiating to replace injured cells of the bronchiolar epithelium. In more severely affected areas, hypertrophy of ECs and of smooth muscle cell nuclei was observed. The marked cellular proliferation might suggest the presence or activation of some growth factor or mitogen in response to MCTP. Edema fluid was observed in the perivascular interstitium, alveolar septal walls, and alveolar lumens, and was accompanied by an infiltration of lympho- mononuclear cells and large foamy alveolar macrophages. This accumulation of fluid resulted in increased wet lung weight and was consistent with increased vascular leak. The wet/dry lung weight ratio was not different from control, perhaps reflecting an increase in both fluid accumulation and cellularity. At day 8 margination of WBCs increased due to treatment, and platelet thrombi were occasionally observed. Alterations of vascular smooth muscle were confined to separation of smooth muscle cells due to edema fluid and occasional hypertrophy of smooth muscle cell nuclei, and were observed in 2 of 3 treated rats with elevated PAP and in 1 of 2 treated rats with normal PAP. Detailed morphometric analysis of vessel size and medial thickness was not performed; however, it did not appear that smaller pulmonary vessels of MCTP-treated rats were thicker than those of controls. Perhaps minor changes in medial thickness would become apparent if detailed morphometry was performed. In seeming contrast to many pathology studies performed with MCT, 8 days after treatment with MCTP vascular alterations were not severe, intimal changes were minor, and the most prominent changes were observed in the airways and interstitial tissue. Two weeks after administration of MCTP, biochemical and histological evidence of severe lung injury was observed. Increases in wet lung weight, wet/dry lung weight ratio, and vascular leak correlated with severe and extensive 195 perivascular and alveolar edema. Marked cell injury accompanied increases in lavage protein concentration and LDH activity. As has been previously reported (Butler gt 91., 1970; Plestina and Stoner, 1972), numerous alterations in bronchio- lar and alveolar epithelial cells were also observed. Also consistent with a previous report (Turner and Lalich, 1965), the bronchiolar epithelium extended to alveolar ducts and sacs, an alteration which could impair gas exchange. At day 14, pulmonary hypertension and RVE were apparent. At this time, EC injury was a frequent finding, as were separation and hypertrophy of medial smooth muscle cells of pulmonary vessels. In addition, some vessel walls appeared thickened, although this was not confirmed by morphometric analysis. To summarize the development of MCTP-induced toxicity, following a single intravenous injection of MCTP, biochemical and physiological indices of lung injury as well as lesions identified histopathologically increase in number, extent, and severity with time. The first indices of injury observed were increases in lavage LDH activity and vascular leak at day 3. This was accompanied by perivascular edema and a mild infiltration of lymphomononuclear cells. Two days later, lung injury had progressed. By day 8, three of five treated rats had elevated PAPs, although vascular lesions were not prominent. The most pro- nounced alteration was the proliferation of many cell types in the airways and interstitium. It was not until 14 days after treatment that vascular lesions, including alterations of ECs and smooth muscle cells, were more pronounced, although still not the most prominent change observed. At this time pulmonary hypertension was evident in all treated rats. A few observations resulting from this study differ from those reported previously for MCT or MCTP. EC changes were noted by electron microscopy within 24 hours (Valdivia e_t _a_l., 1967) and by light microscopy (Turner and Lalich, 1965) and electron microscopy (Merkow and Kleinerman, 1966) somewhat later 196 after treatment with MCT. EC changes were also noted by electron microscopy within one week after treatment with MCTP (Butler e_t_ 11:, 1970). Only minor alterations in ECs were observed in this study, and not until day 14 after treatment with MCTP. Likewise, marked vascular smooth muscle changes and medial hypertrOphy were not observed in this study, in contrast to previous reports for MCT (Turner and Lalich, 1965; Ghodsi and Will, 1981; Kay e_t_ 2.1., 1982a) and MCTP (Chesney e_t Q” 1974a). The most impressive vascular changes observed in this study occurred in the adventitia, and the most pronounced alterations in the pulmonary tissue were observed in the alveolar septae and spaces, and in the perivascular interstitium. However, minor injury was observed in the pulmonary vasculature, and more subtle lesions may have been present but not detectable by light microscopy. Conceivably, more subtle vascular injury could cause a chronic, low-level release of mediators from platelets that may promote injury and pulmonary hypertension. In retrospect, it would have been of interest to examine a time earlier than day 3, before any injury was apparent (as had been planned), and to examine a time between days 8 and 14 when all rats had elevated pulmonary arterial pressure but when the alterations in the lung were less severe than at day 14. Although detailed morphometry is not available at this time, it appears as though pulmonary hypertension developed in some rats at day 8 before any remarkable medial thickening or smooth muscle extension was apparent. This may suggest a role for vasoconstriction in MCTP-induced pulmonary hypertension. Vessel leak and the resulting edema were also apparent well before any elevations in pulmonary arterial pressure were observed, so that vessel leak may also contri- bute to MCTP-induced pulmonary hypertension. 197 III. Influence of Diet Restriction on MCTP-Induced Cardiopulmonary Toxicity The observation that diet reduction attenuated the cardiopulmonary toxicity of MCT (Hayashi gt _a_l_., 1979) prompted an investigation to determine whether this protection was due to an effect of decreased bioactivation of MCT induced by diet restriction. In rats treated 14 days earlier with MCTP, which does not require bioactivation (Bruner e_t a_l., 1986), restriction of food intake reduced the MCTP-'mduced elevation of lung weight and lavage fluid protein concentration and prevented the development of RVE (Figure 7). The elevation in lavage LDH activity caused by MCTP was not affected by diet restriction, however. This lack of effect on LDH activity may be explained by the observation that lavage fluid LDH activity is maximal 10 days following a single injection of MCTP, but by 14 days returns rapidly toward control values (Bruner e_t 11., 1983). Due to this rapid change, the variation in lavage LDH values in MCTP-treated rats is large at this time. Although there was a trend toward decreased LDH activity in lavage fluid of diet-restricted rats (Figure 7), this did not attain statistical significance. These results indicate that reduction of food intake attenuates the toxicity of MCTP, and they suggest that the protective effect of diet restriction seen in MCT-treated rats was not due to decreased bioactivation of MCT. The mecha- nism by which diet restriction lessens effects produced by MCTP remains imknown. Alterations in intake of specific dietary components produce a variety of physiological and biochemical changes. For example, dietary fat content can affect platelet function in a number of ways. Certain variations in dietary fat composition have been shown to increase thrombin-induced platelet aggregation and to decrease aggregation responses to ADP (McGregor and Renaud, 1978). A change in dietary fat has also been associated with an increased concentration of cyclic AMP in platelets, which would inhibit platelet aggregation, and with increased serum concentrations of PGE1 (Fine gt 2.1:, 1981), which downregulates 198 platelets. Furthermore, alterations in dietary fat can also affect the pulmonary vasculature: isolated lungs and isolated pulmonary vessels of rats raised on an essential fatty acid-deficient diet are decreased in responsiveness to some vasoconstrictive stimuli (Morganroth gt a_l., 1985). Another possible explanation for the observed protective effects of dietary restriction relates to the suppression of weight gain. MCTP-treated rats eating ad E1 did not gain weight, and treated rats restricted to 9 g food/rat/day lost weight over the two-week period following treatment (Figure 6). Hayashi and coworkers (1979) reported that in MCT-treated rats pulmonary alterations pro- gress in association with increases in body weight. It is possible that some of the MCT- or MCTP-induced cardiopulmonary responses (for example, RVE) require nutrition adequate for growth, so that toxicity is attenuated in animals which cannot grow. Along these lines, it has been reported recently that dietary reduction attenuates the increase in polyamines and their biosynthetic enzymes in lungs of MCT-treated rats (Hacker and Isaghulian, 1986). Since polyamines are thought to be necessary for cell growth and proliferation, and since dietary reduction inhibits polyamine biosynthesis, this may suggest that reduction of food intake attenuates MCTP-induced toxicity by decreasing production of the neces- sary requirements for cell growth and proliferation. There was a trend toward prolonged survival of diet-restricted, MCTP- treated rats as compared to those eating £1 1113; however, beyond 30 days following treatment there were no significant differences in the number of animals surviving in the two groups (Figure 8). It was noticed that ad li_b animals died more quickly once they became sickly in appearance, while diet-restricted animals seemed to survive in this weakened condition for a longer time. It is not known whether MCTP-treated animals die from pulmonary complications or right heart failure. In this study, the post mortem RV/(LV+S) determined in animals 199 which did not survive 40 days was not significantly different in ad & and diet- restricted animals (data not shown). However, among the surviving animals, the diet-restricted group had a lower RV/(LV+S) than animals allowed to eat ad _1i_b_ (Table 6). This could indicate that animals which develop RVE despite reduction in food intake may not be protected from MCTP-induced lethality. However, the significance of RV/(LV+S) measured after death has not been established, and this observation should be interpreted with caution. The modest effect of diet restriction on survival after MCTP treatment differed in degree from the marked protection observed after MCT administration (Hayashi e_t _a_l., 1979). No mortality was observed in MCT-treated rats on reduced food intake for 90 days. However, when MCT-treated rats which had been diet- restricted for 30 days were allowed free access to food again, they began to die after 10 days. This suggests that MCT may produce a condition which is not lethal until aggravated by food consumption, some specific dietary component, or growth. In a separate series of experiments not presented here, the possibility that sodium was the specific dietary component responsible for the protective effect of diet restriction was tested (Ganey g 31., 1985). Increasing or decreasing the sodium intake of MCTP-treated rats did not alter the cardiopulmonary response to MCTP. Therefore, the protective effects of diet restriction are not due to an alteration in sodium intake. In summary, reduction of food intake attenuates the pulmonary toxicity of MCTP and prevents deve10pment of RVE. Diet-restricted animals survive for a longer time, but this protective effect is short-lived. The mechanism by which reduction of food intake attenuates the toxic response to MCTP is not known. However, many drugs given subacutely retard body weight gain in animals, so that the results of these studies indicate that changes in body weight and food ZOO consumption must be considered when evaluating the effect of drug treatments on the toxicity of MCTP. IV. The Effect of ThrombocytoRenia on MCTP-Induced Pulmonary Hypertension The observation that antibody-induced thrombocytopenia reduced the sever- ity of RVE in MCTP-treated rats (Hilliker _e_t £11., 1984a) suggested that blood platelets may be involved in the pulmonary hypertensive response to MCTP. The observation that lung injury assessed 14 days after treatment with MCTP was not different in normal and thrombocytopenic rats might suggest that reducing the number of circulating platelets either delayed the toxic effects of MCTP or did not affect lung injury. One interpretation of the second possibility is that lung injury and pulmonary hypertension in this model are dissociable, and perhaps not causally related. This idea is not firmly supported in the literature: although there are a few reports of interventions which ameliorate the pulmonary hypertensive response and not lung injury, the converse has never been reported. Another interpretation is that lung injury initiates platelet involvement in the development of pulmonary hypertension. The purpose of the series of experiments performed here was to provide direct evidence that reducing the circulating platelet number attenuated MCTP-induced pulmonary hypertension at day 14, and to explore the possibility that thrombocytopenia delayed the deve10pment of MCTP toxicity. To accomplish the first objective, direct measurement of pulmonary arterial pressure (PAP) was performed in thrombocytopenic rats 14 days after administra- tion of MCTP. MCTP-treated rats with normal numbers of circulating platelets developed pulmonary hypertension and RVE, while in MCTP-treated rats with platelet numbers approximately 24% of control, this response was abolished (Figures 14 and 15). MCTP-induced lung injury was not altered by reduction of 201 platelet numbers (Tables 13 and 14). Thus, these results confirm the finding that thrombocytopenia protects against MCTP-induced RVE, and extend that effect to protection against elevation of pulmonary arterial pressure as well. This also supports the contention that RVE develops in response to pulmonary hypertension in this model. This particular experiment did not address the possibility that the protective effect was observed at day 14 because thrombocytopenia delayed the onset of MCTP-induced toxicity. In rats with normal platelet numbers, increases in lavage LDH activity and vascular leak are first observed by day 3, and lung injury is evident with all indices we used by 5 days after administration of MCTP (Table 3). Pulmonary hypertension, however, does not develop until day 8 or later. Thus, if the onset of toxicity were delayed by as long as one week, 11mg injury would still be apparent by day 14, yet pulmonary hypertension would not have developed. To test whether thrombocytopenia delayed MCTP-induced toxicity, lung injury and pulmonary hypertension were to be assessed in PAS-treated rats 8 days after administration of MCTP. However, in the course of these experiments it was discovered that the degree of thrombocytopenia achieved was critical for the protective effect. MCTP-treated rats in which platelet number was reduced below 10% of normal developed RVE and pulmonary hypertension by day 14 which was not different from MCTP-treated rats with normal numbers of platelets (Figures 12 and 13). The disparate effects of this severe thrombocytopenia and the more modest reduction of platelet numbers which prevented MCTP-induced pulmonary hypertension may be explained by some protective function of the platelet in the latter but not the former situation. That is, platelets may be necessary for some homeostatic mechanism, and it may be that circulating platelet numbers above 10% of normal are required for this function. For example, it is believed that platelets support vascular endothelium and maintain 202 vascular integrity by a mechanism separate from hemostasis or vascular repair (Gimbrone e_t_ a_l., 1969; Roy and Djerassi, 1972). In dogs made thrombocytopenic, vascular integrity was restored after infusion of far fewer platelets than were required to return bleeding time to normal (Roy and Djerassi, 1972). Human thrombocytopenia (Kitchens and Pendergast, 1986) and thrombocytopenia experi- mentally-induced in animals (Kitchens and Weiss, 1975) is associated with hemorrhage, vessel leak, and alterations in capillary endothelium, including thinning of the endothelial cell cytoplasm and the appearance of fenestrations. Although hemorrhage and vessel leak are consistent findings in thrombocytopenic animals, other studies have demonstrated that red blood cells leave the vessel lumen through normal endothelium (Van Horn and Johnson, 1966; Dale and Hurley, 1977; Shepro gt_: 31., 1980). In either case, the fact that vascular integrity is preserved at lower levels of circulating platelets than are other platelet functions may explain the different effects of severe and modest thrombocytopenia on MCTP-induced pulmonary hypertension. For example, it might be that in rats for which the degree of thrombocytopenia was severe (and not in rats with modest thrombocytOpenia), vessel leak caused an increase in pulmonary arterial pressure which obscured any protective effect due to decreased numbers of platelets. By what mechanism is thrombocytOpenia affording protection against the pulmonary hypertension caused by MCTP? In experiments performed here and elsewhere (Figures 14 and 15 compared to Tables 11 and 12; Hilliker g3 31., 1984a), the response to MCTP was attenuated to the greatest extent when rats were thrombocytopenic from days 6-8. In rats with normal platelet numbers, 8 days after treatment with MCTP a generalized proliferative response is observed in the lung, and cell injury is severe. If cell injury and repair are related to the development of pulmonary hypertension, this might suggest that the platelet is providing a stimulus for cell growth or a mediator which injures cells. This would 2.03 fit well with the finding that inhibitors of polyamine biosynthesis attenuate the response to MCT (Olson gt 31., 1984b). The platelet contains and releases a number of growth factors which are mitogenic for smooth muscle (Deuel and Huang, 1984) and endothelial cells (King and Buchwald, 1984), and also releases TxAz, which can cause injury to endothelial cells (DeClerck gt 31., 1985a). Alternatively, the platelet may be providing a vasoconstrictive mediator, the actions of which are confined to the pulmonary vascular bed because that is the site of vascular injury and, therefore the site of platelet activation. Intravascular platelet aggregation and mediator release have been associated with increased pulmonary vascular pressure and pulmonary edema (Bo and Hognestad, 1972; Vaage 53 31., 1974; 1976). Two vasoconstrictive mediators released by platelets are S-hydroxytryptamine and thromboxane AZ’ and their roles in MCTP- induced pulmonary hypertension are addressed in the next section. Another possible mechanism by which thrombocytopenia may afford protec- tion is dependent not on the period of reduced platelet numbers, but on the reappearance and overshoot of platelets observed after treatment with the anti- platelet serum (Figure 16). When rats are made thrombocytopenic from days 6-8 after MCTP treatment, the overshoot of platelet number occurs from days 11-14. Platelets from MCTP-treated rats not co-treated with anti-platelet serum were hyporesponsive to aggregation by adenosine diphosphate, collagen, and arachidonic acid 14 days after MCTP (Hilliker gt 31., 1983b). If this decreased responsiveness is involved in MCTP-induced pulmonary hypertension, it is possible that the presence of newly-formed (and presumably normally-responsive) platelets, or the presence of a greater number of platelets, could be protective against pulmonary hypertension. The time course of these effects may argue against this possibility, though. In rats with normal numbers of platelets, pulmonary hypertension begins to develop by day 8 and is well established by day 14. Platelets respond normally 204 to most aggregating agents at day 7 but are hyporesponsive at day 14 (Hilliker gt 31., 1983b). It seems unlikely that an effect on platelets at day 14 may be a cause of the pulmonary hypertension which began 6-7 days earlier. Platelet numbers in thrombocytopenic rats do not increase above the numbers seen prior to the administration of the anti-platelet serum until day 11, a time when pulmonary hypertension already exists. Therefore, it seems more likely that the reduction in platelet number (and not the platelet rebound) is responsible for the protective effect observed in MCTP-treated rats made thrombocytopenic. In summary, reduction of platelet number to approximately 20% of normal prevented MCTP-induced pulmonary hypertension and RVE observed at day 14. This effect was not observed when the degree of thrombocytopenia was more severe. It is not known by what mechanism thrombocytopenia protects against MCTP-induced pulmonary hypertension, but the platelet may be providing a vasoconstrictive mediator. This possibility is discussed in detail below. V. The Role of Platelet Mediators in MCTP-induced Cardiopulmonary Toxicity The platelet stores or synthesizes a number of vasoactive mediators which can be released during platelet activation. Two vasoconstrictors released by platelets, 5-hydroxytryptamine (5HT) and thromboxane A2 (TxAz), were consi- dered as possible participants in MCTP-induced pulmonary hypertension. A. S-Hydroxytryptamine It seemed reasonable that 5HT may be involved in MCTP-induced pulmonary hypertension for a number of reasons. First, it has been reported that endothelial cell damage is prominent in the pulmonary vessels of rats treated with MCT (Turner and Lalich, 1965; Merkow and Kleinerman, 1966; Valdivia gt 31., 1967) or MCTP (Butler, 1970; Chesney gt fl” 1974a). Relatively high doses of MCTP produce pulmonary edema within 18 hours (Hurley and Jago, 1975), 205 suggesting that MCTP is also capable of causing EC injury. Although no marked alterations in EC were observed by light microscopy after a relatively low dose of MCTP in the present study, more subtle vascular lesions may not have been detected. Platelets activated by interaction with injured vessels release 5HT which can cause vascular contraction of isolated vessels (Cohen 3 31., 1981; DeClerck and Van Nueten, 1982; Mullane g a_l., 1982; McGoon and Vanhoutte, 1984; Ogunbiyi and Eyre, 1984), can increase vascular pressure in isolated, perfused lungs (Figure 20), and can cause pulmonary hypertension :13 mg (Breuer gt 3., 1985). Subtle vascular lesions may cause a low-level release of 5HT which could increase pulmonary vascular pressure chronically. In addition, 5HT can potentiate the response to aggregating agents (DeClerck gt 31., 1982a; Glusa and Markwardt, 1984) or to other vasoconstrictors (DeClerck and Van Nueten, 1982; Van Nueten g 3., 1982). Therefore, elevated release of 5HT in the lungs of MCTP-treated rats could contribute to pulmonary hypertension. Secondly, 5HT is removed from the pulmonary circulation by carrier- mediated uptake into endothelial cells, followed by intracellular inactivation by monoamine oxidase (Gillis and Pitt, 1982; Roth, 1985). Removal of 5HT is depressed in isolated, perfused lungs of rats following treatment with MCT or MCTP (Gillis gt a_l., 1978; Hilliker e_t Q" 1982, 1983c). 'Ihis decreased inactiva- tion of 5HT by the pulmonary endothelium could cause an elevated pulmonary concentration of this amine, and again, pulmonary vasoconstriction. Platelets also actively accumulate 5HT and thereby remove free 5HT from the pulmonary circulation (Born gt Q” 1972; Steinberg and Das, 1980; Given and Longenecker, 1985), and decreased accumulation by platelets could thus result in an increased pulmonary concentration of free 5HT. It is unlikely that decreased accumulation of 5HT by platelets contributes to the pulmonary response to MCTP because fawn-hooded rats, which have a congenital defect in 206 which the ability of the platelet to take up and release 5HT is decreased (Raymond and Dodds, 1975), were no more susceptible to MCTP-induced toxicity than Sprague-Dawley rats (Hilliker gt 31., 1983a). Conversely, since platelets compete with the pulmonary vascular endothelium for uptake of 5HT (Steinberg and Das, 1980), and 5HT removal by endothelium is impaired following treatment with MCTP (Hilliker gt 31., 1983c), platelets could contain a greater amount of 5HT which would be available for release upon activation, thereby causing greater pulmonary vasoconstriction. This also does not appear to be true, as platelets from MCTP-treated rats contain no more 5HT than do platelets from control rats (Figure 17). In addition to elevated pulmonary concentrations of 5HT causing vasocon- striction, it was hypothesized that greater accessibility of smooth muscle receptors or increased responsiveness of vascular smooth muscle to 5HT following MCTP treatment could contribute to pulmonary hypertension. Pulmonary vessel leak precedes pulmonary hypertension in MCT-treated (Sugita fl 3., 1983a) or MCTP-treated (Table 3) rats. This increased vascular permeability could increase the accessibility of smooth muscle receptors to 5HT originating from the blood. Conversely, it also seemed possible that 5HT could contribute to the observed vessel leak (DeClerck _e_t_ Q” 1984, 1985b). Pressor responses to 5HT were elevated in isolated, perfused ltmgs from MCTP-treated rats compared to control rats (Hilliker and Roth, 1985a), suggesting that MCTP treatment increases vascular responsiveness to 5HT. Therefore, 5HT could cause pulmonary vasoconstriction in MCTP-treated rats through greater interaction with 5HT receptors on vascular smooth muscle cells or through enhanced vasoconstriction due to hyperresponsiveness of the vasculature. If 5HT were contributing to MCTP-induced pulmonary hypertension, co-treatment with an inhibitor of 5HT synthesis or with a 5HT receptor antagonist 207 may have reduced the resonse to MCTP. Co-treatment with para-chlorophenyl- alanine (PCPA), a 5HT synthesis inhibitor, decreased PAP and RVE in MCT- treated rats (Carrillo and Aviado, 1969; Tucker gt 31., 1982; Kay _e_t_ 3., 1985). It is possible that PCPA acted in these studies by decreasing 5HT-induced pulmonary vasoconstriction and/or lung leak. However, there are some potential problems with these three studies. One is that drug effectiveness (i.e., decreased synthesis of 5HT) was never confirmed. Secondly, animals treated with PCPA gained less weight than control animals, and it has been demonstrated that diminished body weight gain is associated with prevention of RVE and amelioration of lung injury in MCT— or MCTP-treated rats (Hayashi gt a_l., 1979; Figure 7). It is also possible that in these studies PCPA might have exerted its protective effect by inhibiting the mixed function oxidase enzyme system, thereby inhibiting bioactivation of MCT. In support of this, PCPA was ineffective in reducing the severity of pleural effusion or in prolonging survival in MCTP-treated rats (Plestina and Stoner, 1972). To examine whether 5HT was contributing to MCTP-induced pulmo- nary hypertension, MCTP-treated rats were co-treated with a 5HT receptor antagonist. 5HT-induced contraction of vascular smooth muscle is mediated by a population of 5HT receptors termed 5HTz receptors (Cohen _e_t 3., 1981); therefore, the effect of ketanserin, which specifically antagonizes 5HTz receptors (Leysen gt a_l., 1980; Laduron fl _a_l., 1982), was determined in MCTP-treated rats. Ketanserin inhibited 5HT-induced contraction in isolated vessels (Van Nueten gt 31., 1981; Ogunbiyi and Eyre, 1984) without altering 5HT uptake into platelets (De Clerck gt 31., 1982a). Ketanserin has also been shown to reduce pulmonary arterial pressure in humans (Vincent gt 31., 1984). At the dosing regimen used in the present study, ketanserin inhibited the 5HT-induced shape change in platelet- rich plasma (Figure 19) and the increase in perfusion pressure induced by 5HT in 208 isolated lungs from MCTP-treated rats (Figure 20). However, ketanserin did not protect against the alterations in lung weight or vascular leakage of 125I-albumin observed in MCTP-treated rats (Figure 21). RVE in MCTP-treated rats was also not attenuated by co-treatment with ketanserin. Because many 5HT receptor antagonists have other pharmacological actions which do not involve 5HT receptors, in studies not presented here the effect of a structurally different 5HT receptor antagonist was also examined. and 5HT Metergoline, which has been reported to bind to both 5HT receptors 1 2 (Leysen gt 31., 1980; Peroutka and Snyder, 1983), did not protect against MCTP- induced changes in lung weight or RVE (Ganey gt a_l., 1986). The results of these studies indicate that MCTP treatment does not alter platelet 5HT content, and that co-treatment with the 5HTz receptor antagonist ketanserin does not attenuate the response to MCTP. This suggests that 5HT does not mediate lung injury or RVE in this model. Because RVE is presumed to be the result of sustained pulmonary hypertension in this model, these findings also suggest that 5HT is not responsible for the increased pulmonary vascular pressure due to MCTP. B. Thromboxane A, A role for TxAz in MCTP-induced pulmonary hypertension was hypo- thesized based on a number of observations. Activation of platelets by interac- tion with injured vessels causes release of TxAz which can cause pulmonary vasoconstriction (Svensson gt 31., 1977; Salzman e_t 31., 1980; McDonald gt 31., 1983; Farrukh fl 3., 1985). Thromboxane has been implicated in the pulmonary hypertension produced in isolated lungs by staphylococcal a-toxin (Seeger gt fl” 1984) and by platelets activated with platelet-activating factor (Heffner _e_t 31., 1983) or Staphylococcus aureus (Shoemaker gt a_l., 1984). Additionally, both the stable metabolite of TxAz (Tsz) and the endOperoxide precursors to TxAz (PGG2 209 and PGHZ) are vasoactive. Tsz causes pulmonary vasoconstriction in isolated lung lobes (Kadowitz and Hyman, 1980) and i2 _vi_v_o_ (Friedman g 31., 1979). PGHZ, which may also act at thromboxane receptors, contracts vascular smooth muscle (Hamberg gt 31., 1975a; Malmsten gt 31., 1976), and increases pulmonary vascular resistance (Kadowitz gt 31., 1977; Hyman gt 31., 1978). The endoperoxide analog and thromboxane mimic U46619 increased right ventricular pressure in this study, and the increase was greater in MCTP-treated rats than controls (Table 35). TxA2 could also increase pulmonary vascular resistance by causing vessel leak independent of its prOperties of venoconstriction (Garcia-Szabo gt 31., 1983). In addition to contributing to MCTP-induced pulmonary hypertension through increasing pulmonary vascular pressure or pulmonary vessel leak, it seemed possible that TxA2 could promote platelet aggregation and release of other vasoactive mediators, permeability modifiers or growth factors. TxAz (or PGGZ and PGHZ) is involved in the platelet aggregation response to some agents, although this involvement varies with the species from which platelets are obtained (Hamberg gt 31., 1974; Malmsten gt 31., 1975; Meyers gt 31., 1979; Leach and 'Ihorburn, 1982; Parise gt 31., 1984). TxAz does not appear to be a necessary requirement for aggregation of rat platelets in response to arachidonic acid, collagen, or thrombin (Nishizawa gt 3_l., 1983; Emms and Lewis, 1986; Huzoor- Akbar and Anwer, 1986). It also seemed possible that the vasocontrictive and pro-aggregatory properties of TxA2 might be more effective in the face of decreased PGIZ synthesis due to MCTP-induced endothelial cell injury. Injury to cultured endothelial cells by exposure to a variety of agents has been associated with altered synthesis and/or release of both P612 and TxAz. For example, injury induced by an immunologic stimulus (Goldsmith and McCormick, 1984) or by radiation (Rubin gt 3., 1985) stimulated release of 6-keto PGFla from endothelial 210 cells. Concentrations of hydrogen peroxide which were not cytotoxic either inhibited production of 6-keto PGF 0. from arachidonate in endothelial cells 1 (Whorton fi 31., 1985) or caused a transient increase in 6-keto PGF (1 followed by 1 diminished release in response to subsequent stimuli (Ager and Gordon, 1984). Endotoxin caused release of both 6-keto PGFlo. (Nawroth g 31., 1984) and Tsz (Nawroth _et 31., 1985) from cultured endothelial cells. In isolated human umbilical veins, production of Tsz was stimulated to a greater extent than PGIz by balloon trauma (Mehta gt 31., 1982), and 13 yiyg atherosclerosis is associated with increased PGIz biosynthesis (FitzGerald _et 31., 1984). So, if MCTP-induced endothelial cell injury either depressed production of PGrI2 or did not stimulate PGI2 synthesis to an extent capable of opposing the action of increased concen- trations of TxAz, then TxAz could contribute to pulmonary hypertension. In addition to having vasodilatory and anti-aggregatory properties which oppose the activities of TxAz, it has been reported that PGIz decreased thromboxane production in response to endotoxin (Flynn and Demling, 1982) and that, in isolated lungs perfused with platelets, Tsz was only produced when PGIZ synthesis was inhibited (Boyd and Eling, 1980). These results suggest that PGIZ may in some way regulate TxA2 synthesis as well as oppose the actions of TxAZ. 'Ihus, TxAz may contribute to MCTP-induced pulmonary hypertension by causing pulmonary vasoconstriction or pulmonary vascular leak, by promoting platelet activation and mediator release, or because of an imbalance in the relative activities of PGIz and TxAz. In pr0posing a possible role for TxAz in pulmonary hypertension due to MCTP, it was important to demonstrate that TxAz was present in the lungs of treated rats. MCT-induced pulmonary hypertension is associated with an eleva- tion of Tsz and 6-keto PGFl minces (Molteni gt 3., 1984, 1986). It was of interest to examine vascular a in lavage fluid (Stenmark gt a_l., 1985) and lung 211 production of these prostanoids. Therefore, Tsz and 6-keto PGF1 a release from isolated lungs of MCTP-treated rats was measured at the onset of pulmonary hypertension (day 7) and once pulmonary hypertension was well established (day 14). In isolated lungs perfused with a cell-free buffer, the release of 6-keto PGFla was not different in lungs from rats treated 7 days earlier with MCTP and control rats (Figure 22A). The release of Tsz was also similar in limgs from treated and control rats at this time (Figure 223). Pulmonary injury was evidenced in treated rats at this time by an increase in lavage LDH activity and in lung weight (Table 19), and inflow perfusion pressure was also elevated (Table 20), reflecting the pulmonary hypertensive condition of treated rats. However, these changes were not associated with altered production or release of 6-keto PGF1 a or Tsz. Fourteen days following treatment with MCTP, lung injury was still apparent and RVE was observed (Table 21) suggesting that pulmonary hypertension was established. In support of this, inflow perfusion pressure was higher in lungs of MCTP-treated rats (Table 22). At this time, 6-keto PGF a release from 1 isolated, buffer-perfused lungs of MCTP-treated rats tended to be higher than from lungs of controls, although this difference did not reach statistical significance (Figure 24A). When presented with arachidonic acid, lungs from both control and treated rats released more 6-keto PGF suggesting that MCTP la’ treatment did not impair the lung's ability to synthesize PGIZ. Tsz release was greater from lungs of rats treated with MCTP 14 days earlier than from control rats, both prior to and during arachidonate infusion (Figure 24B). What was the source of Tsz in these buffer-perfused lungs? Tsz could have derived from platelets adhered to vessels, and greater platelet adherence due to endothelial cell damage may explain the elevated Tsz release 212 in lungs of MCTP—treated rats relative to controls. Alternatively, Tsz could have derived from endothelial cells (Ingerman-Wojenski, 1981; Goldsmith and Needleman, 1982) or fibroblasts (Ali gt 31., 1980; Ody gt 31., 1982; Menconi gt 3., 1984), both of which may be proliferating in lungs of treated rats at this time. Results from lungs deliberately made edematous by increasing outflow pressure (Figure 25) suggest that a propensity to accumulate fluid, such as is seen in lungs from MCTP-treated rats (Table 21), may contribute to elevated release of Tsz from isolated lungs. In response to arachidonate stimulus, release of TxBZ increased in lungs from both control and treated rats, suggesting that MCTP treatment did not impair the lung's capacity to synthesize TxAz. These results suggested that MCTP-induced pulmonary hypertension was associated with elevated production in the lung of TxBZ and not 6-keto PGF1 (1' This supported the possibility that the balance of TxA and PGIZ favored the 2 vasoconstrictory prostanoid TxA2 in lungs of MCTP-treated rats. Since the platelet is the major source of TxAZ _i_1_1_ m (Needleman _e_t_ a_l., 1976), and since platelet/vessel wall interaction can promote TxAz release, it was of interest to examine release of Tsz and 6-keto PGF1 a in lungs perfused with blood. At day 7, lung weight was elevated in MCTP-treated rats, but RVE was not apparent (Table 23). Inflow perfusion pressure tended to be higher in lungs from treated rats, but this difference was not significant (Table 24). Similar to results in buffer-perfused lungs, at day 7 there were no differences in the effluent concentrations of either Tsz or 6-keto PGF1 a between lungs from control and treated rats (Figure 26). The change in platelet number between the inflow and effluent perfusates was not different from zero for either group (Table 24), suggesting that significant numbers of platelets were not adhering to the vasculature during perfusion. 213 At day 14, lung weight was elevated and RVE was observed (Table 25). Inflow perfusion pressure was higher in lungs from MCTP-treated rats, reflecting the pulmonary hypertensive condition of these rats (Table 26). At this time, the effluent concentration of 6-keto PGFla was not different in lungs from MCTP- treated rats and control rats (Figure 27A). However, the concentration of TxBZ in the effluent of lungs from MCTP-treated rats was significantly greater than controls by 3 minutes of blood perfusion (Figure 27B). Tsz in the effluent of lungs from rats treated 14 days earlier with MCTP increased during the perfusion. This increase was not observed in controls. The reason for this 3-minute delay until the observed increase in Tsz is unclear. One possible explanation is that a period of time may be required for activation of platelets encountering injured endothelium. Platelets in the blood perfusate were probably not adhering to the vasculature, as the platelet number was not different in the inflow and the effluent perfusate (Table 26), however activation of platelets in the absence of aggregation and adherence may have occurred. Besides platelets, other sources of Tsz, such as endothelial cells (Goldsmith and Needleman, 1982), leukocytes (Goldstein g 31., 1978), or fibro- blasts (Menconi gt 3.1., 1984) may also have contributed to the plasma effluent Tsz concentration. There are two suggestions that the blood was a partial source of Tsz. The first is that the release of Tsz was not different in lungs from treated and control rats during the buffer pre-perfusion (data not shown). The second is drawn from a comparison of the magnitude of increase in Tsz release in blood- and buffer-perfused lungs (Figures 27 and 24). The Tsz concentration in the effluent of lungs from rats treated 14 days earlier with MCTP was 0.2 ng/ml greater than that of controls when lungs were perfused with buffer (Figure 24B). When perfused with blood, the concentration of Tsz in the plasma effluent of lungs from MCTP-treated rats was 1.6 ng/ml greater than 214 control (Figure 27B). Thus, substantially more Tsz was released when lungs were perfused with blood. This argues against lung tissue as the sole source of increased release of thromboxane caused by MCTP treatment. It also seems unlikely that the elevated Tsz concentration in lungs from MCTP-treated rats could be explained by less efficient removal of TxBZ by these lungs because the initial extraction of Tsz was not different from controls (Figure 28). Thus, MCTP-induced pulmonary hypertension was associated with an increased release of Tsz and unaltered release of 6-keto PGF1 a from isolated lungs perfused with either a cell-free buffer or blood. The magnitude of the increased release of TxB2 relative to controls was greater in lungs perfused with blood, suggesting a source of the TxB2 was within the blood. This possibly could be the platelets. These findings indicate that, in isolated lungs of MCTP-treated rats which have deve10ped RVE (and presumably elevated pulmonary arterial These pressures), the balance of TxA and PGIz is tipped in favor of TxA 2 2' results suggest that if events occurring in the isolated lung reflect events occurring i_n; y_i_vg, TxAZ could be contributing to MCTP-induced pulmonary hypertension due to vasoconstriction from elevated pulmonary concentrations of TxAz. In addition to increased release of TxAz in isolated lungs, it was of interest to examine whether MCTP treatment E yi_v_o_ altered platelets so that they released more TxAz upon activation. Some human diseases that involve vascular complications, such as diabetes (DiMinno _e_t a_l., 1985) and Kawasaki disease (Hidaka gt 31., 1983) are associated with increased platelet production of TxAZ. Platelets from spontaneously hypertensive rats also synthesize more TxA2 than platelets from normotensive rats (DeClerk g 31., 1982b; Huzoor-Akbar and Anwer, 1986). Therefore, it seemed possible that MCTP might alter platelet production of Tsz. 215 The concentration of TxB2 was higher in unstimulated, platelet-rich plasma (PRP) from MCTP-treated rats relative to controls at day l, but not at any other time after treatment (Figure 29A). Although lung injury was not assessed in the present study at this time, in a different study, vascular leak was not elevated one day after treatment with MCTP (Bruner gt 31., 1986). In addition, injury reported here at day 3 (Table 3) or 4 (Table 27) was relatively mild so that major injury would not be expected at day 1. Thus, the basal concentration of Tsz in PRP was likely elevated before the onset of major injury. This could be caused by an MCTP-induced alteration in the platelets such that they released TxA2 more readily during the preparation of PRP. Alterna- tively, the observed increase in TxBZ in unstimulated PRP could be derived from other cellular sources of Tsz in the blood. The release of '1‘sz in response to arachidonic acid-induced platelet aggregation was not higher in PRP from MCTP-treated rats at any time after treatment (Figures 29B and 29C). Thus, administration of MCTP 13 _v_iyg does not increase platelet production of TxAz. Relatively high concentrations (1 mg/ml) of MCTP added directly to PRP i_n_ gig; abolished the aggregation response and decreased Tsz production, but lower concentrations of MCTP did not affect either the aggregation response or the release of Tsz (Table 29). This indicates that moderate concentrations of MCTP do not directly affect platelets. In contrast to previous findings (Hilliker gt 3., 1983b), the aggregation response to arachidonic acid was not different in PRP from treated and control rats (Table 28). Hilliker and coworkers (1983b) demonstrated a 36% decrease in maximal aggregation and a 40% decrease in the rate of aggregation in response to arachidonic acid in PRP from rats treated 14 days earlier with MCTP. The reason for the different results obtained here is not obvious. 216 Thus, although MCTP treatment resulted in elevated Tsz release by isolated lungs, it did not enhance Tsz production by platelets and did not alter platelet aggregation responses. If TxAz is involved in MCTP-induced pulmonary hypertension, co- treatment with a drug which inhibits the biosynthesis of TxAZ or interferes with its activity may have reduced the pulmonary hypertensive response to MCTP. Some drugs which inhibit prostaglandin biosynthesis, but also have other actions unrelated to prostaglandins, have afforded partial protection against the toxicity of MCTP. Hydralazine, which inhibits platelet thromboxane synthesis (Greenwald gt 31., 1978), attenuated the development of RVE and the increase in lavage protein concentration in MCTP-treated rats (Hilliker and Roth, 1984). Sulphin- pyrazone, which also inhibits platelet prostaglandin biosynthesis (Livio gt 31., 1980), prevented the development of RVE, but did not alter the indices of lung injury (Hilliker and Roth, 1984). However, inhibition of prostaglandin biosynthesis was not confirmed in these studies, and protective effects may have been due to some other action (for example, vasodilation) of these drugs. In addition, sulphinpyrazone depressed body weight gain, and this effect may have contributed to the observed protection. To examine the involvement of TxAz in MCTP- induced pulmonary hypertension more closely, rats were co-treated with either ibuprofen, Dazmegrel, or L-640,036. Ibuprofen inhibits the enzyme cyclooxygenase (Longenecker gt 31., 1985) which converts arachidonic acid to the prostaglandin endoperoxide which is then converted to TxAz, PGIZ, and prostaglandins of the A-F series (Figure 2). Ibuprofen has also been reported to inhibit platelet function (McIntyre and Philp, 1977). Treatment with ibuprofen significantly decreased pulmonary hypertension in a porcine model of acute respiratory failure (Kopolovic gt 31., 1984). However, co-treatment with doses of ibuprofen which decreased platelet function (Table 30) 217 and circulating plasma thromboxane concentration did not alter MCTP-induced RVE or increased lung weight (Table 31). This is consistent with the finding that co-treatment with indomethacin, another cyclooxygenase inhibitor, did not alter MCT-induced toxicity (Stenmark gt 31., 1985). Inhibition of cyclooxygenase also suppresses synthesis of PGIZ (Figure 2), the vasodilatory and anti-aggregatory action of which may be beneficial in relieving pulmonary hypertension. Treatment with PGIz has afforded some relief to patients suffering from primary pulmonary hypertension (Higenbottom gt a_l., 1984). In addition, PGIZ infused into the pulmonary artery acutely reduced pulmonary arterial pressure in monocrotaline-treated rats (Bowdy gt 31., 1986) and dogs (Czer g 31., 1986). PGEl, another product of the cyclooxygenase enzyme, has also been shown to reverse the pulmonary hypertension in rats fed 9. spectabilis seeds (Roum gt 3.1., 1983). Inhibition of the effects of PGIZ or PGE1 by treatment with ibuprofen could have masked any protective effect of inhibition of thromboxane synthesis, therefore, the effect of a specific thromboxane synthe- tase inhibitor was determined in MCTP-treated rats. Treatment with Dazmegrel (UK38485) decreased serum Tsz levels in animals (Parry gt 31., 1982) and man (Fischer gt 31., 1983), but had no effect on the plasma concentration of 6-keto- PGFla (Fischer gt a_l., 1983). Similar results were seen here (Table 32). Despite a depressed plasma concentration of TxBZ, co-treatment with Dazmegrel did not alter MCTP-induced lung injury at day 7 (Table 33) or 14 (Table 34), or MCTP- induced RVE at day 14 (Figure 31). This is consistent with the recent report that Dazmegrel did not alter toxicity caused by administering the parent compound, monocrotaline (Langleben gt 31., 1986). The endOperoxide precursor to TxAz, PGHZ, has been reported to have pro-aggregatory (Hamberg gt 31., 1974) and pulmonary vasoconstrictive actions (Kadowitz g; g, 1977) similar to TxAz itself, and is thought to act at the same 218 receptor as TxAz. Therefore, treatment with a thromboxane synthetase inhibitor may not completely prevent this activity. Accordingly, the effect of co- treatment with a thromboxane receptor antagonist on MCTP-induced toxicity was determined. L-640,035 inhibits platelet aggregation in guinea pigs and rabbits i_n_ 3133, and human platelet aggregation i_n_ 3333 in response to the prostaglandin endoperoxide analogue U44069, to arachidonate, or to collagen, but not to ADP (Chan gt 31., 1986). The U44069-induced increase in pulmonary resistance in guinea pigs and dogs i_g gi_v_o_ was also inhibited by L-640,035 (Carrier gt a_l., 1984). Treatment with L-640,035 inhibited the increase in right ventricular pressure induced by U46619 in anesthetized rats (Figure 32, Table 35). Co-treatment with L-640,035 did not attenuate the MCTP-induced elevation in lung weight or LDH activity and protein concentration in lavage fluid (Table 36), nor did it affect the MCTP-induced elevation in pulmonary arterial pressure at day 14 (Figure 33). An interesting finding was the greater response to U46619 in MCTP/VEH-treated rats relative to DMF/VEH controls (Table 35). This is consistent with the finding of increased vascular responsiveness in isolated, perfused lungs from MCT— (Gillespie gt Q” 1986) or MCTP-treated rats (Hilliker and Roth, 1985a). Co-treatment with L-640,035 essentially returned the response to U46619 in MCTP-treated rats to that seen in DMF control rats. This raises the possibility that, if there were an elevated concentration of TxA2 in the pulmonary vasculature of MCTP-treated rats, then the greater vascular responsiveness might allow thromboxane to contribute to pulmonary hypertension despite the degree of receptor antagonism achieved in this study. It is unclear whether the pulmonary concentration of TxAz is elevated in MCTP-treated rats. Results in isolated, perfused lungs would suggest that pulmonary concentrations of Tsz may be elevated 14 days after treatment with MCTP, however, plasma concentrations of 219 Tsz were no greater in MCTP-treated rats at day 14 than in DMF controls (Table 32.). In summary, administration of MCTP to rats was associated with elevated release of Tsz from isolated, perfused lungs at day 14 when pulmonary hypertension was established, but not at day 7 during the onset of pulmonary hypertension. The MCTP-induced increase in TxBZ was greater in lungs perfused with blood than in lungs perfused with buffer, suggesting that part of the Tsz was derived from the blood. The release of 6-keto PGF1 a was not affected by treatment with MCTP. Platelets from MCTP-treated rats did not produce more Tsz during aggregation with arachidonic acid than platelets from control rats, nor were they altered in responsiveness to arachidonic acid. Co-treatment with drugs which inhibit the biosynthesis of TxAz (ibuprofen or Dazmegrel) or with a TxAz receptor antagonist (L-640,03S) did not attenuate the lung injury or pulmonary hypertension caused by MCTP. These results suggest that TxAz is not necessary for the pulmonary hypertensive response to MCTP. SUMMARY AND CONCLUSIONS These studies were undertaken to examine the role of the platelet and platelet mediators in the pulmonary hypertension caused by MCTP. When rats treated with MCTP on day 0 were moderately depleted of platelets from days 6-8, pulmonary hypertension and RVE did not develop by day 14. MCTP-induced lung injury at this time was not affected by platelet depletion. These results suggested that the platelet was contributing in some way to the pulmonary hypertensive response to MCTP. One mechanism by which the platelet may have been involved was through release of vasoactive mediators. Two such mediators, 5HT and TxAZ, were examined as possibly playing a role in the response to MCTP. Although 5HT causes pulmonary vasoconstriction in rats and increases vascular permeability, co-treatment with a 5HT receptor antagonist did not attenuate the lung injury, vessel leak, or RVE caused by MCTP. Treatment with MCTP also did not alter platelet content of 5HT. These results suggest that 5HT is not a necessary contributor to MCTP-induced toxicity. Release of Tsz, a stable metabolite of TxAz, was elevated in lungs of MCTP-treated rats relative to controls. In addition, the Tx mimic U46619 produced pulmonary vasoconstriction in anesthetized rats, and this response was enhanced in rats treated 14 days earlier with MCTP. These results suggested that the concentration of TxA may be elevated in lungs of MCTP-treated rats, and 2 that these lungs may respond to TxAz with greater vasoconstriction than controls. Treatment with MCTP did not alter arachidonic acid-induced aggregation or 220 221 release of Tsz from platelets, suggesting that in treated rats platelet activation would not be accompanied by a greater release of TxAz. Co-treatment with either a cyclooxygenase inhibitor (ibuprofen), a throm- boxane synthetase inhibitor (Dazmegrel) or a thromboxane receptor antagonist (L- 640,035) did not alter the lung injury, pulmonary hypertension, or RVE caused by MCTP. These results suggested that TxA was also not necessary for the 2 development of pulmonary hypertension due to treatment with MCTP. By what mechanism does the platelet contribute to MCTP-induced pulmo- nary hypertension? At this time, the answer to that question is unknown. One potentially interesting area of investigation was not addressed in these studies: the possible contribution of platelet-derived growth factors, including PDGF. 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