University of Manitoba Faculty of Graduate Studies Tetrahydrocannabinol and Lang Surfactant Metabolism in Isolated Fetd Type II Alveolar CelZs BY Tracy C. Cherlet Submitted to the Faculty of Graduate Studies In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE Department of Hurnan Anatomy and Ce11 Science University of Manitoba Winnipeg, Manitoba National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wollingîm OliawaON K1AôN4 OttawaON KIAW Canada canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Biblioîhèque nationale du Canada de reproduce, loan, distribute or seîi reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur fomat électronique. The author retains ownership of the L'auteur conserve la propriété du copwght in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. THE UNIVERSITY OF iMANITOBA FACULTY OF GRADUATE STUDIES ***** COPYRIGHT PERMISSION PAGE Tetrahydrocamabinol and Lung Surfactant Metabolism in Isolated Fetal Type ïï Aiveolar CeUs BY Tracy C. Cherlet A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of Master of Science TRACY C. CHERLET O 2000 Permission has ken grrnted to the Library of The University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfiim tus thesidpracticum and to lend or sel1 copies of the fdm, and to Dissertations Abstracts International to publish an abstract of this thesis/practicum. The author reserves other publication rights, and neither this thesis/practicurn nor extensive extracts from it may be printed or othenvise reproduced without the author's written permission. 1 would like to take this opportunity to thank Dr. LE. Scott, my advisor, for al1 his patience and invaluable advice over the 1st three years. 1 would also like to thank the University of Manitoba and the Department of Human Anatomy and Ce11 Science for their financial support. Finally, 1 would like to thank my husband, parents and brother for their emotional support and encouragement throughout this project. LISTOFFIGURES............................................... 7 ABSTRACT .....................................................10 INTRODUCTION ...............................................12 2.1 Structure of distal ainuays and alveoli ............................ 13 2.1 A) Distal airways 2.1.a.i) Clara cells .................................... 13 2.1. a.ii) Ciliated cells .................................. 14 2.1 B) Alveoli 2.1. b.i) Fibroblasts .................................... 14 2.1 .b.ii) Macrophages ................................. 15 2.1 .b.iii) Alveolar type 1 cells ........................... -15 2.1 .b.iv) Alveolar type II cells........................... -16 2.2 Development and maturation of the lungs ............................ 17 2.3 A) Function ........................................... -19 2.3 B) Synthesis ........................................... -20 2.3 C) CTP:Phosphocholine cytiylyltransferase (E.C.2.7.7.15)....... -23 2.3 D) Secretion ............................................ 28 2.3 E) Clearance of surfactant fiom the alveolar space ..............34 2.4 A) Cellular affects of cannabinoids.......................... -40 2.4.a.i) Specific lipid and protein interactions................ 40 2.4.a.ii) Cannabinoid receptors ...........................42 MATERIALS AND METHODS ...................................-47 4.1Materials ....................................................47 A) Isolation of rabbit pre-type II cells ........................ -47 B) Synthesis and release of surfactant by isolated fetal rabbit typencells........................................... 49 C) Isolation of DSPC ..................................... 49 D) Pulse-chase experiment................................. 50 E) Detennination of cellular THC toxicity .................... 52 F) Cytosolic and microsomal fractionation of rabbit hg......... 52 G) Protein quantification ................................... 53 H) Enzyme assays ....................................... -53 I) Synthesis of surfactant by freshly isolated type II cells ........ -54 J) Secretion of surfactant DSPC hmisolated fetaf type II cells ...55 K) Release of surfactant DSPC fiom freshly isolated fetal typencells .......................................... 55 L) Statistical analysis ..................................... 56 RESULlTS ......................................................57 DISCUSSION ...................................................91 CONCLUSIONS ............................................... 104 FUTUREDIRECTIONS .........................................105 REFERENCES .................................................106 TrgEL; Lipid Biosynthesis ..................................... .25 F: 2: Secretion of surfactant fiom type II cells. ................... .36 URE: 3; Accumulation of radiolabeled DSPC in cultured fetal rabbit type II cells and release of [3HlDSPCby these cells following exposure to 10"- 10 JM THC over 24 hours. ......................................-60 FIGllpE 4; Radioactivity associated with choline in isolated fetal rabbit type II cells following exposure to I04M THC for 30 minutes, 1, 3,9, 20 and 48hours ......................................................... 62 GIW5: Accumulation of ['Hlcholine in phosphocholine in isolated fetal Rabbit type II cells following exposure to 104M THC for 30 minutes, 1, 3.9, 20and48hours................................................... 64 GURE 6; Accumulation of [3H]choline in CDP-choline in isolated fetal rabbit type II cells following exposure to 104M THC for 30 minutes, 1, 3,9, 20and48hours ................................................... 66 URE 7: Accumulation of [3H]choline in PC in isolated fetal rabbit type II cells following exposure to 104M THC for 30 minutes, 1,3,9,20 and48hours ..................................................... 68 IGURE 8; Accumulation of ['Hlcholine in DSPC in isolated fetal rabbit type II cells following exposure to 104MTHC for 30 minutes, 1,3,9,20 and 48hours ......................................................-..70 GIRE 9; Lactate dehydrogenase activity in culture medium collected from control ce11 cultures or cultures exposed to 10-4 M THC over a period of 48hours......................................................-..72 Im10; Specific activity of CPCT associated with whole adult lung cytosolic fiactions ............................................... .76 IGW11; Specific activity of CPCT in fetal and adult lung cytosols ......78 1- 1- Specific activity of CPCT in cytosolic and membranous fractions fiom fetal rabbit lungs of 24 gestational days in the presence of 100ug PG, 104M THC or a combination of the two ...................... .80 1- 1- Total CPCT activity in cytosolic and membranous fhctions fkom fetal rabbit lungs in the presence of lOOug PG, 104M THC,or a combination of PG and THC ....................................... -82 GUBE 14; Accumulation of radiolabeled DSPC in fkshly isolated fetal rabbit type II cells following exposure to 10"-10~MTHC over 1,3, 8 and 18hom......................................................... 86 15; Release of radiolabeled DSPC by cultured fetal rabbit type II cells following exposure to 1 0-'M TPA or 1O-'- 1 04M THC over 3 hours ........................................................... 88 16; Release of radiolabeled DSPC fiom fieshiy isolated fetal rabbit type II cells following exposure to lO"M TPA or 1 04M THC for 1, 3 and 5hours .......................................................... 90 PC Phosphatidylcholine DSPC Disaturated phosphatidylcholine DPPC Dipalmitoylphosphatidylcholine PE Phosphatidylethanolamine PS Phosphatidyiserine PI Phosphatidylinositol PG Phosphatidylglycerol CPCT CTP :phosphocholine cytidylyltrans ferase LMW Low molecular weight (refemng to CPCT) r-mlw High molecular weight (referring to CPCT) PKC Protein kinase C THC A~-tetrahydrocannabinol CB, Cannabinoid receptor 1 -2 Cannabinoid receptor 2 LDH Lactate dehydrogenase TPA 12-O-tetradecanoylphorbol- 13-acetate SEM Standard error of the mean PLC Phospholipase C MEM Minimal essential medium CDP-choline Cytidine diphosphocholine DAG Diacylg 1ycerol PA Phosphatidic acid 2-AG 2-arachidonylglycerol Anandamide N-arachidonylethanolamide DHAP Dihydroxyacetone phosphate G-3-P Gl ycerol-3-phosphate 1-AG-3 -P 1-acylglycerol-3-phosphate PLA, Phospholipase A, FPF Fibroblast pneumocyte factor Type il alveolar cells are the primary site of synthesis of disaturated phosphatidylcholine (DSPC), the major component of pulrnonary surfactant. DSPC is thought to convey the surface tension lowering properties of surfactant in preventing alveolar collapse at end-expiration. Recent research indicates that lipid soluble agents may affect fetal lung development following materna1 exposure. Since surfactant is primarily lipid in nature, potential exists that any