CYTOSKELETAL MODULATION OF SURFACTANT TRAFFICKING
Nice Cojocaru
A thesis submitted in conformity with the requirements for the Degree of Master of Science, Graduate Department of the Instihite of Medical Science University of Toronto
O Copyright by Alice Cojocam (1998) National Library Bibliothèque nationale 1*1 ofCanada du Canada Acquisitions and Acquisitions et Bibliographie Senices seMces bibliographiques 395 Wellington Street 395. rue Wellington ûttawaON K1AûN4 OttawaON KIAW Canada Canada
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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 fkom it Ni la thèse ni des extraits substantiels may be printed or otherwïse de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Cytoskeletal Modulation of Surfactant Secretion Degree of Master of Science 1998 Alice Cojocaru Graduate Department of Institute of Medical Science University of Toronto
Abstract
Alveolar type II cells are responsible for synthesis, secretion, and reuptake of pulmonary surfactant. There is strong evidence that the cytoskeleton, primarily the actin filaments, is involved in surfactant trafncking. The purpose of our study was to examine the temporal relationship be~eencytoskeletal changes and surfactant secretion and uptake. Type II cells in primary culture were stimulated with agonists for different time periods. At the same the points, cells were pretreated with actin modulating agents, cytochdasin D (CD), a drug that prevents actin fkom polymerizing, thymosin a, a highly specific actin monomer binding protein, or control vehicle. CD, thymosin, and/or secretagogues increased secretion especially in the £ktfive minutes. These findings were correlated with cytoskeletal changes. None of the agents modulated the uptake of surfactant-like liposomes under conditions of these experùnents. We conclude that corncal actin is disrupted during secretion and that this may be an early requisite step in
exocytosis. Acknowledgements
This project is a story of a journey whose paths were rough at tirnes, a story about
fiiendship and collaboration, which involved the hard work of many people, to whom 1'11 be forever grateful.
1 would like to thank rny supervisor, Dr. Jeff Edelson, for giving me the
opportunity to work on this challenging project, for his trust, support, and patience. From
the beginning of my graduate program to the present the, 1 felt privileged to work under
his supervision.
I am deeply grateful to my cornmittee rnembers, Drs. Greg Downey, Martin Post,
and Michal Opas. 1 am especially thankful to Greg, for his continuous support, intelligent
suggestions, and astute remarks. 1 am very indebted to Dr. Post, whose directions,
expertise and support have been crucial in finishing this project. Also, 1am grateful to
Dr. Opas for his suggestions and help with the irnaging part of this project. Furthemore,
1would like to thank Dr. Keith Tanswell for allowing me to prepare liposomes in his
laboratory and Rose Belcastro for excellent technical assistance.
1 would like to express my gratitude to Jonathan Plumb, for introducing me to
most of the experimental procedures 1 used in this project, for his enormous patience and
willingness to share know ledge.
1 am very indebted to my fnends who supported me. In particular, 1 am thankful
to Hedy Ginzberg for being always there with an encouragement or a new suggestion for
my project. 1would like to thank Dr. Downey's team for their niendship and for creating
one of the nicest working environments: Vera Cherepanov, Kin Chan, Benjamin
Matthews, Theodoros Tsakiridis, Qin Dong, Ba Tu, Joshua Kruger, Kathleen Sang, and Arik Bergman. 1 would also Like to thank my Romanian fi&, whose "joie de vivre" aliowed me to get through some hard times with a smile.
1 would also like to thank Battista Calvieri and Steven Doyle, for their he$ and expertise in confocal microscopy.
In conclusion, I would like to thank my husband, Tony, for his continuous support and understanding, and to rny parents for their love and encouragements throughout these
Y-- Table of Contents
Ab breviations IX
List of figures XII
List of appendices XIV
Chapter 1. INTRODUCTION
1.1. General Introduction
1.2. Histologie Description of Alveolar Type II Ceiïs
1.3. Ultrastructural and Biochernical Cbaracterization of Lameilar Bodies
1.4. Composition and Biop hysical Properties of Surfactant
1.4.1. Surfactant Lipids
1.4.2. Surfactant Subfractions and Apoproteins
1 A.2.l. Surfâctant Protein A (SP-A)
1.4.2.2. Surfactant Protein B (SP-8)
1.4.2.3. Surfactant Protein C (SP-C)
1.4.2.4. Surfactant Protein D (SP-D)
1.4.3. Biophysical Properties of Surfactant
1.5. Other Functions of Surfactant
1.6. Synthesis of Surfactant
1.6.1. Regulation of Phosphatidylcholine Synthesis
1.7. Surfactant secretion
1.7.1. Methods of Investigating Mechanisms of Surfactant Secretion
1.7.2. Regulation of Secretion
1.7.2.1.Physiological Stimuli 1-7.22. Pharmacologicai Factors 35
P-adrenergic Stimuli 35
Purinergic Stimuli 37
Calcium 38
Phorbol Myristate Acetate (PMA) 39
Acid Base Status 40
Pros taglandins, Leukotrienes and Purines 41
Other Secretagogues 42
1J.2.3. Current Secretion Models 43
1.7-2.4. Inhibitors of Surfactant Secretion 46
1.8. Clearance of Surfactant 51
1.8.1. Role of Pneumocytes 53
1.8.2. Role of Alveolar Macrophages 54
1.8.3. Intra-alveoiar MetaboIism 55
1.9. Implications for Pulrnonary Disease 56
1JO. Cytoskeleton: A Brief Review 60
1.10.1. G-actin Assembles into Long F-actin Polymers 62
1.10.2. The Actin Cytoskeleton is Organized into Bundles and
Networks of Filaments 63
1.10.3. Cortical Networks ofActin Filaments Stiffen Ce11 Membranes
and Immobilize Integral Membrane Protehs 64
1.10.4. The Dynamics of Actin Assembly 65
1.10.5. Fungal Toxins Dismpt the Monomer-Polymer Equiiibrium 68 1.1 0.6. Actin-Binding Pro teins ControL the Length of Acth Filaments
1.10.6.1. Sequestration of Actin Monomers by Small
Actin-Binding Pro teins
1.1 1. Cytoskeletal involvement in Surfactant Metabolism
1.12. The Cytoskeleton and Exocytosis in other Ce11 Systems
1.12.1. The Cytoskeleton and Exocytosis
1.12.2. Spatial Compartmentalization: Reserve and Releasable Pools
1.12.5. Transport and Fusion
1.13. Summary
Chapter 2. Rationale and Study Hypothesis
Chapter 3. Materials and Methods
3.1. Materials
3.2. Methods of Procedure
3.2.1. Type II Ce11 Isolation
3.2.3. Quantitation of F-actin
3 -2.3. Measurement of SuI-factant Secretion
3.2.4. Liposome Preparation
3.2.5. Liposomal Uptake by Type II Cells
3 -2.6. Ce11 Permeabilization
3.2.7. Cytotoxicity Evaluation
3.2.8. Fluorescence Stainùig of F-actin and G-actin Chapter 4. ReJuIts and Discussion
4.1. Infiaence of Secretagogues on Surfactant Secretion:
Earty Time Points
4.2. Effect of Secreiagogues on Cytoskeletal Actin
4.3. Role of Secretagogues in Surfactant Uptake
4.4. Effects of Cytoskeletal Disruption on Surfactant Uptake
4.5. Influence of Actin Cytoskeleton Disrupting Agents on Early
Surfactant Secretion
4.6. Surfactant Secretion Induced by Cytoskeletal Disrnpting Agents
Modalates the Distribution of F-actin and Gactin in Alveolar Type II Ce11 126
Chapter 5. Summary and Significance 134
Chapter 6. References
Appendices Abbreviations a-MEM: a-modified Eagle's medium a-MEM-AB: a-modified Eagle's medium without antibiotics
AIR: Al subtype Pl purinoceptors
A2R: A2 subtype P 1 purinoceptors
ALI: acute lung injury
Anx IIt: annexin II tetramer
ARDS: adult respiratory distress syndrome
BHT: butylated hydroxytoluene
Ca-CM-PK: ca2'-calmodulin dependent protein kinase
Cc: critical concentration
CD: cytochalasin D
CLSE: Calf Lung Surfactant Extract cm: choline-phosphate cytidylyltransferase
DAG: diacylglycerol
DMEM: Dulbecco's modified Eagle's medium
DMEM-LG: Dulbecco's modi fied Eagle' s medium - low glucose d-MVB : electron-dense rnatrix multivesicular bodies
DPPC: dipalmitoylphosphatidy lcholine
DSPC: disaturated phosphatidylcholine
EGTA: ethylene-bis-oxyethylenenitnlo-tetraaceticacid
F-ach: filamentous actin
fattv acid svnthase FBS: fetal bovine sem
G-actin: globular actin
Hepes: 4-(2-hydroxyethy1)- 1 -piperazineetha.esulfonicacid
IP3: inositol trisphosphate
LDH: lactate dehydrogenase
1-MVB : electron-lucent matrix multivesicular bodies
MB: meth y lene b lue
MVB: rnultivesicular bodies
NAME: N S2-nitro L-arginine methylester
NO: nitric oxide
NSF: N-ethylmaleimide-sensitive fusion proteins
OAG: 1 -01eoyl-2-acetylglycerol
PA: phosphatidic acid
PBS: phosphate bu ffered saline
PC: phosphatidylcholine
PC-PLC: phospholipase C acting on phosphatidylcholine
PGE2: prostaglandin E2
PK: protein kinase
PKA: CAMP-dependent protein kinase
PKC: protein kinase C PLD: phospholipase D
PM: plasma membrane
PMA: phorbol 12-rnyristate 13-acetate
PPI-PLC: phosphoinositide-specific phospholipase C
PR: p-adrenergic receptors
Ems: respiratory distress syndrome
SM: sphingomyelin
SNAP: soluble NSF attachment protein
SNARE: SNAP receptors
SNP: sodium nitroprusside
SP-A: surfactant protein A
SP-B: surfactant protein B
SP-C: surfactant protein C
SP-D: surfactant protein D
SV: synaptic vesic le ma: tumour necrosis factor a t-SNARE: target membrane-associated SNARE
TP4: thymosin PJ
VAMP: vesicle-associated membrane protein
V-SNARE: vesicle-associated SNARE List of Figures
Chapter 1.
Figure 1. Schematic of DPPC monolayer. 8
Figure 2. Diagram of the major pathways of surfactant phosphatidylcholine
within the type iI ce11 and within the alveolus. 20
Figure 3. Three level modulation of surfactant secretion. 43
Figure 4. Summary of control mechanisms of pulmonary surfactant
secretion. 51
Figure 5. The three phases of actin polymerization. 65
Chapter 4.
Figure 1. Standardized surfactant secretion in control type II cells. 94
Figure 2. Role of PMA on surfactant secretion in type II cells. 95
Figure 3. Role of PMA on surfactant secretion in type II cells (compared to
contro 1). 98
Figure 4. The-dependent variations in the amount of F-actin with PMA
treatment. 1O0
Figure 5. Variations in the amount of F-actin with time in control and PMA
treated alveolar type II cells. 102
Figure 6. Variations in the amount of G-actin with time in control and PMA
treated alveolar type II cells. 103
Figure 7. Uptake of liposomes prepared hmsynthetic phospholipids
([3~]-~~~~)during incubation with isolated granular pneumocytes
XII der24 h in prirnary culture, as a hction of tirne and
temperature. 109
Figure 8. Standardized speci fic uptake of liposomal phosphatidylcholine by
control and PMA stimulated granular pneumocytes, as a hction of
time. 110
Figure 9. Standardized specific uptake of liposomal phosphatidylcholine by
conaol and PMA stimulated granular pneumocytes, as a function of
time and different ce11 treatments. 113
Figure 10. Effects of CD on surfactant secretion. 118
Figure 1 1. Effects of PMA and CD on surfactant secretion. 119
Figure 12. Stimulation of exocytosis with PMA and terbutaline at defined
[ca2']. 124
Figure 13. Changes in the arnount of F-actin with CD i PMA treatment. 129
Figure 14. Changes in the arnount of G-actin with CD ir PMA treatment. 130
Figure 15. Effects of thymosin on F-actin. 132
Figure 16. Effects of thymosin on G-actin content. 132 List of Appendices
Appendix A.
Appendix B.
Appendix C.
Appendix D. Chapter 1
Introduction 1.1. General Introduction
Recent rapid advances in the understanding of the puhonary sdactant system have occurred at the molecular, cellular, physiological, and clinical levels. Physiological obsewations by Kurt von Neergard in 1929 led to his prediction of the existence of a material that reduces intraalveo lar surface pressure. Further experimentation in the late
1950s by Pattlel and Clements' rnarked the beginning of the contemporary era of surfactant research. The field has now matured to the point where it has led to new standards of therapy for neonatal lung disease with potential investigational promise for the treatment of acute lung injury in adults.
hilmonary surfactant is a surface-active material lining the alveolar surface of the lung and, as a lipid-protein cornplex, consists of about 90% lipids and 540% surfactant- specific proteins. This material is synthesized by alveolar type II cells, which cover ody about 5% of the alveolar surface, while the rest of the alveolar surface is represented by alveolar type 1 ceIls.
The principal function of surfactant, and the one most widely investigated, is the maintenance of low surface tension at the air-liquid interface and the prevention of alveolar collapse on expiration. Surfactant is essential for normal lung Cunction. Forces generated at the interface between gas phase and aqueous subphase (i.e. surface pressure) tend to diminish alveolar surface area and constitute a net driving force for the influx of fluid from the interstitium into the aiveolar lumen. Surface pressure rises as the aiveolus decreases in diameter. Without surfactant, collapse of parts of the lung would occur regularly during deflation and development of alveolar edema would be greatly facilitated. The existence of an adequate amount of surfactant is an essential prerequisite for nomal ventilation, and ventilation itself greatly influences the regulation of the surfactant system.' In addition, evidence suggests that surfactant also has a role in the host defense mechanisms and immune fûnctions of the lung.'* '
Lung immaturity with insuflicient surfactant can Iead to respiratory distress syndrome (RDS), a major cause of illness in prematme infants, whereas surfactant deficiency is also believed to play a contributory role in addt acute lung injury and
ARDS.~ A number of homones accelerate lung maturation and stimulate surfactant production in the late gestation fetus; and newbom RDS can often be prevented by materna1 administration of glucocorticoids and other hormones. Onset of RDS in newbom infants can be prevented or attenuated by the administration of exogenous surfactant preparations, some of which are now available commercially.' Exogenous surfactant therapy is of potential benefit in the management of acute lung injury in adults;
When the lipid composition of lung surfactant was established,' Io it becarne possible to predict neonatal respiratory distress before birth by a low lecithinlsphingomyelin ratio in amniotic fluid," markhg the application of basic science to clinical practice in this field.
1.2. Histologic Description of Alveolar Type II Ceiis
Type II cells are cuboidal cells with a diameter of about 9 Pm. They usually occur singly and only occasionally are small groups of two or three such cells found in the alveolar epithelium. They are characterized by 0.1 pm microvilli, which lie on their apical surface. In general, the type II cell has the same morphologic appearance and presurned physiological fùnctions in all mammalian cells. The functions of type II cells include the synthesis, storage, and secretion of surfactant, the reepithelization of the alveolar wall after lung injury, and transepithelid solute transport to limit the volume of alveolar fluid and perhaps regdate the composition of the alveolar fluid Type II cells are also the progenitors of termindly differentiated squamous alveolar type 1 cells.
A distinct morphologic feahire of these cells is the presence of large larnellar bodies (0.2-2 pm diameter), which constitute 18-24% of their cytoplasrn. These lamellar bodies likely fùnction as intracellular reservoirs for newly synthesized and intemalized surfactant.
1.3. UItrastructural and Biochemical Characterization of LameUar Bodies
Lamellar bodies are characterized on ultrastmctural examination by concentric or striated lamellated structures contained within a lirniting membrane. Their lamellated appearance re flect s the organization of p hospholipids, whereas their appearance as transverse or concentric in electron micrographs is a reflection of the plane of section of the bell-shaped
Biochemical characterization and pulse-chase studies of lung phosphatidy Icho line
(PC) synthesis have coafirmed that lamellar bodies are the intracellular storage organelles of surfactant. The phospholipid composition of these organelles is similar to that of surfactant with phosp hatidylcholine (PC) representing 70-80% of p hospholipid and significant amount of phosphatidylglycerol (PG). Lamellar bodies have a relatively high amount (3-5- 13%) of phosphatidylethanolamine (PE), possibly as a component of the limiting membrane of the lamellar bodies.I3 Lamellar bodies also contain surfactant protein A (SP-A)" and the mature fom of surfactant protein B (SP-B). The localization of SP-Chas been more difficult, shce it has been very difficult to raise antibodies to the processed protein. In situ hybridization provides the opportunity to localize the site of production of SP-C. SP-Chas been recently localized in the adult and fetal rat lung tissues, with results generally correlatùig with the localization of SP-A." The same researchen found that rnRNA for SP-C is found in adult and fetal rat type II cells, but is absent fiom bronchiolar cells and alveolar macrophages.
In addition to sdactant, the lamellar bodies also contain enzymes that are normally considered of lysosomal origid6. Recent evidence indicates the presence of hydrolytic enzymes and H' and ca2' transport processes, suggesting a more complex role for this unique organelle.
It is now established that larneilar bodies of identical periodicity and ultrastructural geometry are present in lung (type U pneurnocytes), serosal mesotelium
(perineum, pleura, and pericardiurn), and joints (type A and type B synoviocytes).l7 Not only pulmonary larnellar bodies, but also those at extrapulmonary sites are found in association with SP-A, while SP-A or SP-A-like proteins are present in mesurable quantities in normal serosal fluids. These findings have disclosed totally new avenues of research into multisystem disorders, where for the fkst time an organelle and associated protein provide a liak between diverse tissues affectecl by rheumatoid disease. Evidence of shared epitopes between SP-A and mycobacterïal65-kD heat shock protein indicates a possible etiologic mechanism.
A hitherto ultrastmcturally biological system of lamellar body secretion is now being revealed as a major biological system that appears to subserve surfactant, lubricant, surface protection, and sealant functions in body surfaces and tissue interfaces. This new fi-ontier was bom out of investigations into the mesotelium, research that was vital for advancing the therapy of peritoneal dialysis and for preservation of the dialyzing membrane.
1.4. Composition and Biophysical Properties of Surfactant
Surfactant is a heterogeneous material that exists in several different physical forms'' and is more easily dehed functionally than stmcturally. It is readily obtained nom the lungs by bronchoalveolar lavage and can be purified by differential centrifugation. Isolated surfactant preparations consist of approximately 90% lipid, 10% protein, and small amounts of carbohydrate.
1.4.1. Surfactant Lipids
Of the lipids in surfactant, 80-90% are phospholipids. Cholesterol is the most abundant neutral lipid. There is remarkably little inter-species variation in the phospholipid composition of lung tissue and surfactant. l9 SurFactant phospholipids contain much higher proportions of phosphatidylcholhe (PC) and phosphatidylglycerol (PG) than lung tissue phospholipids, whereas the latter are richer in phosphatidylethanolamine (PE), phosphatidylserine (PS) and sphingomyelin
(SM). Phosphatidylcholine (PC) is the major phospholipid class Ui surfactant and about
60% of the PC molecules have saturated acyl chains in the sn-1 and sn-2 positions. The disaturateci PC (DSPC) in lung sUTf'tant is largely dipaimitoylphosphatidylcholine
(DPPC). This lipid is the major surface-active component of pulmonary surfactant that is responsible for decreasing surface tension to values below 10 mPJ/rn2 at low lung volumesm. The structural features contribute to the molecule's surface-tension-lowering properties (fig. 1). DPPC
Hyclrophobic Fatty Acid Tai1
Water
Hydrophobie Polar Head
I Figure 1. Surfactant is mostly phospholipid, and predominantiy phosphatidylcholine, most of which in turn is disaturated with palmitic acid. The structure of the resulting molecule, dipalmi toy lphosphatidy lchoiine (DPPC),is crucial for surfactant function. When the alveolar surface area is reduced at low lung volumes, DPPC forms a monolayer, with the hydrophilic polar head group aligned along the air-iiquid intedace and the hydrophobic fatty acid taii extending into the air space. Because the tait is fully saturateci, it is arranged in a straight line, and this ailows close packing of DPPC. With close packiog, îhere is mutual repulsion of DPPC molecules, which lowers surface tension and prevents alveoiar collapse. (after Caminiti SP and SL Young. The pulmonary surfactant system. Hospital Practice, Jan 15, 1991.) The fully sahirateci palmitate residues can be ananged in a straight line, allowing close packing of DPPC molecules. The hydrophobic long fatty acid tail extends above the alveolar air-liquid interface when the surface area is reduced at low lung volumes.
The polar head group, choline, has a net charge and is attracted to water molecules in the alveolar hypophase. Close packing of DPPC results in mutual repulsion among the molecules and thus a lowering of surface tension. The polar head of the phospholipid is immersed in the aqueous subphase, while the hydrophobic rest protrudes into the gas phase, tending to be aligned next to a phospholipid molecule. Thus, water molecules are displaced fiom the interface and the high Wace tension is lowered. During the process of deflation of the Iung, compression of the phospholipid film with decreasing aiveolar sucface enhances its capability to lower surface tension. This enhanced ability to lower surface tension when under pressure is a characteristic feature of natural surfactant, not observed in industrial or other man-made detergents, which bears importance for the functioning of the system. Under compression, the surface tension Ui the presence of surfactant can actually approximate O dyn/cm2. Given the significant compression required for the reduction in surface area, surfactant loss occurs due to squeezing out of material during deflation. During the subsequent inflation of the lung and reexpansion of the phospholipid film, new material is adsorbed to the interface. It is presumed that newly secreted surfactant is integrated into a uniquely shaped structure cdled tubular rnyelin, and that it is preferably with this structure that the complicated process of adsorption of surfactant phospholipids to the interface is started.'
Surfactant is also enriched with a phospholipid that is uncornmon in animals but very comrnon in bacteria, phosphatidylglycerol (PG). Compared with other tissues, the lung's PG content is unusually hi&: 5-10% of sudactant phospholipids. Its role in surfactant fiinction is controversial. PG has been shown to promote the association of surfactant apoproteins with phospholipids, and data suggest that it may also play a role in the recycling of surfactant by alveolar epithelium. It is known that amund term the percentage phosphatidylglycerol in the sinfactant synthesized by the type II cells increases, while at the same tirne the percentage of phosphatidylinositol shows a dec1ine."- '2 This late gestational increase suggests that
PG might play a role in the key fûnction of surfactant at the omet of breathing. However, myo-inositol-treated newbom rabbits, in which phosphatidyhositol (PI) replaces PG have no notable defect in surfactant fiinction. Other surfactant lipids, which constitute
10- 15% of the total iipid component, have no known role in surfactant function at this the.
Phosp hatidylcholine (PC) and phosphatidylethanolamine (PE) are zwitterionic phospholipids and have been treated as interchangeable in many experimental designs.
However, there are significant and important differences in chemistry and properties between these two lipids. At physiological pH, PE is mdynegatively charged. PE has a smaller head group, can hydrogen bond through its ionizable amine, and has the unique property of undergoing bilayer-to-nonbilayer physical transition influenced by its fatty acid content and temperature." Both PC acd PE possess a dipole moment across their respective head groups that can be oriented by the membrane electrochemical potential?
Since only PE cmhydrogen bond, the response to the electrochernical potential would be influenced di fferently by the immediate environment. It is clear that PC and PE are not exchangeable with respect to their properties and it is also clear that they are not functionally exchangeable in supporthg biological processes. 1.43. Surfactant Snb-fractions and Apoproteins
The other major component of surfactant is a group of apoproteins that are specifically expressed in the lung. They account for les than 5% (by weight), of surfactant, but they have aroused much interest.
Pulmonary surfactant isolated fiom lung lavages can be separated into subhctions based on buoyant density ? These subfkactions differ in morphological appearance, pro tein compositior~,and metaboiic role." In vivo pulse-chase experiments have shown that larger, heavier subfiactions are the metabolic precursors of the smaller, lighter surfactant subtype." Gross and colleague? have identified and characterized a serine protease, convertase, which mediates the conversion of large to small aggregate surfactant under conditions of surface area cycling. In several different animal models of lung injury, the ratio of small (inactive) to large (active) surfactant aggregates is in~reased.~'." Understanding the mechanisms involved in aggregate conversion could lead to treatment strategies for lung-injury, focused on maintaining large-aggregate integrity.
Recent studies have shown that SP-A is important for rnaintaining large aggregate integrity in vitro. SP-A appears to interact in conjunction with SP-B. These observations suggest decreased levels of SP-A in lung injury>' might, in part, be responsible for the increased smdYlarge surfactant aggregate ratio in these lung injuries. 1.4.2.1. Surfactant Protein A (SP-A)
The most abundant SUffactant protein is the water-soluble surfactant protein A
(SP-A). The primary structure has been determined for SP-A fiom several species and it is highly conserved?
The human genome contains two quite similarly transcribed SP-A genes (SP-A 1 and SP-A~)~and an SP-A pseudogene." The human SP-A1 and SP-A2 genes and the
SP-A pseudogene appear to be localized on chromosome By use of in situ hybndization, SP-A mRNA has been localized in type II cells and non-cilliated bronchiolar epithelial or Clara
SP-A, the major sudactant protein is a highly conserved sialoglycoprotein of 228 amino acids, Mr s 29,000-36,000." SP-A contains two distinct domains; the arnino- terminal third of the protein is collagen-like, whereas the carboxyl-terminal two-thirds has the properties of a lectin. The collagen-like domain foxms a triple helix. Withlli the alveolus, SP-A exists as a multimer composed of six triple-helical structures (1 8 polypeptide chahs) with a molecular weight of approximately 700,000?
SP-A has the capacity to bind lipids and carbohydrates and to interact with specific cell-surface recepton. SP-A binds strongly to surfactant phospholipids and acts in the presence of calcium, SP-B,and SP-Cto prornote the structural transformation of the lamellar body to tubular myelin.42 SP-A's role is to promote spreading of phospholipids once they have been secreted into the alveolar space. SP-A also acts in cooperative and calcium-dependent fashion with SP-B and SP-C to promote rapid formation of phospholipid surface films, and thus facilitates the reduction of alveolar sudace tension.' It has been suggested that SP-A mediates the endocytosis and reutilization of secreted surfactant components through binding to specific high affinity receptors on the apical surface of type II cells."
The finding that purified SP-A inhibits surfactant phospholipid secretion by isolated type II cellstgsuggests that, once secreted, this protein might act in a negative feed-back manner to exert a regulatory role in surfactant synthesis and secretion. SP-A also binds with high affinity to alveolar macrophages" and augments endotoxin-activated alveolar macrophage migration and phagocytosi~.~
Neutral lipid cooperates with surfactant-associated protein A to organize dipalmitoylphosphatidylcholine in the surface films and enhance formation of a DPPC- nch reservoir below the air-water interface. Studies with chi~neric'~or mutated foms of
SP-A have demonstrated the involvernent of the COOH-terminal domains of the protein in ligand and receptor binding, the regulation of surfactant secretion and lipid uptake by type II cells. In vitro studies also suggest a role for SP-A in preventing phospholipase induced surfactant degradation. "
SP-A synthesis and secretion may be regulated independently, as part of the alveolar defense system, fkom that of the other surfactant components. Recently, it has been reported that synthesis of SP-A cm be enhanced by interferon-y without affecthg the production of other components of the surfactant system". SP-A appears to be involved in the regulation of surfactant hunove?, formation of hibular rnyelhf9, and the immune regulation within the lmg? However, mice made SP-A deficient by homologous recombination grow and reproduce normally and have normal lung iùncti~n.~The only sdactant abnormalities that were detected were a severe reduction in tubular myelin figures in the airspace and elevated minimum surface tensions at low surfactant concentrations.
1.4.2.2. Surfactant Protein B (SP-B)
Surfactant contains two smaller proteins with unique properties, one of which is their CO-purificationwith lipid into organic solvents. SP-B and SP-C have been isolated and purified fiom such extracts. In situ hybridization studies suggest that type II cells and also bronchiolar cells may be the site of synthesis of human SP-B". The gene for
SP-B is on chromosome 2. The original transcription product of 381 amino acids undergoes significant posttranslational modification and the protein that ultimately co- isolates with surfactant is only 79 amino acids long. The functional protein has two regions with a high content of hydrophobic amino acids, which are presumably sites of phospholipid interaction.
The function of SP-B is not well understood, but it may be involved in the formation of tubular myelin. Recent studies have shown that SP-B contributes to the surface activity of surfactant and that this protein may increase the intemolecular and intramolecular order of the phospholipid bilayer, supporting the concept that SP-B increases the lateral stability of the phospholipid la~er.~'A recent study confirmed that the 25 amino-terminal peptides of SP-B stabilized the phospholipid layer by increasing the collapse pressure of surfactant phosp ho lipids whic h may prevent squeezing out of the phospholipids nom the monolayers of the alveolar-air A specific charge interaction between the cationic peptide and an anionic lipid such as PG may be responsible for this stabilizaîion.
Lung morphogenesis and surfactant phospholipid synthesis in SP-B (4)mice proceed normally pnor to birth." However, lamellar body fcrmation is dimpted, resulting in abnormal inclusions consisting of multivesicular bodies and disorganized lamellae. Neither mahue larnellar bodies, nor tubular myelin were detected in SP-B knock-out mice. SP-B deficiency resulted in aberrant processing of the SP-C proprotein, leading to accumulation of a processing intemiediate Mr = 11,000 and a decrease in mahue SP-C peptide levels. Lack of SP-B results in respiratory failure at birth, and decreased SP-B protein is associated with decreased lung co~npliance.~~These hdings demonstrate the critical role of SP-B in perinatal adaptation to air breathing.
1.UA Surfactant Protein C (SP-C)
SP-C, the other hydrophobic protein fond in the bronchoalveolar lavage fluid, is the product of a gene localized on chromosome 8. The primary translation product contains 197 amino acids and has a molecular weight of 20,000. A small hgment of this, ody 36 arnino acids long, is the mature protein that has been isolated corn lavage.
This "active" SP-C peptide present in the alveolar lavage is perhaps one of the most hydrophobic proteins known, due to its high content in valine residues. Like mature SP-
B, mature SP-Cis highly enriched in lamellar bodies.
Both SP-B and SP-C have dramatic effects on the spreading properties of lipids and markedly accelerate the rate at which a surfiace film is formed at an air-liquid interface, which causes a rapid reduction in dacetension.' SP-B has a greater capacity to reduce surface tension than SP-C,suggesting that SP-B is more efficient than SP-C in squeezing out unsaturated phospholipids fkom the surface?
1A.2.4. Surfactant Protein D (SP-D)
SP-D is a collagenous glycoprotein recently descxibed as a surfactant-specific protein. It is synthesized by alveolar type II cells and is found in alveolar lavage in relatively low concentrations55. In rat lavage, most of SP-D is in the soluble kction and not associated with surfactantM. SP-D is simila. in structure to SP-A as well as to other members of the collagenous C-type lectin family. There is evidence that SP-Dmay function in pulmonary host defense mechanism. SP-D interacts with Escherichia coli in a calcium-dependent manner; binding is saturable and inhibited by saccharides. SP-D also binds to macrophages, and at hi& concentrations causes agglutination of bacteria." in contrast to SP-A, which specifically binds to dipaimitoylphosphatidylcholine, SP-D binds selectively to phosphatidylinositol in a calcium-dependent rnar~ner.~~An 11 kb gene localized on human chromosome 10, at the same locus as the gene for SP-A encodes SP-
D.'~
SP-D is present in secretory granules in Clara cells, as well as in type II cells in the rat. Expression of the SP-Dgene is developmentally regulated in fetal lung; expression of the gene encoding SP-D is initiated in late gestation, after induction of the other surfactant-associated proteins and of sdactant phospholipid synthesis.* 1.43. Biophysicai Properties of Surfactant
In the last few decades, the biophysical properties of surfactant were expIored6', its apoproteins discovered and its ultrastructural forms revealeda. Remarkable features of the surfactant system include: o Type II cells can secrete alrnost their weight in surfactant every day o Surfactant reduces the surface tension of the alveolar aqueous lining to zero (or very
close to it), o The low molecular weight surfactant apoproteins are the most hydrophobic proteins
hown, o The arnazing ultrastmcturai beauty of tubular myelin
In utero, fetal lungs are filled with liquid. Once air breathing commences, an air- liquid interface is formed, and the effects of surface tension then dominate lung mechanics. Surfactants preferentially adsorb to a surface and act to lower interfacial tension. Absence of surfactant would make alveoli collapse at end expiration. If this occurred, a tremendous force, generated with each breath, would be necessary to open collapsed alveoli. This is the overwhelming problem for the infant with respiratory distress syndrome (RDS).
Surface tension for a sphere is given by the Laplace equation, P=2T/R, in which
P=pressu.e, T=surface tension, and R=radius. If surface tension remains constant, pressure must rise with smaller radii. This concept is important in the lung because it applies to each alveolus, and not al1 aiveoli are the same size. If surface tension were constant, mal1 alveoli would ernpty into Iarger ones because of their higher pressure. Obviously, this does not occur. One reason is that surface tension is not constant but varies directly with surface area, thereby keeping inflation pressure relatively constant regardless of dveolar ~ize.~~Another advantage of reduced surface tension may be that it prevents pulrnonary edema. The effect of surfactant is to reduce the hydrostatic forces acting to move semm from alveolar capillaries into the alveolar air space.
1.5. Other Functions of Surfactant
Surfactant also plays a role in lung host defenses, and a number of its effects on the immune system have been demonstrated. SUTfactant enhances the phagocytosis of staphylococci by alveolar macrophages, enhances killing of staphylococci by macrophages via increased bacterial cell membrane permeabilityw, and inhibits B-and T- ce11 proliferation." Surfactant covers and protects the ce11 membrane of the alveolar epithelium, opsonizes ba~teria~~,and coats inorganic dust particles. Cycling variations in surface tension contribute to the alveolar clearance of particles. Surfactant's imrnunologic role in the lung is the subject of ongoing investigation.
1.6. Synthesis of Surfactant
Much attention has focused on the mechanisms and regulation of phospholipid
synthesis by type II cells. Initial studies examineci lipid biochemistry in the whole lung
or subcellular fkactions derived thereof. These approaches were largely eclipsed by the
advent of reliable procedures for the purification of type II cells. The metabolic disposition of the type II cell is that of a ce11 committed to lipogenesis and it is likely that regdatory mechanisns found in other lipogenic cells, such as the adipocyte, the hepatocyte, and the 3T3-LI ce11 he,are relevant to surfactant phospholipid production, where there is a commonality of biospthetic pathways.
Surfactant phospholipids and proteins are synthesized de novo by the alveolar type II cell. These products are packaged inside lamellar bodies and are subsequently secreted into the alveolar air space. In addition, there is evidence that surfactant is recycled and resecreted Using autoradiography, Gaston Chevalier and André J. Collet of
Laval UniversiQF studied sites of surfactant assembly within the type II ceil. Choline and leucine labelled with tritium were injected into rats, and the tirne course of radioactivity in type II ce11 compartments was followed. It was shown that PC was synthesized in the endoplasmic reticulum. After synthesis, the PC was transferred to the
Golgi apparatus, then to multivesicuiar bodies (a part of the lysosomal system) and eventualIy to the lamellar body (fig. 2): Figure 2. Diagram of the major pathways of surfactant phosphatidylcholine within the type II ce11 and witbin the alveolus. (after Lewis JF and AH Jobe. Surfactant and the addt respiratory distress syndrome. Am Rw Resp Dis, 147: 2 18-233, 1993.)
Chevallier and Collet proposed that newly synthesized surfactant phosphatidylcholine was transported directly to immature larnellar bodies but that surfactant protein left the tram-Golgi and entered multivesicular bodies (MVB). Fusion of MVB with lipid was postulated to produce a membrane-bound organelle, the composite larnellar body, which contained lamellated matenal (lipid), plus vesicles Many ceIl types, perhaps dl, have MVBs but they are usually thought to be part of the endosorne-lysosome system and the suggested utilization of MVB for posttranslational assembly of a secretory granule may be unique to the type II cell.
Williams68demonstrateci two populations of MVB in type II cells, larger ones with an electron-lucent matrix (1-MVB) and smaller ones with electron-dense matrix (d-MVB).
The 1-MVBs contain immunoreactive SP-A but not acid phosphatase activity, whereas the d-MVBs contain lysosomal enzyme activity but no detectable SP-A. Like many epithelia, type II cells are polarized;" 1-MVBs are mainiy apical, but d-MVBs and composite bodies are basal. Lamellar bodies are believed to be derived fiom composite bodies, and in perinatal lung, both are localized in the same basal region. In the adult, lamellar bodies are located randomly throughout the type II cell and are secreted by exocytosis in response to physiological stimuli. The phospholipid composition of lamellar bodies is nearly identical to that of surfactant obtained from the alveolar air space. Lamellar bodies have a phospholipid-protein ratio 10 tirnes greater than the average in the isolated type II cells, lung homogenate, or other cellular components.
Transfer of phospholipids fkom the Golgi apparatus to the lamellar body occurs by an unknown mechanism-possibly the action of phospholipid exchange proteins, which rnight direct individual phospholipids to specific sites in the cells. Evidence for such proteins has been found in intact lung and in type II cells. but proof of theu hction is lacking.
The site of PC synthesis, as well as that of PG synthesis have been localized to the endoplasrnic reticulum. In other tissues, PG is synthesized in the mitochondria
(perhaps reflecting a cornmon bacterial ancestry), and it has been suggested that this is the case in the type II cell as well. However, the observation of a switch-over fiom surfactant phosphatidy linosito1 to phosphatidy lglycerol production around term2'.=, together with the evidence that the endoplasmic reticulum is the site in the type II cells where inositol inhïbits phosphatidylglycerol synthesis, indicates that the endoplasmic reticulum is the intracellular site of surfactant phosphatidy iglycero l production.
Synthesis of the other lipid components of surfactant is likely to occur in the endoplasmic reticulum.
Highly unstable, structural foms of surfactant are present in the hypophase of the alveolar linhg fluid. Mer secretion, lamellar body contents form an extended lattice called tubular myelin (figure 2). Tubular myelin formation cm proceed spontaneously, but requires phospholipid, calcium, SP-A, and SP-B;42tubular myelin is likely the precursor of the phospholipid monolayer at the air-liquid interface.
Surfactant protein synthesis also takes place in the type II cell. SP-A has been the most thoroughly studied, since it is the rnost abundant. Translation of SP-A occurs on rough endoplasmic reticulum, and the new protein molecule includes a leader sequence that inserts SP-A into the endoplasmic reticulum. It then moves to the Golgi apparatus and undergoes extensive modification. Before any Mertransport occurs, the protein assumes the structure of a triple helix, and possibly even an octadecamenc structure.
From there, its fate remains controversial: SP-A is concentrated in lamellar bodies, but whether this results fkom transport of oewly synthesized material or kom other pathways is not clear. 1.6.1. Regulation of Phosphatidylchoüne Synthesis
Regulation of SUffactant phospholipid synthesis has been studied in developing fetal lung and isolated type II cells f?om fetal and addt animals. The fetal lung is a particularly suitable mode1 because of the developmental increase in surfactant synthesis and its susceptibility to hormonal regdation. Most studies have focused on phosphatidylcholine, the major lipid component of surfactant, although synthesis of other phospholipids has also been studied.
Choline-phosphate cytidy ly ltransferase (CYT),the enzyme that catalizes formation of CDPcholine fkom choline phosphate and CTP, is a rate-regdatory enzyme in phosphatidylcholine biosynthesis in al1 systems exaznined including adult and fetal lungs and type II cells. Altered CYT activity invariably accompanies altered p hosp hatidylc holine biosynthesis. CYT activity increases in developing lung and is increased by glucocorticoids and other hormones that stimulate surfactant production in fetal rat, rabbif mouse, and human lung both in vivo and in culture."
Developmental increases in de novo fatty acid biosynthesis and fatty acid synthase
@AS) activity occur in the late gestational fetal lung70 and both are accelerated by glucocorticoids. Glucocorticoids increase the rate of fatty acid biosynthesis in fetal rat, rabbit, and hurnan lung and the activity of FAS in fetal rat and human lung."
There are no discemible differences in the time courses of the effects of dexarnethasone on FAS and CYT activities in fetal rat lung explants. Inhibition of FAS activity blocks the stimulation of CYT? Pulmonary surfactant phosphatidylcholine synthesis increases in fetal lung type II cells with advancing gestation." This increase is accompanied by an increase in gene and protein expression of CTPghosphocholine cytidylyltransferase, which catalizes a regdatory step in de novo phosphatidylcholine synthesis by fetal type II cells.
1.7. Surfactant Secretion
The first report of an exocrine bction of type II pneumocytes expressed as release of lamellar bodies in the alveolar lumen was given by Bensch et al. in 1964".
This process was described as " classical" exocytosis7? Extracellular stimuli modulate intracellular chernical events, which result in movement of the lamellar bodies towards the apical sdace of the type II ceil, apposition of lamellar bodies to the apical plasma membrane, fusion of the limiting membrane of the lamellar bodies with the plasma membrane, and extrusion of the lamellar body contents into the alveolus. The lamellar body undergoes regulated exocytosis in response to specific stimuli. This process appears to be calcium, energy, temperature and cation dependent. Details of the exocytosis of lamellar bodies are still unclear. ~ecently?it was suggested that annexin II tetramer (Anx IIt), one of the two forms of annexin II, which belongs to a family of Ca2'- and phospholipid-binding proteins, may play a role in the Fusion of lamellar bodies with the plasma membrane during surfactant secretion.
Intracellular second messmgers, cytoskeletal elements, protein phosphorylation and dephosphorylation, and membrane fusion proteins are al1 involved in exocytosis in type II cells. The sfeps linking the initial stimulus to the secretion of lamellar bodies however, remain incompletely understood. Outside the cell, newly secreted surfactant is transfomecl into tubular myelin, a highly ordered, lattice-like structure characterized by a system of densely packed, rectangular tubules. A key property of tubular myelin is its rapid adsorption to an air-
liquid interface. It is not known exactly how this structure is formed, but calcium is required in vitro.
Tubular myeh is likely the imrnediate precursor of the functional surfactant monolayer. Tubular myelin is rich in SP-A., SP-B, and SP-C,al1 of which are required
for the most rapid adsorption. When surfactant is isolated, only those fractions that are enriched in tubular myelin and SP-A adsorb rapidly. This process is calcium dependent.
Studies of surfactant synthesis and its extracellular life cycle showed that fhctions enriched in tubular myelin were radiolabelled before the other fractions in the alveolar air space, which suggests that tubuIar myelin is the precursor for the other components.
Once in the alveolar air space, surfactant ccanot play its role in rninimizing surface tension until it foms a monomolecular layer at the air-liquid interface. At the very Iow surface tension present at low lung volumes, the monolayer is believed to be composed almost entirely of DPPC. Pure DPPC adsorbs to an air-liquid interface very slowly, whereas lung surfactant does so quickly. Surfactant apoproteins aid in adsorption to the air-liquid interface?
1.7.1 . Methods of Investigathg Mechanisms of Surfactant Secretion
Many different mode1 systerns have been used to study surfactant secretion.
Morphometric techniques at the electron rnicroscopic level have been used to estimate the cellular content of lamellar bodies before and after treatment with stimuli. Physical
methods, Le. measuring surface tension of alveolar lavage liquid, have also been used to
estimate secretion. Chernical measurement of surfactant secretion has generally focused
on secretion of one surfactant component because swfactant is chemically heterogeneous,
and it is difficult to measure dl surfactant components simultaneously. This approach
appears to be valid because it appears most surfactant components can be secreted
together. The majority of studies of control of surfactant secretion have use lipid components as surfactant markers for secretion. Various classes of lipids have been used?
in increasing order of selectivity: the entire lipid pool; the entire phospholipid pool; al1 phosphatidy lcholines; and saturated phosphatidylcholines in which fatty acids on both the
CI and C2positions are saturated. There are very few studies of secretion of surfactant pro teins."
Systems involving whole animais, isolated lungs, lung slices, and cultured type II cells have al1 been used to study surfactant secretion. Each type of mode1 system fiords distinct advantages and disadvantages. Systems involving whole animals afford preservation of cell interactions both within the lung and among various organs, but do not easily permit direct effects on type II cells to be differentiated fkom effects that act
indirectly on other cells, or on other organs. Mode1 systems using isolated type II cells
allow the direct study of type II cells, but it should be kept in mind that isolation and
culture of cells can introduce artifacts and that effects observed in viîro are not
necessady those that are important in vivo, mainly due to absence of the neural and
humoral factors. Unfortunately, it has been difficult to correlate observations made in vitro with those made in vivo. Because the lung is sparsely innervateci at the alveolar level, it seems unlikely that the nervous system directly affects type II cells. Both deep infiation of the lungs and circdating catecholamines probably modulate secretion in vivo.
Unfominately, naturally occ~gsubstances that correspond to many pharmacological agents active in viîro have not been identified.
No transformed ce11 lines maintain bonafide characteristics of type II cells' differentiated fiuictionsn. Most in vitro studies regarding surfactant are done using primary ce11 cultures of type II cells f?om rat or rabbit lungs.
The study of subcellular mechanisms of surfactant secretion and of its control started with in vivo animal models, but in vitro approaches quickly becarne the methods of choice for this purpose. Various incubation or culture techniques, including lung slicesR,primary cultures of type II ce11 monolayersm,and organotypic cultures8'have been used. It has been demonstrated 31 that the material secreted by isolated cultivated cells was simi lar to surfactant obtained by bronchoalveolar lavage.
Electron microscopie studies have provided images of the extrusion of the lamellar body content into the alveolar lumen8', which suggested a fusion of the lirniting membrane, which sunounds the lamellar body, with the apical plasma membrane of the aiveolar type II cells. In a morphomettic analysisUin the rat in vivo, it has been show that lamellar bodies are released through epithelial cell-surface pores averaging 0.2 x 0.4 pin size. Most often, the pores are surrounded by an elevated area devoid of microvilli, which overlays an area of granular cytoplasm. The size of the pores as shown by scanning electron microscopy is about half the average diameter of the lamellar bodies, which implies a distortion of the latter at the time of extrusion. It was suggested that this distortion may be important for the conversion of lamellar body content into functional &actant in induchg dimption of the highly ordered material of the organelle. Secretion of sudhctant has been studied in the intact lung using lung lavage technique and also with isolated type II cells. The rate of basal secretion has been estimated to be as much as 1O%/hour by using kinetic studies employing biochemical and morphometric analysis of intact lungs". Secretion rate as percent of PC released is
-1 %/h for isolated cellsa?
1.7.2. Reguiation of Secretion
Secretion nom cells can occur by both constitutive and regulated pathways.
Constitutive secretion is the process by which substances are secreted continuously without prior concentration and storage. It is possible that one or more components of swfactant are secreted by constitutive, as well as regulated pathways, and that the two pathways rnay be utilized to differing extents during various states of health and disease.
Studies on type II cells have focused on regulated secretion, in which a stimulus causes previously synthesized matenal stored in intracytoplasmatic organelles to be released fiom the cell.
In spite of over three decades of active research on surface-active material, the actual physiological stimulus and biochemical messages for signal transduction in vivo are not kno~n~.Secretion cmbe stimulated by hyperventilation in vivo; the biochemical regulation of this process remains to be discovered. The intracellular mechanisms (Le. the nature of second messmgers and their effects) which trigger the release of larnellar bodies in vivo are not completely clear.
Surfactant secretion is regulated by at least three distinct signahg pathways: a PKC dependent mechanism, a CAMPdependent pathway and activation of a Ca2+-calmodulin dependent protein kinase (Ca-CM-PIS).
The degree of coordination in regulation of surfactant components has been the subject of recent inquiry. Although it has recently been suggested that sorne surfactant components could be secreted separately, the presence of most of them in lamellar bodies, including al1 the phospholipids in the same proportions as in intraalveolar material and also SP-A, allows one to regard the release of lamellar bodies as equivalent to surfactant secretion. The similar time courses for the increase in the specific phospholipids* and SP-An after intravenous in.ection with labeled precursor also argue for simultaneous release of these cornponents. However, in view of more recent information, the observations in this paper are probably incorrect.
For the most part, shidies of the regulation of surfactant secretion have focused on the lipid components, particularly phosphatidylcholine or disahirated phosphatidylcholine. Surfactant phosphatidy lcho line secretion appears to be regulated for the most part, although some constitutive secretion may also occur. The phospholipid composition of isolated lamellar bodies is virtually identical to that of surfa~tant'~and it is clear that surfactant phospholipids are secreted by exocytosis together with lamellar bodies. In contrast, there are confiictuig reports on the cellular distribution of SP-A. Ln some studies, SP-A was reported to be concentrated in lamellar bodies," in others, lamellar bodies were reported to contain relatively little SP-A? In addition, in vivo experiments suggest that SP-A is secreted independently of lameilar bodies.9' Secretion of SP-A in isolated type II celIs is largely constitutive and not regulated." Lamellar bodies are highly enriched in SP-B and SP-CemHowever, secretion of those surfactant proteins has not been investigated.
Surfactant secretion is enhanced in vivo by labour, hyperventilation and by a number of physiological agonis&." In vitro, well-established sdactant phospholipid secretagogues include Padrenergic agonists, A, and P, pu~oceptoragonists, phorbol myristate acetate (PMA), diacylglycerols (that directly activate protein kinase C) and ionophores ionomycin and A23 187 (that promote calcium uiflwc into the cells)." "
Arachidonic acid and its metabolites, vasopressin, histamine, antihi stamines, and serum lipoproteins, also stimulate phosphatidylcholine secretion in type II cells. By contrast,
SP-A, a number of plant lectins, A, purinergic agonists, substance P, and compound
48/80 were reported to inhibit surfactant secretion? " 95
Surfactant phospholipid secretion is mediated by at least three signal-transduction rnechanisms. P-agonists and adenosine A, receptor agonists act at ce11 surface recep ton that are coupled to adenylate cyclase. Activation of adenylate cyclase results in the generation of CAMP,which in turn activates CAMP-dependentprotein kinase (PU).
Type II ce11 CAMP levels increase rapidly in response to terbutaline, isoproterenol, adenosine, and adenosine analog~es.~p-receptors are coupled to adenylate cyclase via the heterotrimeric G protein Gs; cholera toxin, an agent that pemanently activates Gs, stimulates phosphatidylcholine secretion in type II ~ells.'~Increased CAMPin response to terbutaline and forskolin is associated with increased activation of cAMPdependmt protein kinase96and phosphorylation of actin." Phosphorylation of actin and/or other proteins is believed to lead ultimately to phosphatidylcholine secretion.
Activation of other protein kinases also leads to phosphatidylcholine secretion in the type II cell. Thus, PMA, 1-oleoyl-2-acetylglycerol,and dioctanoylglycerol are effective surfactant secretagogues. ATP" and va~opressin~~activate phosphoinositide- specific phospholipase C (PPI-PLC)via a G-protein (possibly GJ. PPI-PLC is an enzyme that acts on phosphatidylinositol bisphosphate to generate the second messengers inositol trisphosphate (IP,) and diacylglycerol. DAG activates protein kinase C (PKC), which in tum activates phospholipase D (PLD). PLD hydrolysis of phosphatidylcholine leads to the formation of choline and phosphatidic acid (PA). Phosphatidate phosphohydrolase converts PA to DAG, which then Meractivates PKC. A phospholipase C acting on phosphatidylcholine (PC-PLC) may also lead to generation of DAG. PMA and 1-oleoyl-
2-acetylglycerol (OAG), as well as other ce11 permeable DAGs such as dioctanoylglycerol, directly activate PKC. ATP also binds to the adenosine A?receptor or to another methylxantine-sensitive P, receptor that is coupled to adenylate cyclase. IP, promotes mobilization of calcium from intracellular stores; the ionophores ionomycin and A23 187 prornote calcium influx into the cell. Calcium activates a Ca2'talmodulin- dependent protein kinase (Ca-CM-PK). Protein phosphorylation by PKA,PKC, or Ca-
CM-PK ultimately leads to surfactant secretion.
Diacylglycerol is known to activate protein kinase C, and activation of that enzyme was reported in type II cells exposed to AT P.'^ The stimulatory effects of ATP and ionornycin on surfactant semetion are decreased by calmodulin antagoni~ts,~ suggesting that a calmodulin dependent step is also involved in regulating surfactant secretion. Whether that is a calcium/calmodulin-dependent protein kinase or another calmodulin dependent step remains to be detemined.
The stimulatory effect of ATP on surfactant secretion appears to be mediated by al1 three signal transduction mechanisms. ATP increases CAMP formation, 'O' activates
PPI-PLC with generation of IP, and diacylglycerol, activates protein kinase C, and increases calcium level~.'~The action of ATP, the prototypical P2 agoni* likely is mediated by more than one receptor. Based largely on the potency order of ATP analogues, P2 purinoceptors are divided into P,P,,, P, P,, and Pz, subtypes.'" Only the P,, and P, receptors are coupled to PPI-PLC in other systems, so the P2 receptor on the type II ce11 is likely to be one of those. There is indication that P,, receptor mediates surfactant secretion in type II cell.'" Taken together, these data suggest that ATP acts through a P,, receptor that is coupled to PPI-PLC via a G-protein. PPI-PLC activation results in formation of diacylglycerol, which in tum activates protein kinase C, an enzyme that is also direc tly activated by PMA and dioctanoylglycerol. Subsequent protein phosphorylation then initiates PC secretion.
1.7.2.l. Physiological Stimuli
The normal pattern of breathing involves relatively shailow breaths at tidal volume punctuated by occasional deep inflations of the lungs, texmed sighslq.
Continuou shallow breathing without sighs causes diminished lung cornpliance, atelectasis, and hypoxe~nia'~~Several different lines of evidence in intact animals and in isolated lung models suggest that an increase in lung volume stimulates surfactant release.
Increased rates of surfactant release were dernonstrated with hyperventilation.59
Nicholas and Barr Ia6demonstratedthat increased tidal volume was an efficient stimulus of surfactant release in the isolated perfused rat lung and that this was unafkted by various inhibitors, including tetrodotoxin, propranolol, atropine and indomethach. Based on the absence of the effect of tetrodoto~in'~',which blocks generation of neural signals, the same authors concluded that intrapulmonary neurons are not required for lung response.
Increased Iamellar body release was observed in sighed rats, compared to their unsighed controls, and this effect was not prevented by bilateral vagotomy"? In their experirnent, the authors have used ventilated rats at normal tidal volume without periodic deep breaths, or with a four-the tidal volume inflation every 5 minutes for 1 hour. The volume density of larnellar bodies in alveolar type II cells was about one-third lower after
60 minutes of ventilation in sighed than unsighed rat, and the effect of sighs was not blocked by bilateral cervical vagotorny. These morphologie data supported the idea that large inflations are potential stimuli for surfactant secretion.
A recent direct demonstration indicated that the release of surfactant was a consequence of the stretch of alveolar type II cells themselve~.~~The hypothesis that mechanical distention directly stimulates alveolar type II cell surfactant secretion was tested by stretching cultured type II cells on silastic membranes. The intracellular calcium concentration was measured in single cells before and fier swtching and a single stretch caused a less than 60 second increase in cytosolic calcium, followed by a sustained 15-30 minute stimulation of surfactant secretion. Both Ca2+mobilization and exocytosis exhibited dose-dependency to the magnitude of the stretch stimulus. Thus, they concluded that mechanical factors can trigger complex cellular events in nonnewon, nonmuscle cells and may be involved in regulating normal lung functions.
Taken together, these data are consistent with the concept of inflation to total lung capacity being a physiological stimulus of SUffactant release into the alveolar space. The simple mechanical distention of the alveolar wall induced by sigh or by the onset of breathing triggers extrusion of lamellar bodies into the alveoli. There are some phenornena, however, which involve receptor-transduced signals. Barr and CO-worker~''~ proposed that two pools of surfactant exist in Iung tissue: a large pool that turns over slowly and is decreased in response to the type II ce11 distortion and a srnall pool that tums over rapidly and is under sympathetic nervous control. The two kinds of stimuli could possibly have different physiological functions and would be set in action in different circumstances, sighs presumably for regular basal secretion and nervous stimulus for acute release.
Cholinergic and adrenergic antagonists inhibit the response induced by mechanical stimulation of surfactant secretion, Le. by lung inflation, hyperventilation, and deep sighs. Recent experirnents have show the sarne phenomenon in vitro with type
II cells grown on stretchable membranest? Increased surfactant secretion in response to mechanical stimuli is thought to involve direct membrane distortion, a calcium-dependent phenomenon. In experiments perfomed on rabbit pups delivered by Cesarean section after 30 minutes air breathing, 30 minutes nitrogen breathing, or 30 minutes of tracheal occlusion, lmg expansion itself was demonstrated to be a stimulus of dâctant release indicating an increase in &actant in those treated with air or nitrogen, but not in those with occluded trachea, assigning a secondary role to catecholarnines, which were most probably similarly increased in both anoxic groups. " '
It is possible that lung distention induces surfactant release through a mechanism not as simple as the passive stretch and deformation of type II cells previously
~u~~ested~~;it may involve a complex cascade of events, including generation of ATP, and possibly also leukotrienes andfor prostaglmdins, and subsequent activation of purinoceptors or receptoa of arachidonic acid metabolites.
1.7.2.2. Pharmacological Factors
P-adrenergic stimuli
The £kt group of well-characterized stimuli is formed by P-adrenergic agonists, which increase surfactant secretion in isolated type II cells, isolated whole lungs, and intact anirnals. Adrenergic antagonists can block this effect. The agonists act directly, via P-adrenergic receptors on the type II cell, to increase intracellular CAMPlevels. As support for this mechanism, CAMPanalogues stimulate surfactant secretion, an effect that cm be augrnented with phosphodiesterase inhibitors. Stimuli capable of increasing cellular levels of CAMP appear to induce surfactant release, and the enhancement of surfactant secretion induced by activation of adrenergic receptors appears to involve the mediation of CAMP.Thus, B-agonists cause a rise in CAMP concentration in type II cell~~~~,CAMP analogs mimic their effects in stimulating surfactant release, and phosphodiesterase inhibitors potentiate theu effects."=
Morphologie studies in rat~~~~demonstratethat injection of p-adrenergic agents causes a decrease in the nurnber of lamellar bodies per type II cell. The physiologie source of P-adrenergic stimuli is thought to be circulating catecholamines; the mechanism of action involves increasing cellular levels of CAMP.
Cholera toxin was also shown to enhance surfactant secretion through an increase of cellular CAMP."^ Its mechanisrn of action, althoug.not yet determined, could be somewhat different kom that of P-agonists since its effects appeared much later after application of the stimulus.
Not surprisingly, other agents that increase cellular CAMPalso stimulate secretion. Analogues of CAMP,"^* methy 1 xanthuies1I6,forskolin%, and c holera toxin"', al1 stimulate secretion in primary cultures of type II cells. Surfactant release induced b y the B-agonist terbutaline or by the diterpene forskolin has been show to correlate with an activation of CAMP-dependentprotein kinase. This activity has been identified in cytosolic preparations of type II pneumocytes, and binding of labeled CAMPto its regulatory subunits has been demonstrated.
Whitsett et al." reported that type II ce11 actin is phosphorylated in viiro through a
CAMP-dependentprocess. Changes in actin phosphorylation that occur during development are not due to changes in cytosolic actin content or CAMPdependent protein-kinase activity, but appear to be related to the presence of factors inhibiting CAMP-dependentactin phosphorylation in fetal lung cytosol. Resumably protein kinase
A is involvecl in the stimulus-secretion pathway mediated by CAMP. Increased CAMP in response to terbutaline and forskolin is associated with increased activation of CAMP- dependent protein kinase and phosphorylation of actin. Phosphorylation of actin and/or other proteins is believed to lead ultirnately to phosphatidylcholine secretion. However, distal steps in the surfactant secretory pathway have been poorly characterized to date.
Purinergie Stimuli
Adenosine compounds are potent secretagogues for phosphatidylcholine in culhired type II cells. Both aden~sine~l~'~~and ATP119*'S stimulate secretion. ATP and
PMA (see below) are the two most potent secretagogues for surfactant in vitro. Gilfillan and Rooney showed that the stimulatory effect of adenosine could be mimicked by non- metabolizable adenosine analogues and could be blocked by 8-phenyltheophylline. The effects of adenosine analogues were nor additive to those of terbutaline. Adenosine analogues also increase cellular CAMPcontent. These findings suggest that the stimulatory properties of adenosine occur via a P I-purinoceptor mechanism. Recent observations Mersuggest that both Al and A2 subtypes of the PI-receptor may regulate surfactant secretion, at lest in culhued cells. The A2 subtype appears to stimulate secretionlla,while the Al subtype may inhibit secretion?
ATP, which can act via both PIand P2purinergic receptors, may act via several intracellular pathways. Treatment with ATP stimulates cAMP.Ir However, ATP also causes a rise in intracellular fiee ca2+l", which may be mediated b y phosphatidylinositol hydrolysis and subsequent generation of IP3. Rice and ~o-workef3~~a.lsorecently provided evidence that protein kinase C may be involved in mediating the stimulatory effect of ATP. Pz receptor-mediated signal appears to be responsible for protein kinase C activation through phosphatidylinositol-bis-phosphate hydrolysis to inositol-bis- phosphate and release of calcium Eom interna1 storestc, whereas PIreceptors, like P- adrenergic receptors, appear to act through an increase in CAMPproduction.
Calcium
In a wide variety of ce11 types12', the release of secretory granules and their content is triggered by a rise in intracellular calcium concentration.
Since in various ce11 types protein kinase C is involved in transduction events which implicate the rnediation of diacylglycerol or inositol phosphates and calcium ions, it appears reasonable to speculate that the latter rnay play a role in kinase C-mediated surfactant secretion. In fact, although inositol triphosphate formation has indeed been show to be stimulated by ATP,13 and although increasing evidence exists for involvement of calcium movements in the surfactant secretion process, it is much less clear whether they are linked primarily with activation of protein kinase C or of CAMP- dependent kinase.
Early experimentP documented that the calcium ionophore A23 187 stimulates surfactant secretion in cultured type II cells. In later experiments, both ionophores
A23 187 and ionomycin were shown to stimulate secretion in vitro, with approximately equal potency to P-adrenergic agent^^^'^"'^*. Treatment with ionophores caused a rise in cytosolic calcium, as measured by fluorescent probes, although results with such probes must be interpreted with caution because some probes apparently are not evenly distributed throughout the cytoplasm. Warburton and CO-workersIMshowed that verapamil, a calciumchannel blocking agent, has a multiphasic concentration effect on in vitro secretion, with a maIl stimulatory effect at low concentrations. Interestingly,
CAMPconcentrations were increased by verapamil. The stimulatory effects of both P2 purinergic agents131and mechanical distortion of type II cel1si3=appearto be mediated by mobilization of intracellular calcium.
Phorbol Myristate Acetate (PMA)
The enhancement of surfâctant secretion by a phorbol ester, phorbol myristate acetate (PMA), without associated elevation of cellular levels of cAMP1I6indicates the existence of another triggering mechanism of surfactant secretion. Since PMA is known to activate protein kinase C and since this event has effectively been shown in PMA stimulated type II ~ells,'~~this second mechanisrn appears to involve protein phosphorylation mediated by protein kinase C. Protein kinase C phosphorylates many proteins in virro. Some of these substrates may be calcium- or calmodulin-binding proteins. ' '' Phosphorylation of these high-ety calrnodulin-binding proteins by PKC in intact cells could cause displacement of bound calmodulin, leading to activation of Ca2'- calmodulin-dependent processes, particularly PI turnover and surfactant secretion in type
ïï cells."' The inhibitor of protein kinase C, tetracaine, inhibiteci PMA-induced sdactant secretion but had no effect on terbutafine-induced secretion. Like CAMP-dependent kinase, protein kinase C in type II cells is mainly cytosolic, but under stimulation with
PM& 40% of the activity in cytosol was translocated to the particdate hction.
PMA was one of the ktsubstances found to act as a secretagogue for type II
~ells."~PMA and ATP (see above) are the two most potent secretagogues for surfactant secretion in vitro,
Agents that activate protein kinase C represent the most potent group of single agonists on surfactant secretion leading one to ascribe a major role to protein kinase C and the mediators that activate this enzyme. The signal transduction mechanism which sets protein kinase C in action in type II cells, however, is not as well understood as the role of P-receptors for CAMP-dependentkinase activation. Nahiral analogues of PMA have not yet been identified. Many investigators consider PMA as being an analogue of
DAG.
Acid Base Status
Studies with isolated perîûsed lungs suggest that intracellular allcalosis stimulates surfactant se~retion.'~Findings with isolated type II cells are somewhat different in that varying the pH of the extracellular rnedi~rn'~'doesnot modulate secretion in vitro.
Furtherrnore, the regulation of intracellular pH by amiloride-sensitive N~'RI' antiporter does not appear to be involved with stimulated surfactant secretion in vitro.13* Prostaglandins, Lenkotrienes, and Purines
Studies with hypmentilated rabbitstJ9,evidence suggests that arachidonic acid metabolites may be important in mediating stimulateci secretion. Rooney and CO-workers have presented evidence that leukotrienes play a role in stimulating surfactant secretion, both in and in vivot4I.
Prostaglandins may play a physiological role in surfactant release, particularly in the perinatd period. Exogenous prostaglandin E2 stimulates the rate of phosphatidylcholine release from rabbit lung slices and the inhibitors of prostaglandin synthesis, indomethacin and flufenamic acid, inhibit the secretion. In addition, labour was shown to be a potent stimulus of surfactant secretion at birth, an effect that could be blocked by ind~rnethacinl~~.However, the prostaglandin-mediated surfactant release does not seem to involve elevaîion of intracellular CAMP."^
There are strong suggestions that arachidonic acid derivatives synthesized through the lipoxygenase pathway are implicated in the surfactant secretion process.'"
More recently, Gilfillan and Rooneylu demonstrated that lenkotrienes E4, Dé and C4
(decreasing order of potency) were stimulatory agents of PC secretion by cultured type II pneumocytes. Much more recently than adrenergic mechanisms or lipid-mediator effects, purïnergic rnechanisms have been shown to be involved in the control of surfactant secretion. Adenosine 5'-triphosphate (ATP) and AMP are among the most potent surfactant secretagogues identified thus fari". Other Secretagogues
Histamine. antihistamines. Both histamine'4sand antihistaminic agentsHghave been reported to stimulate surfactant secretion in cultured type II cells. These results are difficult to interpret, particulariy because there is no uniform agreement about whether histamine stimulates secretion.
Vasopressin. Brown and Wood14ecentlyreported that vasopressin is a potent stimulant for sdactant secretion in vitro.
Although these experiments have ccntributed to progress in the understanding of intimate intracellular mechanisms which trigger surfactant release, the complete elucidation of these mechanisrns and of the exact role of CAMP-dependentprotein kinase, protein kinase C, and calcium ions still requires additional research. There appear to be three levets of regulation of surfactant secretion. The basal Ievel is represented by mechanical stimulation while two other main routes of influence can be distinguished: chemical agonists presumably reaching the aiveolar epithelium via the bloodstream, and locally generated factors and mediators. A mode1 of regulation with three distinct levels can be outlined (figure 3). Level Stimulation Inhibition
lack of substratum - padrenergic compoinads - Systernic factors - imrriaturity of type Li - oüier humoral secretagogues cells I - systdc inhibitors?
- compounds - hypoxia? irnlaiown inhibitors? Local e.g. ATP - factors - sulfidapeptide-leukotrienes (inhaled substances, particles etc.) and prostaglandin E2 - changes in pH?
Local
Secretion
Figure 3. Three level mode1 of regdation of surfactant secretion. Detailed explanation in text. (after Wirtz H and M Schmidt. Ventilation and secretion of pulmonary surfactant. Clin Invest, 70: 3- 13, 1992)
1-7.2.3.Current Secretion Models
The current view on secretion emphasizes the basic resemblance between constitutive and regulated models of secretion, as well as the importance of specific proteins in these pro cesse^.'^' In this view, the exocytotic machinery relies on a small number of interacting proteins. Most of the research was so far done in neuronal cells and the study of the synapse. However, some of the following mechanisms might be present in dveolar type II cells as well, and future investigations remain to establish their existence. Protein fdiies associateci with intracellular trafic and synaptic transmission have been found to be highly consend throughout evolution.'" Among these proteins are proteins of the vesicle membranes [e.g., synaptic vesicle (SV)] and of target membrane [e.g., the plasma membrane (PM)] and the soluble proteins N-ethy haleimide- sensitive fusion proteins (NSF) and soluble NSF attachment protein (SNAes). The proteins on the membranes serve as receptors for SNAPs and NSF, with which they form complexes characteristic of both regulated and constitutive se~retionl~~and are generally termed SNAREs (for SNAP receptors). The SNARES assemble with SNAPs and NSF into a 20s fusion particle, a competent protein complex that is essential for vesicular traficking within subcellular comparûnents, e.g., between the Golgi stacks.lMIn the nerve terminal, the SNAREs consist of vesicle-associated membrane protein
(VAMP)/synaptobrevùi fiom the SV and syntaxin/HPC-l and synaptosornal-associated protein of 25 kD (SNAP-25) fiom the PM. Based on these findings, the SNARE hypothesis proposes that vesicle targeting and fusion, as weil as specificity, are govemed by interactions between vesicle-associated SNAREs (v-SNARE; such as VAMP) and target membrane-associated SNAREs (t-SNAREs; such as syntaxin or SNAP-25). The
SNARE hypothesis also suggests that NSF, an ATPase, disrupts the SNARE complex via
ATP hydrolysis, thus operating as a molecular switch that leads, perhaps, to membrane fu~ion.'~'The three SNAREs form a stable core complex, also called the heat sensitive/sodium dodecyl sulfate-resistant complex.'" However, these proteins interact transiently with additional proteins, such as synaptotagmin, the putative Ca2+sensor for release.ls3Based on in vitro cornpetition assays, it was suggested that a-SNAP may diçplace synaptotagmin fiom the c~rnplex,'~~which is then probably rehted back to operate at iater steps in exocyto~is.~~
A separate line of research has identified the synaptic proteins VAMP, syntaxin, and SNAP-25 as the target proteins of clostridial toxins, Le., tetanus and botulinum toxins, which block neurotransmitter relea~e."~
The SNARE hypothesis'~'~proposes that the donor-acceptor membrane recognition is based on an interaction of a complementary set of v- and t-SNAREs that reside on distinct membranes. According to this view, it is the activation of the SNARE complex by the aSNAPINSF that eventually leads to fusion. The interaction between the
SNARE proteins and their interacting protein synaptotagmin, denoted as the exocytosis core complex, occurs in fast releasing synapses at the active zone, in close vicinity to vo ltage-dependent Ca2+channels.
Much of the current howledge on potential interactions among proteins of the exocytotic apparatus is based on in vitro studies. Two main features cornmon to al1 proteins discussed are that they bind multiple proteins, and that they sense signals and respond to them. Protein binding leads to conformational changes in the protein and, consequently, additional binding sites are either excluded or enhanced. It is an important challenge to offer a better structural explanation of the interplay between binding sites in exocytic proteins. This will be a major step toward the edification of the sequence of events that constitute secretion. 1.7.2.4.Inhibitors of Surfactant Secretion
The basic level of regulation appears to act independently of systemic influences through the interaction of stretch-induced stimulation if ventilation is augrnented or if a deep breath is taken antagonized by the concurrent accumulation of SP-A. At this level of regulation every fuactional unit in the lung is independent fkom the adjacent one.
Surfactant itself appears to exert a negative control on its proper secretion. Ln other words, there would exist a short feedback contrd of surfactant secretion. From a teleological point of view, this would prevent the accumulation of excessive amounts of surfactant in the alveolus and exhaustion of alveolar type II cells with larnellar bodies after potent stimulus.
~ice~~~showedthat compound 48/80 inhibits both basal secretion and secretion stimulatecl by either forskolin or cytochalasin D. Compound 48/80 is a mast ce11 secretagogue and induces histamine release. It is a condensation product of N-methyl-p- methoxy-phenethylamine and formaldehyde. The mechanism by which 48/80 inhibits surfactant secretion remains uncertain. High concentrations of substance P, a small neuropeptide, also inhibit secretion in vitro. Its effect may be related to receptor occupancy of type II cells.
Together with the putative role of SP-A in modulating the reuptake of alveolar surfactant components, the secretion-inhibithg activity of SP-A points out the major role that this protein appears to play in alveolar surfactant homeostasis and the regulation of the pool size. SP-A,the major surfactant-associated protein, is a potent inhibitor of phosphatidylcholine secretion in vitro.ln'l3 ' The mechanism by which SP-A inhibits secretion is UnICIlown. Cellular CAMP levels are not etedby SP-AI5=.Because SP-A inhibits secretion stimulateci by every agonist thus far tested in vitro, its seems likely that its effects occur distally in the exocytic process, although effects on very early secretory events have not been excluded. Interest has also focused on what portion of the molecule confers its inhibitory properties. Because SP-A contains a lectin-like domain and has lectin-like propertiesly Rice and Singlet~n~~exarninedthe effects of various lectins on surfactant secretion. Several plant lectins, including concanavalin A, w heat germ agglutiain, and Maclura pomifera, al1 inhibited both basal and stimulated secretion.
Like with SP-A, the inhibitory effects could be reversed by washing of the ~ells'~or by competing with appropriate sugars, N-acetyl -D-glucosamine and methyl a-D- rnann~side'~',suggesting that these lectins bind to the carbohydrate domains on the ce11 surface. Since the lectin effect, like that of SP-A, is not agonist specific and is easily revened by washing or by preventing binding of lectin with appropriate sugars, the mechanism of inhibition of PC secretion is most likely at the ce11 membrane level.
Local factors. Several inhibitory factors seem to influence surfactant semetion at this level of regulation. Both local hypoxia and oxygen radicals may damage the type II ce11 and result in decreased surfactant secretion. The effect of pH was also investigated.
Methylamine, a weak base, inhibits PC packaging by inhibithg tdficking of PC to lipid- rich light subcellular fktions suggesting that the tdlicking of surfactant into lamellar bodies might be sensitive to changes in the pH of lamella. bodies. A number of inhalatory toxins also act by this rne~hanism.'~'Special interest was given to cigarette smoke. Its innuence on surfactant system has not been weU established, although early studies have found decreased amounts of surfactant-associated phospholipids in the bronchoalveolar lavage fluid of ~mokers.~"Similar results were reported from animal studies.'@Nevertheless, synthesis appears not to be affécted'" and the number and size of alveolar type II cells tend to be larger in smokers than non-smokers.'" The fact that the amount of total surfactant-associated phospholipids in the lung is not diminished but the amount of extracellular surfactant is aiso pointed to a disturbance somewhere dong the process of secretion or to enhanced ~ptake.'~'
Factors that decrease surfactant secretion exist on a systemic level. The infant respiratory distress syndrome is one representative, yet specid, situation of a systemic inhibitory factor for surfactant secretion. The underlying mechanism here is the lack of mature type II cells due to irnmaturity of the fetus at the time of buth. The alveoli do not open easily upon the first breath, requiring hi&-pressure ventilation support and high oxygen tension, which leads to barotrauma and fùrther deterioration of the lung. Timely application of exogenous surfactant is extremely helpful'" in this situation and is now perfomed in neonatai centers around the world.
Lack of substrate, although a logical choice, does not appear to be a likely mechanisrn for surfactant deficiency. This may be due to recycling capabilities and to the ubiquitous nature of the basic building blocks: glycerine, fatty acids, choline, and inositol. They seem to be available even after prolonged penods of fasting.
Other known systemic inhibitors include acidosis and States of toxic agents generally used in experimental settings or ingested with suicida1 intent: paraquat, 3- rnethylindole, chlorphentennine, butylated hydroxytoluene (BW,bleomycin,
(especially in addition to high oxygen tensions), N-nitroso-N-methylurethane,cadmium chlonde, semicarbazide, ethanol, and irradiation in doses fiom 10-30 Gray. A review of these and other agents has been published.'" In generd, these conditions and agents act indirectly through damage to the type II cell.
Recently, it was demonstrated that p henothiazines inhibit agonist stimulated surfactant secretion and amexin II tetramer (Anx-Ut)-mediateci membrane fusion.'*
It is known that turnour necrosis factor a,TNFa, induced inhibition of suffactant synthesis participates in the pathogenesis of the acute respiratory distress syndrome.
Recently, Vara et al.'" examined the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNFa. Human type II pneumocytes in culture showed decreased incorporation of radioactively labeled glucose into phosphatidylcholine by TNFa, PGEZ, sodium nitroprusside (SNP), or 8-bromoguanosine 3',5'-cyclic monophosphate. The effect of TNFa was attenuated by indomethacin, N R-nitro L- arginine methylester (NAME) or methylene blue m).The effect of PGEZ was attenuated by NAME,while that of SNP was revened by MB, but not by indomethacin.
TNFa induced an increase in PGE2 and guanosine 3' ,5 'cyclic monophosphate ce11 content and in the NO release to the medium. NAME did not affect PGEZ production, while indomethacin blunted NO generation. The results suggested that NO generation, secondary to PGEZ production, is responsible for the TNFa-induced inhibition of PC synthesis by human type II pneumocytes. NO seem to exert this effect through activation of guanylyl cyclase. '" Inhded NO may modify surfactant either by interacting with the surfactant complex, or by changing the capacity of the proteins of the epithelial lining fluid to inhibit sudace activity.'"
Inhibitors of various cytoskeletal elements such as colchicine and Wiblastine inhibit secretion stimulated by various secretagogues in vitro"? These same agents may achially stimulate basal secretion in viîro. Cytochalasins cm either sti~ndate~~~or inhibitl"secretion. Although there is no direct evidence to support such hypothesis, it may be that stimulation of basal secretion occurs because cytoskeletal elements interposed between lamellar bodies close to the surface of the ce11 and the plasma membrane are disrupted, which allows fusion and exocytosis to occur. In contrast, stimulated secretion may be inhibited because cytoskeletal elements involved in larnellar body transport are disrupted.
Although it is clear that surfactant secretion can be inhibited in vitro, it remains a matter of conjecture to what extent sirnilar mechanisms operate in vivo. Whether or not the processes have correlates in vivo, the study of the mechanisms by which secretion can be inhibited is likely to provide new insights into exocytic processes in type II cells. The following is a schematic of the control mechanisms involved in surfactant secretion. Ionophore PMA
CP(?) DAG (?) IP3 (?)
I ATP CAMP+
I Protein phosphowlations b-
Cytoskeleton l Surfactant release
Figure 4. Sumrnary of control mechanisms of pulmonary surfactant secretion. Abbreviations: BR = f3- adrenergic receptors; PIR, P2R = purinoceptors; AIR = A 1 subtype Pl purinoceptors; A2R = A2 çubtype P 1 purinoceptors; SP-A R = receptors to surfactant protein A; PMA = phorbol myristate acetate; DAG = diacylglycerols; iP3 = inositol triphosphate; PK = protein kinase.
1.S. Clearance of Surfactant
Since su-factant is continuously synthesized and secreted by the type II cell, a mechanism for its removal must exist or it would accumulate within the lung. Several different mechanisms may contribute to intraalveolar metabolism of surfactant: uptake, recycling, and reutilization: (a) recycling, in which components are not degraded, but are reutilized by being taken up by the type II cell, Uicorporated into lamellar bodies and then resecreted; (b) degradation and reutilization of components to synthesize new surfactant
Lipids or proteins; (c) uptake and catabolism by phagocytic ceIls, or by the various epithelid cells lining alveoli or mal1 airways; (d) mucociliary-dnven movement to the upper airways; (e) losses via lymphatic vessels and the pulmonary circulation; and/or (f) intra-alveolar removal fiom the surfactant system, either as intact molecules or as degradation products such as fatty acids (Figure 2).
The protein and lipid components of surfactant are separated upon enhy of the lipid into the surface monolayer, which may be a consequence of the high surface pressures generated at low lung volumes, which force protein out of the surface. Mer fûnctioning as a surface tension-reducing agent, surfactant is removed nom the alveolus and there is active secretion but the pool size is constant. The presence of a modulable and coordhated mechanism for surfactant uptake and clearance in suggested by the relative constancy of surfactant pool size in the face of active and stimulable secretion.
Abundant biochemical data demonstrate that surfactant is recycled and most estimates indicate that the majority of phospholipid leaving the alveolus reenters the type II ~ell.~~
Bi-directional surfactant flux between lamellar bodies and alveolar surfactant has been demonstrated. ln
Although initial studies suggested a major role of alveolar macrophages in clearance of s~rfactmt,~"their role appears to be quantitatively srna11.l~~The possibility that alveolar type 1 and Clara cells may participate in the clearance of surfactant has been con~idered."~The intra-alveolar metabolism of neutral lipid, particularly cholesterol, is poorly understood. l9
1.8.1. Role of Pneumocytes
Geiger and CO-workersl1°demonstrated that alveolar epithelial cells take up and recycle surfactant. Phosphatidylcholine May be recycled directly to lamellar bodies or it may be degraded and reutilized for synthesis. Direct incorporation appears to be the principal route of phospholipid recycling. They showed that type II cells recycle surfactant material for subsequent resecretion without major biochemical alteration.
Recent demonstration of lipoprotein-mediated signal transduction in isolated type
II cell~~~and clearance studies of radiolabelled cholesterolfalsuggest complex mechanisms regulate surfactant clearance. Specific receptors on type II cells are involved in clearance of both neutral and phospholipid cornponents of surfactant.
In addition to lipid, SP-A, SP-B,and SP-C also reenter the type II ceIl,'". lS3.lbl but recycling of SP-D has not been reported. Receptor-rnediated endocytosis involving coated pits was reported for SP-A in cultured type II ~ells'~'but was not for SP-BmJin cultured type II cells. These findings are compatible with the demonstration that type II cells express a high-affinity receptor for SP-A, but not for SP-B." Electron microscopic autoradiographs show that both type II cells and macrophages ingest detectable amounts of radiolabelled SP-A or pho~phatidylcholine.'~Other alveolar septal cells, as well as
Clara cells of the terminal airway, apparently do not participate in recycling of surfactant proteins. Mer reuptake by the type II cell, phosphatidylcholine, SP-A, and SP-B are initially localized to MVBs and, a few minutes Iater, in lamellar bodies. lg6 Recycled SP-
A is found in I-MVB, but not in d-MVB, and recycled phospholipid is found in bath?
Thus, SP-A and phosphatidylcholine separate during recycling. It is possible that d-
MVBs are part of a degradation pathway and l-MVBs are involved in recycling to lamellar bodies. SP-A targets recycled phospholipid into lamellar bodies, apparently protecting the lipid from degradation.* In recent in vivo experiments, 75% of liposome phosphatidylcholine bound to SP-A entered a lamellar body-rich subcellular hction compared with 35% when the lipid was admuüstered with bovine serum albumin (S.P.
Caminiti, unpublished observations).
Intracellular catabolism of internalized surfactant also occurs, although the intracellular location and biological significance of this route remain unclear. The cornpartment (or compartments) within type II cells that rnight receive surfactant destined for degradation has not been established. The usual structure of a lysosome has no readily identifiable counterpart in type II cells, although the d-MVB has lysosomal features, and even lamellar bodies contain lysosornal enzymes and a mildly acid pH.lJ6
1.8.2. Rote of Alveolar Macrophages
Surfactant-like lipidsLg7and surfactant apoproteins14have been identified within alveolar macrophages, suggesting that macrophages may participate in surfactant clearance. Macrophage phagocytosis of tubular myelin is mediated, at least in part, by
SP-A,'~'perhaps analogous to reuptake of surfactant components by alveolar type II cells (see below). The role of macrophages in surfactant clearance is rather limiteci. Within one hour of instillation, macrophages' clearance accounted for approximately 15% of disappearance fiom the alveolar ~ornpartment.~"
1.8.3. Intra-alveolar Metabohm
There is littie evidence for the clearance of significant amounts of alveolar surfactant via the airways to the upper respiratory tract.'" Estimates of clearance of endogenously synthesized and secreied DPPC through the upper airways approximate 3% of surfactant flux through the alveolar pool in the rabbit?' Some clearance also occurs through the lyrnphati~s,'~'but does not appear to be a major component of the clearance pathways (~0.5%).A small portion of instilled lipids appears to exit the lung by direct transfer fkom the alveolar surface to the blood. This is consistent with a previous report"' of a predominant clearance of albu.directly hmthe alveolus to the blood, with smaller amounts exiting via lymphatics.
Limited degradation of alveolar phospholipids appears to take place in situ through the action of intraalveolar enzymatic activity.'" The lysosomal enzymes contained in and released wiîh lamellar bodies are possibly involved in this phenornenon.
Phospholipases, with both acidic'" and alkaline'" optimal pH, a variety of &ee fatty acids and lyso-PC are al1 constituents of the bronchoalveolar lavage fluid.
The intra-alveolar surfactant metabolism was reviewed recently by Edel~on."~
Intra-alveolar surfactant includes high rnolecular weight, tubular myelin nch, large aggregate forms, and lighter, small aggregate for~ns.'~~The large aggregate forms have relatively nomal biophysical activity and appear to be the precursor of the small aggregate forms. Gross et al? proposed a mode1 of extracellular surfactant metabolism in which larnellar (large aggregate surfactant) is converted into tubular myelin and vesicular (small aggregate) forms after repeated cyclic expansion and contraction
(" cycling" ) of the air- 8uid interface of surfactant. Serine protease activity is required for the conversion of large to srna11 aggregate foms; a labile, 75 kD protein that requires cycling for optimal activity has been identified as a candidate protease.lg8 The authors postulated that the convertase enzyme might be produced by alveolar type II cells, and may be a constituent of normal alveolar fluid.
SUTfactant associated proteins appear to be important modulatory proteins of uitra- alveolar surfactant metabolism. SP-B and SP-C are required for aggregate conversion in vitro,'99and SP-B is degraded during the conversion pro ces^.'^ SP-A inhibits subtype conversion.20'Ueda et al." documented that the presence of serum proteins accelerates the rate of conversion of large to small aggregate foms, suggesting an additional mechanism by which protein in the alveolar space may inhibit surfactant activity.
1.9. Implications for Pulmonary Disease
Alveolar epithelial cells are a key structural determinant of the physical
separation between gas and blood that characterizes the normal functional anatomy of the
hg. The alveolar epithelium is the primary high-resistance barrier to the passage of
fluid and electrolytes, and is an early and consistent site of structural damage in acute
lung injury (ALI)? Adult respiratory distress syndrome (ARDS) is a clinically and pathophysiologically complex syndrome of acute lung infiammation characterized by the abrupt omet of respiratory failure, diffuse bilateral radiographie infiltrates, arterial hypoxemia, and mortaiity of 40-60%.'*~ Features of acute lung injury fi-equently include interstitial and alveolar edema, hyaline membrane formation, development of atelectasis because of disturbed alveolar surfactant function with consequent gas-exchange impairment, and an increased pulmonary vascular resistance resulting fiom vasoconstriction, rnicroembolization, and/or microthmbi.
Restoration of an intact epithelial function is a major determinant of survival after acute lung injury in patients with increased permeability pulmonary edema The prognostic significance of intact alveolar epithelial function, measured by the ability of the epithelium to remove liquid nom the air spaces of the hg,was investigated by
Matthay and Wiener-~ronish.~'~They have demonstrated that active ion transport across the epithelial ce11 barrîer is the primary mechanism for clearance of the edema fluid nom the air spaces of the hg.
The hypothesis that physiologic abnormalities are present in the lungs of individuals with ALI was suggested by Ashbaugh et al.20sSubsequent investigators have documentai abnormalities of surfactant biophysical activity, alterations in phospholipid composition, and the presence of protein and lipid inhibiton of surfactant activity that are relevant in the context of human ALI.
Biochernical study of surfactant fkom injured lungs generally reveals a decrease in total phospholipid; decreases in proportional phosphatidylcholine (PC), saturated PC, and p hosphatidylglycerol content; with increases in free faîty acids, sphingomyelin, and lysophosphatidylcholine?' Biophy sical studies generally demonstrate that surfactant obtained fiom injured lungs fiinctions abnormally, with lighter buoyant density, increased compressibility, and a decreased ability to reduce surface tension to a minimum alveolar
Gregory et al.'" noted decreased concentrations of surfactant protein A (SP-A) in patients with the adult respiratory distress syndrome (ARDS). Changes in surfâctant pool size, proportional phospholipid composition, apoprotein concentrations, and minimum surface tension also occurred in individuals at nsk for (but who did not develop) ARDS.
in the context of ALI, conversion of large to small surfactant aggregates may be abnomal.'" Significant increases in the smalI aggregate hction and decreases in SP-A,
SP-B,and SP-C levels in an adult sheep mode1 of lung injury following cecal ligation and perforation have been described. In addition to reduced synthesis, reduced secretion or accelerated degradation, adoruptake of surfactant nom the alveolus, surfactant inhibition may be a key functional determinant of lung fûnction in the context of acute injury. In neonatai respiratory distress syndrome, wherein quantitative surfactant defects are clearly predominant, the chical superiority of surfactant-replacement therapy given before the ktbreath suggests the presence of inhibitors of surfactant fun~tion."~
Surfactant replacement therapy has gained widespread acceptance for the treatrnent of neonatal respiratory distress syndrome, leading to improvements in neonatal
morbidity and m~rtaIity.=~*Major constituents of therapeutic surfactant are: DPPC, PG,
fatty acids, hexadecanol (this substance is metabolized to palmitic acid, which is later utilized in the synthesis of phospholipids), tyloxapol (detergent used to disperse DPPC),
SP-A, SP-B, SP-C, and SP-D. During the past few years, a wealth of information on the composition and sequence of pulmonary surfiactaat proteins has been published In the presence of surfactant proteins SP-B and SP-C, SP-A enhances the surfâce activity of the phosph~lipids.~'' SP-A may play a key role in the regulation of surfactant tum-over by facilitating uptake and inhibithg secretion of phospholipids b y type II pneumocytes." '.
When Calf Lung Surfactant Extract (CLSE, Rochester, NY) was compared with CLSE +
SP-A in the presence of aibumin, the latter was more resistant to inacti~ation."~
Cockshutt and coworkerP also observed an increase in resistance to inactivation in the presence of SP-A. SP-A has also been shown to stimulate macrophage migration in a concentration-dependent manner by enhancing cher no taxi^.^^^ SP-A collaborates with SP-
B in transfomllng surfactant fiom the intracellular storage form to tubular myelin?
Granted the effects of SP-A on phenomena that might aect risk of infection, and SP-A regulation of the intra-dveolar surfactant pool, it is not yet clear whether SP-A would be helpfbl or detrimental as a constituent of therapeutic surfactants.
Based on the evident quantitative and qualitative surfactant abnormalities and encouraging data fiom pilot, preliminary and animal studies, the role of surfactant- replacement therapy for ALI is the subject of active in~estigation.'~~ 1.10. Cytoskeleton: a Brief Review
The cytoskeleton is an intemal network of three types of filaments: 7- to 9-nm- diameter microfilarnents, 10-nm-diameter intermediate filaments, and 24-nm-diameter microtubules.
Microfilaments and microtubules grow by the polymerization of actin and tubulin subunits, respectively; intermediate filaments are more complex polymers built with a comrnon a-helical subunit. By means of cross-luiking cytoskeletal fibres, complex structures such as bundles, geodesic-dome-like networks, and gel-like lanices are formed of simple components. Ce11 shape itself is at least in part a function of bonds between the plasma membrane to cytoskeletal supports. Ce11 motility relies on the ability of motor proteins to hydrolyze ATP to navigate a scaffold composed of micro filaments or microtubules. Other motor proteins cause the fibres to slide past each other. The other mechanism for motiiity, which is responsible for many of the changes in a shape of the cell, results fiom the polymerization of tubulin and acth and their assembly into bundles and networks. A few movements involve both the actin and motor proteins and cytoskeleton rearrangements.
The entire actin cytoskeleton can be visualized by fluorescence rnicroscopy der staining the actin filaments with fluorescent dyes. It is larger than any organelle, filling the cytosol with actin filaments. Radidly oriented actin filament bundles lie at the leading edge of the cell, while longitudinal and axial bundles (called stress fibres), run along the entire length of the cell. In addition, a network of filaments fills the rest of the cell, but the individual filaments of this network are difficult to resolve in the Light microscope.
Actin is the most abundant intracellular protein in an eukaryotic cell, is a rnoderately sized protein and consists of approximately 375 residues, encoded by a large, highly conserved gene family. h muscle cells, actin comprises 10% by weight of the total ce11 protein, and in non-muscle cells actin makes up 14% of the cells' protein. A typical concentration of actin in the cytosol of human platelets is 0.5 mM."' Even higher concentrations of actin exist in special structures like microvilli, where the actin concentration is 5 mM.
At least six different actins have been identified in birds and mammals. Three are called a-actins; each one is unique to a different type of muscle. Two other actins, termed non-muscle pactin and yactin, are found in nearly dl non-muscle cells. The sixth actin, another pactin, occurs in the intestinai smooth muscle. The sequencing of actin nom different organisms has revealed that it is one of the most conserved proteins in a cell, comparable with histones, the structural protein of chromatin.*"
a-actins differ in only four to six amino acids: nonmuscle p- and y-actin differ fiom each other at just a single position and differ fiom smated muscle a-actin in only 25 residues. Recently, a farnily of actin-related proteins sharing 50 percent similarity with actin has been identified in many eukaryotic organisms. In addition to the actin cytoskeleton itselc actin-related proteins are dso commonly found in association with microtubules and cytoplasmic dynein, a microtubule motor protein. Actin exists as a globular monomer caiied Gactim and as a filamentous polymer called F-actin, that is a string of G-actin subunits. In electron mimgraphs, F-actin filaments, and any proteins bound to hm, are seen as cytoskeletal microfilarnents. Each actin molecule contains a M~~'cation cornplexed with either ATP or ADP. Thus, there are four states of actin: ATP-G-actin, ADP-G-actin, ATP-F-actin, and ADP-F-actin. Two of these forms, ATP-G-actin and ADP-F-actin predominate in a cell.
Although G-actin has a globular appearance at electron microscopy, X-ray crystallographic analysis reveals that G-actin is separated into two lobes by a deep cleft.
The Lobes and the cleft comprise a.ATPsse fold, the site where ATP and M~'' are bound to amino acid side chahs. The floor of the cleft acts as a huige that allows the lobes of the proteins to flex relative to one another. When bound to the cleft, ATP becomes a latch that holds the lobes together, suggesting a mechanism for modulating conformational changes in the molecule.
1.10.1. G-actin Assembles into Long F-actin Polymers
In the presence of M~~+, K', ~a+, G-actin polymerizes into F-actin filaments.
This is reversible: F-actin depolymerizes into G-actin in solutions of low ionic strength.
The assembly of G-actin into F-actin is accompanied by the hydrolysis of ATP to
ADP and Pi; however, ATP hydrolysis not necessary for polymerization to occur.
The ability of G-actin to polyrnerize into F-actin and of F-actin to depolymerize into G-actin are perhaps the most important properties of actin. 1.10.2. The Actin Cytoskeleton is Organized into Bundles and Networks of
Füaments
Actin bundles and networks are the most common cellular arrangements of actin filaments, providing a network that supports the plasma membrane, determining the cell's shape. In bundles, the actin filaments are closely packed in parallel arrays, whereas in a network the actin filaments crisscross, often at right angles, and are loosely packed A network associated with the plasma membrane is planar or two-dimensional, like a net or a web. Within a ceil, the network of actin filaments is three-dimensional, giving the cytosol gel-like properties.
In all bundles and networks, the filaments are held together by actin-cross-linking proteins. To connect a pair of filaments, a cross-linking protein must have two actin- binding sites, one site for each filament. The length and flexibility of a cross-linking protein critically determine whether bundles or networks are formed. Short cross-linking proteins will hold actin filaments closely together, tending to force the filaments into parallel alignment, a bundle. In contrast, long, flexible, cross-linking proteins are able to adapt to any arrangement of any actin filaments and thus will tether orthogonal oriented actin filaments in networks. The shortest actin-binding proteins such asfimbrin and a- actinin, are found in actin bundles in a cell, while the longest -filamin, spectrin, and dystrophin -,are found in actin networks. Many cross-linking proteins contain a calrnodulin-like calcium-binding domain. The role of calcium is regulatory; it prevents these proteins fiom cross-linking actin filaments. 1.10.3. Cortical Networks of Actin Filaments SWen CeU Membranes and
Immobüize Integral Membrane Proteins
The richest area of cellular actin filaments are found in the cortical actin network, a narrow zone adjacent to the plasma membrane, that generally excludes most organelles
Eom the cortical cytoplasm. In the erythrocyte, the actin filaments form a two- dimensional network adjacent to the plasma membrane. By contrast, platelets, epithelial, and muscle cells have more complicated cortical cytoskeletons, in which actin filaments are part of a three-dimensional network that fills the cytosol and that anchors the ce11 to the substratum. For example, the cytoskeleton of an inactivated platelet consists of three components: a rim of microtubules (the marginal band), a cortical actin network, and a cytosolic actin network. A critical difference between the erythrocyte and platelet cytoskeleton is the presence of a second network of actin filaments, organized by filamin cross-links into a three dimensional gel.
The surface of a ce11 that is exposed to the surroundhg medium is not srnooth, but is studded with numerous membrane projections, the most comrnon being the microvilli.
A phospholipid membrane is fiagile and without support such projections would be unable to withstand the stresses encountered by cells in an organism. However, the underlying cytoskeleton supports these cell-surface projections by providing a bundle of actin filaments to which the membrane is anchored. Because it is held together by protein cross-links, the actin bundle is stiff and provides a rigid structure that reinforces the projecting membrane, enabling it to maintain its long, slender shape. 1.10.4. The Dynamics of Actin Assembly
In a cell, the cytoskeleton is dynatRic - microfilaments are constantly shrinking or growing in length and bundles and meshworks of microfilaments are continually forming and unformïng. Changes in the organization of actin filaments result in changes in ce11 shape. Several mal1 actin-binding proteins control actin polymerization. The polymerization of actin filaments proceeds in three sequential phases (figure 5).
Nucleus, f
Nucleation Elongation
Nucleus
F-actin Steady state
?&re 5. The three phases of actin polymerization. During the polymerization of G-actin in vitro, the m&s of actin filamen& increascs fieran ktial lag period and eventually reaches a steady state. In the fïrst phase, G-actin monomers bound with ATP (open circles), form nuclei - stable complexes of ach (black cucles) which in the second phase are elongated by the addition of subunits to both ends of the filament. In the third phase of assembly, steady state, the ends of actin filaments are in equilibrium with monomeric ATP-G-actin. After their incorporation into a filamenf subunits slowly hydrolyze ATP and become stable ADP-F-actin (scattered circles).
The fint phase is marked by a lagperiod in which G-actin aggregates slowly into short, unstable oligomers. Once the oligomer reaches a certain length (three or four subunits), it cm act as a stable seed, or nucleus, which in the second phase rapidly elongares into a filament by the addition of actin monomers to both of its ends. This growth phase is enhanceci by the spontaneous and random breakage of the growing filaments, producing additional filament ends that also act as nuclei for elongation.
Adding a smdl number of F-actin nuclei to the solution of G-actin can eliminate the lag period.
As F-actin filaments grow, the concentration of G-actin monomers decreases until it is in equilibrium with the filament. This equilibrium condition, the third phase, is called steady stnte because G-actin monomers exchange with subunits at the filament ends, but there is no net change in the mass of filaments. The equilibrium concentration of monomen is called the critical concentration (C,). This value is important because it measures the ability of a solution of G-actin to polymerize. Under typical in vitro conditions, the Ccis 0.1 FM. Above this value, a solution of G-actin will polymerize; below this value, a solution of F-actin will depolymerize.
Each acth molecule contains a Mg'* cation complexed with either ATP or ADP.
Thus, there are four states of actin: ATP-G-actin, ADP-G-actin, ATP-F-actin, and ADP-
F-actin. Two of these forms, ATP-G-actin and ADP-F-actin, predominate in a cell.
Although G-actin appears globular in the electron microscope, x-ray crystallographic analysis of G-actin has reveaied that it is separated into two lobes by a deep cleft. The lobes and the cleft comprise an ATPase fold, the site where ATP and MCare bond by ionic and hydrogen bonds to amino acid side chains. A clue to the hction of ATP and
MCcame nom the discovery that the ATPase fold is also found in other ATP-binding proteins, including hsp70 (a molecular chaperone), sugar kinases such as hexokhase, and sorne bacterial phosphatases. By cornparhg these structures, x-ray c~ystallographers noted that the floor of the cleft acts as a hinge that dlows the lobes of the proteins to flex relative to one another. When bound to the cleft, ATP becomes a latch that holds the lobes together. This brings a better understanding of how the presence of ATP or ADP in the cleft will affect the conformation of the actin molecule; in fact, without a bound nucleotide, actin denahires very quickly .
The addition of ions - Mc,K*, or Na' - to a solution of G-actin will induce the polymerization of G-actin into F-actin filaments. The process is also reversible: F-actin depolymerizes into G-actin when the ionic strength of the solution is lowered. The F- actin filaments that form in vitro are indishguishable fiom microfilaments isolated fiom cells. This indicated that other factors such as accessory proteins are not required for polymerization.
Actin polymerization is accompanied by ATP hydrolysis to ADP and Pi. ATP-G- actin monomers add to the ends of a filament; once they are incorporated into the filament, the ATP bound to the actin subunit is slowly hydrolyzed to ADP. As a result of
ATP hydrolysis, most of the filament consists of ADP-F-actin, but ATP-F-actin is found at the ends. However, it is known that hydrolysis is not essential for polymerization to occur, because actin containhg ADP or a non-hydrolysable ATP analog is able to form filaments. 1.1 OS. Fungai Toxhs Disru pt the Monomer-Polymer Eqailibriom
Cytoskeleton disrupting drugs have been part of the ce11 biolo@sts7 armamentariun since the 1970s. The hgal alkaloids, cytochalasins B and D were among the ktsuch drugs studied. Cytochalasin causes actin filaments in a ceil to depolymerize by binding to the (+) end of F-actin, where it blocks fiirther addition of subunits. The critical concentration then shifts to that of the (-) end, which causes subunits to dissociate from the filament. When cytochalasins are added to some live cells, the actin cytoskeleton disassembles and cell movements iike locomotion and cytokinesis are inhibited.
A second toxin, phaiioidin, has the opposite effect on actin: it poisons a ce11 by preventing actin filaments nom depolymerizing. Isolated fiom Amanita phalloides (the
"Angel of Death" mushroom), phalloidin binds at the interface between subunits in F- actin, where it is an ideal position to lock subunits together. Even when actin is diluted below its critical concentration, phalIoidin-stabilized filaments will not depolymerize.
Phalloidin binds only F-actin and when conjugated to fluorescent dyes is commonly used as a means for visualking F-actin. Tagged with fluorescent dyes, phalloidin binds any actin filament in a cell, and has proven useful to measure the distribution of actin by fluorescence microscopy. 1.10.6. Actin-Binding Proteins Contml the Length of Actin Füaments
A variety of cellular mechanisms are us& to regulate actin polymerization.
Regulatory actin-binding proteins that either promote, or inhibit actin polymerization have been identified,
1.1 0.6.1. Sequestration of Actin Monomers by Smail Actin-Binding Proteins
Biochemical calculations considering the Cc of actin (0.1 FM) and ambient intracellular ionic conditions would predict that most cellular actin (0.5 CrM) should exist as filaments: there should be very Little G-actin. Actual measuements, however, have show that as much as 40% of actin in a ce11 is in the unpolymerized G- form. It, therefore, appears that cytosolic proteins sequester actin, holding it in a form that is unable to polyrnerize. Two candidate proteins are thymosin ,û4 (Th)and proflin. Both proteins are abundant in the cytosol and bind actin monomers with a 1: 1 stoichiometry.
TP, (MW5,000) binds an actin monomer in 1: 1 complex but is unable to bind F- actin. When complexed with TP,, actin cannot polymerize. The concentration of TP, in platelet cytosol is 0.55 mM,approximately twice the concentration of unpolymerized actin (0.25 mM). On this basis alone, one predicts that approxirnately 70% of the monomeric actin in a platelet should be sequestered by TP,. TP, huictions like a buffer for monomerk actin. The 1: 1 complex of actin and TP, is in equilibrium with unbound
TB,, monomeric G-actin, and F-actin. When cytosolic concentration of TB4 was arti ficially increased, the number of F-ach filaments was dramatically reduced, compared to control cells, which showed a normal distribution of F-actinT9 TB, has found application as a relatively specific means of interferhg with cellular levels of F- ach220 ,although its high molecular weight mandates the availability of methods for introducing it into the cytosol of the ce11 under examination.
Biochemical analysis of profilin (MW 15,000) showed that it preferentially binds
ATP-actin monomers as a stable 1 :1 complex. On the basis of its binding constant and cellular concentration, one would predict that profilin could buffer up to 20% of the unpolymerized actin in the cytosol, suggesting that it played a secondary role to thymosin
p, as an actin sequestering protein. Its cellular role may be to shuttle actin between G- actin and F-actin pools. Because its binding aflïrity for actin is intermediate between that of thymosin P, and the (+) end of actin filaments, profilin may regulate the store of unpolymerized actin by drawing actin from thymosin P4 and releasing it to actin filaments. Thus, profilui's actin-sequestering properties may be part of a complex membrane-based actin assembly system.
1.1 1. Cytoskeletal Involvement in Surfactant
Metabolism
Both indirect and direct evidence support the notion that the cytoskeleton of the
alveolar type II ce11 is involved in secretion of surfactant. The presence of actin has been
demonstrated near the lamellar bodies and plasma membrane, particularly in the
microvilli.'~Surfactant secretion induced in vivo by the P-adrenergic agonist isoproterenol was correlated with an increase in the number of actin filaments wociated with lamellar bodies or the cellular apex.=' Intratracheal instillation of cytochalasin D, a drug that disrupts actin filaments, reduced the secretory response of type II cells in an organotypic culhue system." Similar results were obtained with cytochalasin B in lung slices?
Rice provided direct support for a role of cytoskeletal elements in the modulation of surfactant secreti~n.'~~Cytochalasin D enhanced surfactant release with a biphasic dose response relationship. Cytochalasin D maximally stimulated release at concentrations of
0.1 - 1-0 pM with release approaching control levels above those concentrations. At concentrations above 4 pM, B-agonis t induced surfactant release was completely inhibited. Some cytochalasins affect glucose transport in addition to microfilament bction in erythrocyte~~~'~It is possible at low concentrations of cytochalasin in type II cells, microfilaments are disrupted, stimulating release, while at higher concentrations, glucose transport and cellular metabolism are inhibited, inhibiting release of surfactant.
Release of I~H]PCproduced by cytochalasin D is additive to release induced by terbutaline or forskolin, although the agents are present at concentrations producing maximal release when present singly, as detemiined by dose-response curves. One interpretation of this observation is that cytochalasin D and CAMPcause release by independent mechanisms. Another interpretation is that the two agents affect two different steps of the same pathway, each of which is augmented, producing an additive effect. However, cytochalasin D has no effect on CAMP levels. Microtubules also appear to be implicated in the secretory process of the lamellar body. This was illustrated, for instance, by inhibition of the basal secretion of phosphatidylcholine f?om lung slices induced by tubulin-binding dmgs, colchicinemand vinbla~tine.~'Studies conducted with isolated type II cells demonstrated that colchicine or vinblastine inhibited agonist-stimulated secretion,In whereas the basal secretion was stimulated rather than inhibited by these substances. These differences in response between basal and stimulated secretion have been interpreted as reflecting effects on different steps of surfactant release. The above mentioned study by Brown et al. produced an additional argument for the involvement of microtubules: compounds that stimulate surfactant secretion also increase the degree of polymerization of microtubule protein in isolated pemised rat lung.
In fetal type II pneumocyte organoid c~lhires:~it has been demonstrated that sUTfactant synthesis, but not secretion, was inhibited by colchicine, whereas secretion, but not synthesis, was inhibited by cytochalasin B. The latter did not prevent the formation of lamellar bodies in fetal cells isolated nom fetal lung prior to differentiation, but it induced the appearance of large vacuoles with small residues of lamellar body material, probably reflecting an intracellular turnover of non-secreted surfactant. By contrast, only the treatment with colchicine prevented ce11 differentiation and inhibited completely the occurrence of lamellar bodies. The authors concluded that an intact microtubular system is necessary for surfactant synthesis and lamellar body formation, but not for secretion of previously synthesized lamellar bodies, while functional actin filaments are not required for surfactant synthesis, but are essential for secretion processes. 1.1 2. The Cytoskeleton and Exocytosis in other CeIl
Systems
1.12.1. The Cytoskeleton and Exocytosis
There has been considerable interest in the relationship of cytoskeleton changes to exocytosis in cultured chromafi cell, mast tells,= pancreatic P-cells and parotid achar ceus. '26 Early actùi depolymerization after stimulation appears to be a common feature of stimulated exocytosis. Cheek and ~urgoyne~~reporteci intense labeling of the cortical zone beneath the ce11 membrane of resting cells by rhodamine phalloidin, a specific fluorescent stain for F-actin. Mer nicotine stimulation, the fluorescence was markedly reduced for a bnef interval (c 1 min), then reappeared, in association with a burst of exocytosis. The depolymerization of F-actin, with generation of G-actin, was confirmed with the deoxyribonuclease @Nase) method for detecting G-actin. Vitale et al."' showed that the disruption or depolymerization of rhodamine phalloiclin-labeled F-actin (after nicotine stimulation) took place in a patchy, rather than a continuous fashion and those areas of F-actin disassembly corresponded to cellular sites of exocytosis.
Thus, F-actin was originally viewed as a more or less uniform barrier to granule fusion and its depolymerization as prornoting fusion. This had first been suggested by
Orci et al?' in studies of pancreatic P-ce11 secretion. However, the idea of a simple uniform barrier that is opened before exocytosis does not account for the discontinuous distribution of F-actin noted above. 1.1 2.2. Spatial Compartmentalization: Reserve and Releasable Pools
A two-cornpartment model has been used to explain the biphasic pattern of neurotransmitter vesicle release observed der neural stimulation. Such a model predicts the presence of a mal1 releasable pool in close proximity to the ce11 membraneU0and a second, larger reserve pool linked to the cytoskeleton. Following stimulation, the reserve cornpartment dissociates hmcytoskeletal tethers, moves to the membrane, and replenishes the releasable pool. Recent studies in both the nerve terminals1 and the chromafnn cellD2provide support for this model. In their analysis of the chromaffin cell, von Ruden and Neher (cited above) proposed that the replenishment of the readily releasable pool, and exocytosis itself, are Ca2' dependent. This model of spatial compartmentaIization may also apply to other ce11 types ='.
Support for such a model of compartmentalization was provided by Nakata and
Hirokawam who noted considerable nonuniformity in the distribution of subcortical actin filaments in cultured adrenal chromaffin cells. Regions with thick filamentous layers between granules contrasted with other areas showing few actin filaments, with the granules very close to the membrane. Actin filaments were rarely observed in the cytoplasm. The authors suggested that the granules closest to the membrane, where actin was sparse, were the most likely to undergo immediate exocytosis, with actin depolymerization under local regulation at each exocytic site. By contrast, reserve granules, cross-linked to the cytoskeletal network, were thought to be released to advance to the cell membrane after stimulation. 1.12.3. Tramsport and Fusion
Once the granules are released fiom the cytoskeleton, they move to the plasma membrane. It has been suggestedUsthat the reduction of cytosol viscosity that follows actin reorganization allows vesicle movement toward the plasma membrane. Conversely, a more directed form of transport, such as actin-myosin interaction rnay be re~ponsible.~~
Vesicles attach to the membrane by a process of d~cking.~'There are several models for the events that take place at and after the dockuig step. A protein scaffolding between the grande and the plasma membrane is formed; and actin filaments rnay be part of the scaffolding."'
Both traficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a smdl number of proteins, i.e. the exocytotic core complex. In nerve terminais, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP-25 at the plasma membrane.
These mechanisms were described previously in this chapter.
1.13. Summary
The type II alveolar epithelial ceil synthesizes and secretes pulrnonary surfactant.
A variety of in vitro and in vivo surfactant secretagogues have been identified. The enhanced release of surfactant fkom the type II ce11 is associated with a substantial reduction in both the number and volume density of lamellar bodies per cell."' Morphologic evidence indicates that secretion of lamellar bodies is accomplished by exocytosis.
Actin exists in most cells in both a globular, or G form, as well as filamentous, or
F forrn. The relative pool sizes of F- and G-actin, and mechanistic relationship to surfactant secretion remain unclear. Shi& between F and G - actin pools may occur rapidly concomitant with changes in cellular function. Acth filaments appear to be involved in both secretion and uptake of surfactant. Cytochaiasin D, which disrupts filamentous actin, has been shown to augment surfactant phospholipid release firom type
II cell~'~~.In fetal lung, Whitsett et al." have shown that actin is phosphorylated by a
CAMP-dependentprotein kinase in vitro. thus suggesting that actin may be an intracellular site of action of surfactant secretagogues.
Surfactant uptake is inhibited by cytochalasin D by 49%, suggesting a dependence on actin, and by 88% with combined cytochalasin D and hypertonicity, or nearly the same level as ATP depletion, when type II cells in culture were incubated with fluorescent surfactant-like liposomes for 30 or 60 minutes.'-'9
The actin network regulates secretion of surf'actant and this process could take place as early as after 30 seconds of exposure to different secretagogues or actin filament disrupting agents. The early effects of secretagogues and actin disrupting agents on surfactant secretion and uptake in rat alveolar type II cells in primary culture could establish an intereshg area of investigation. Chapter 2
Rationale and Study Hypothesis The purpose of our sntdy was to examine the temporal correlation between cytoskeletal changes and early changes in surfktant handling by type II cells.
Given the demonstration of the presence of a sub-plasma membrane cortical actin
" shell" ,that appears to be actively rnodulated early in the course of stimulated secretion in other ce11 systems, the goal of the present study was to examine the distribution of F- and G-actin in alveolar type II cells, and whether it is actively modulated early in the time course of stimulated secretion in these cells.
Based on these data we attempted to examine the possibility that surfactant secretion in vitro is associated with depolymerization of subcortical actin filaments as a necessary first step in exocytosis. The relationship between modulation of the actin shell and surfactant secretion and uptake were examined over these early time frames following stimulation. Chapter 3
Materials and Methods 3.1. Materials
Dulbecco's modified Eagle's medium - low glucose (DMEM-LG), a-modified
Eagle's medium without antibiotics (a-MEM- AB), and phosphate buffered saline without calcium and magnesium (PBS - Ca, Mg) were prepared in the Tissue Culture
Media Preparation laboratory at the Faculty of Medicine, University of Toronto. Fetal bovine senun (FBS) and Gentamycin Reagent Solution were purchased from Gibco BRL
(Burlington, ON). NBD-phallacidin m-(7-nitrobenz-2 oxa- 1-3 oxa-diazol4yl) phailacidin] and deoxyribonuclease 1, Buorescein conjugate were purchased f?om
Molecular Probes (Eugene, OR.,USA). Porcine panaeas elastase was purchased from
Worthington Biochemical Corporation (Freehold, NJ,USA), polypropylene cuvettes were purchased nom Sarstedt (Germany), scintillant fkom ICN (Costa Mesa, CA, USA), ready- to-use Dako fluorescent medium fiom Dako Corporation (Carpinteria, CA, USA),
[methyl 'Hl-choline chloride and phosphatidykholine La-dipalmitoyl-2palmitoyl-9,lO-
'H (N) fiom Mandel Scientific (Mississauga, ON), Supercell eight-chamber slides fiom
Fisher Scientific (Whitby, ON). The following were purchased from Avanti Polar Lipids
Inc. (Alabaster, AL, USA): 1,2 diacyl-sn-glycero-3 phosphocholine (palmitoyl) saturated;
L-a-Lecithin-(Phosphatidylcholine) Egg Yolk; Phosphatidyl-DL-glycerol (PG) (sodium salt) and cholesterol. Thymosin P, was kindly provided by Dr. Allan Goldstein fiom The
George Washington University, Washington, DC, USA. Ali of the following chernicals were purchased fiom Sigma-Aldrich (Oakville, ON): purified rat IgG, reagent grade, fiom senun; bovine albumin; phorbol 12-myristate 13-acetate (4B, 9a,128, 13a,2O- pentahydroxytiglia-1,6 dien-3 one 12P-myristate 13-acetate), PMA; streptolysin O (SLO) fiom Sheptococcur pyogenes; formddehyde; paraformaidehyde; Sigma Enzyme Control
2E; f3-nicotinamide adenine dinucleotide, reduced fonn, disodium salt; L-glutamine gamma-irradiated, ce11 culture tested; fibronectin nom bovine plasma; Phdoidin,
TRITC-labeled.
3.2. Methods of Procedure
3.2.1. Type II celi isoiation
Alveolar type II cells were isolated fkom the lungs of adult male Sprague-
Dowley rats (225-250 g, supplier Harlan, South Carolina) by a modified elastase digestion and immunoglobulin panning method of Dobbs et al. 240 In this technique, alveolar macrophages are separated fiom the cnide lung homogenate by means of adherence to IgG coated Petri dishes. The lungs of anesthetized rats were cleared of blood in situ, removed nom the thorax, lavaged, and incubated with elastase. Tissue was minced and filtered through progressively smailer Nitex gauze. Cells were then "panned" on bacteriologic plastic Petri dishes pre-coated for 3 hours with rat IgG. The supematants containing unattached cells were centrifuged. Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% of fetal bovine senun (FBS). The fieshly isolated cells were plated at different densities as required by different experiments ushg 2-3 wells per condition and I labeied with 1 pCi/ml 3 H-choline in
Dl0, for the Bligh and Dyer lipid extraction experiment. They were then cultured for 18-
20 hours at 37OC in a humidified atmosphere of 95% air and 5% C02. At least 95% of the cells were identifiable as type II cells after this period of culture. Cells were identified using the alkaline phosphatase method 24'
3.2.2. Quantitation of F'-actin
Day-1 cells' (i.e. harvested 20 hours af€erisolation) media were discarded; cells were washed 3 times in DMEM at room temperature. DMEM + albumin + 100 nM TPA was added to stimulated cells. DMEM + albumin (1 mg/ml) was added to control wells and incubated for l', 3 ', 5'' 30' and 60' respectively, at 37OC. After the incubation period, media were collected for measurement of surfactant secretion (see below).
Culture wells were washed 3 tUnes with PBS at room temperature, fixed in 3.7% formaldehyde in DMEM + albumin and incubated for 15' at room temperature. The media were aspirated and the cells were added 100 pg lysophosphatidylcholine (to permeabilize the membrane) in PBS + 1.65~1O"M NBD-phallacidin in 300 pVwell. The cells were left in the dark for 20' at room temperature. Following this, media were aspirated, cells were rinsed once in PBS and 1 ml of methanol was added to each well to extract bound NBD-phallacidin. The plates were foi1 wrapped incubated in the dark for
15' at 37OC. Cells were scrapped kom tissue culture wells and the suspension transferred to 1.5 ml Eppendorf tubes and centrifuged 100% (12x3g) for 5 minutes. The supematants were collected into polypropylene cuvettes and added lm1 methanol to the pellet, resuspended and incubated in the dark for 15' at 37OC. The cells were centrifuged as before and the supernatants were collected and added to the previous supernatant. The cuvettes with methanol were used as control to determine the background fluorescence using a Hitachi Spectrophotorneter. Samples were excited at 488 nm and the absorbency read at an emission wavelength of 522 nm. Control readings were subtracted nom each sample reading. The results were expressed as a ratio of F-actin content of stimulated cells to unstimulateci cells.
This biochemical method uses the fluorescence of NBD-phallacidin bound to F- actin to quanti@ F-actin levels. NBD-phallacidin is a phalloidin derivative, tagged with a fluorescent dye. Phalloidin is a toxin that poisons a cell by preventing actin filaments f?om depolymerizing. Isolated from Amanita phalloides (the " Angel of Death" mushroom), phalloidin buids at the interface between subunits of F-actin, where it is an ideal position to lock subunits together. Even when actin is diluted below its cntical concentration, the phalloidin-stabilized filaments will not depolymerize. Phalloidin binds only to F-actin and its most cornrnon use is as an F-actin-specific stain for light microscopy. Tagged with fluorescent dyes, phalloidin binds any actin filament in a cell, and the arnount of F-actin cm be measured spectrophotometrically. Although a straightforward method, it has some disadvantages: first, NBD-phallacidin bleaches relatively quickly, and second, it is highly F-actin specific, detecting al1 lengths of actin filaments, even if there were changes in their distribution inside the cell. It does not bind to G-actin, although changes in this actin pool might occur, with disruptions in the actin filaments. 3.2.3. Memurement of surfactant secretion
Cells prelabelled with 1 pCi/mi 3~-cholineof a ImCi/ml solution (for 18-20 h) immediately after plating were stimdated with IO-' M TPA the following day. The supernatants were centrifuged at 800 rpm for 10 minutes, to pellet loose cells. 50 pl of the supematants were collected for the (LDH) cytotoxicity assay. The samples of supematants were used as aqueous phase for a Bligh and Dyer lipid extraction (see below). Chloroform and rnethanol were added such that the final 1-phase system had a chlorofom-methanol-aqueous ratio of 1:2:0.9 (Le. chloroform 2 ml, methmol4 ml, and aqueous solution 0.85 ml and supernatant 0.95 ml). To the monophase were added 2- phase amounts of chloroform (2 ml) and aqueous solution (2 ml) and the two phases mixed. The samples were centrifuged at 1,500-rpm (300 g) for 10' at SOC. The upper phase was aspirated and replaced and then vortexed. We centrifùged them as mentioned above. The lower phase was collected, and air-dried for 18-20 hours. The next day, scintillant was added and the tritium radioactivity (disintegrations per minute) was detemiined with a Bechan p-scintillation counter. The percentage secretion of phosphatidyl choline was rneasured as disintegrations per minute media / (disintegrations per minute media + disintegrations per minute cells). The extraction efficiency was approxirnately 90%.
3.2.4. Liposome preparation
Liposomes were prepared frorn lipid mixtures containhg DPPC:egg-
PC:PG:cholesterol in a molar ratio of 10:5:2:3. ['Hl-PC was used as a tracer for all studies and mixed with other lipids in chlorofotm before the solvent was evaporated under nitrogen. Lipids were then suspended in 1 ml phosphate buffered saline, pH 7.4, and sonicated at 50% under nitrogen with three one minute bursts hma probe sonicator
(Branson Sonifier, mode1 250/450, Danbury, CT). The vesic les were stored ovemight under nitrogen at 4OC. Before use, the vesicles were centrifbged at 1,000 g for 15 minutes, to remove aggregates. The translucent supmatant, containhg liposomes between 40 and 200 prn diameter, was used for uptake studies.
3.2.5. Liposomal uptake by type II ceUs
Alveolar type II cells were isolated as described in section 3.2.1. and plated ont0
35 mm dishes at a concentration of 1 x 1O6cel1s/rni,2 mVdish and incubated ovemight.
20 hours after isolation, cells were washed 3 heswith a-MEMand incubated in a-
MEM +/- 0.2 pM cytochalasin D for 30'. Cells were then washed 3 times with a-MEM and 20 p1 of liposomal suspension *1W7 M TPA, 10.2 pM cytochalasin D to 2 ml a-
MEM were added to each well and incubated at 37OC, 5% CO2 for 1', 3 ', s', 30', 60',
120'. Supernatants were collected, cells were washed 3 times in PBS, and the washings collected for radioactivity counts. Aftewards, cells were scraped 3 x 0.6 pl 1% TX- 100 and collected. 1000 pl of each sample was collected and counted. The counts for each hction were compared to the initial counts of the liposomal suspension. Phospholipid liposomes containing [3H]-dipalmitoylphosphatidylcholinewere incubated with rat dveolar type II cells in phary culture in the absence or presence of
PMA &CD. Cells incubated for different theperiods and the profile of radioactivities were assessed for media; cells, trypsinized and non-trypsinized hctions; and washings.
Total liposome uptake was partitioned into trypsin-releasable and trypsin-resistant radioactivity. After removing the supernatants and washing the cells for five times, the radioactivities associated with the pellet remained between 1-3% of total radioactivities, even with ce11 treaûnents known to stimulate liposome uptake.'" Some of the liposomes bound to the tissue culture plates.
3.2.6. Ce11 permeabiüzation
Day 1 alveolar type II cells in primary culture, plated at a concentration of 2x106
/ml, and prelabelled with 1 pCi/ml of 3H-cholinechloride were washed five times in intracellular buffer 1 permeabilizùig medium (140 mM KCI, 10 mM glucose, 10 mM
Hepes, 1 mM EGTA, 1 mM MgCl,, 189 pMCaCl,, pH to 7.4 with KOH) and incubated
for 30 minutes. Fresh baer was added to cells and some of the wells were treated with
0.4 unitdm1 SLO f concomitant addition of 10 FM thymosin P,. Cells were incubated at
37OC for 10 minutes to allow formation of pores and penetration of thymosin inside the
cells. After 10 minutes, cells were immediately placed on ice for 8 minutes to enable
closure of pores and intracellular entrapment of thymosin. Wells were washed with wann
medium once and fiesh medium was added to cells. Secretagogues were added two
minutes after the cells were placed in the incubator at 37T, 5% CO,. Cells were incubated for one hou afier which supematants were coliected, cells washed tbree times in PBS and then scrapped in ice-cold methanol. Fractions hmboth cells and supematants were reserved for the cytotoxicity assays. The lipid extraction assay was performed as described in section 3.2.3).
3.2.7. Cytotoxicity evaluation
Release of the constitutive cellular enzyme, lactate dehydrogenase (LDH)was used as an estimate of cytotoxicity. LDH release was measured fluorometrically and expressed as the percentage of cellular total enzyme released into the supernatant. LDH activity in the cells and medium were assayed by mesuring disappearance of NADH at
340 nm. Each value represents the average of two paired determinations made on material pooled fiom three wells per sarnple in each experirnent. Experimental conditions were compared in cells obtained fiom at least four separate ce11 isolations.
None of the agents utilized in these experiments resulted in statistically significant release of lactate dehydrogenase above control levels.
3.2.8. Fluorescence staining of F-actin and Gactin
Type II cells were plated at a concentration of 1O6ce1ls/ml on bovine plasma
fibronectin precoated (50 pg/ml dissolved in PBS) chamber slides (Fisher Scientific,
Whitby, ON). Chamber slides exposed to fibronectin were incubated for 60 minutes at
room temperature in sterile conditions. Mer 1 hou, fibronectin was aspirated and the
slides air-dried for 15 minutes, under sterile conditions. Freshly isolated type II cells were added to chambers at the above mentioned concentration and incubated for 18-20 hours in a humidified atmosphere, at 37OC, 95% air, 5% CO*. The next day, the medium was aspirated, cells washed 3 x 1 ml DMEM and incubated for 30 minutes with(out) 0.2
p.M cytochalasin D. Supernatants were then aspirated and cells washed lx DMEM. The cells in primary culture were added DMEM + 10% FBS f 0.2 pM cytochalasin D,
stimulated with 1O-' PMA, and incubated for 3O", 1', 3', Y, 1O,, 30', 60', and 1207, at 37
OC, then washed and fixed with 4 % paraformaldehyde for 30 minutes on ice. The membrane was dissolved with cytoskeletal buffer containing 0.1 % Triton X- 100,500
mM PIPES, 5 mM EGTA, 100 mM KOH, 2 mM MgCI,, pH 6.8. Further, the fixed cells
were stained with fluorescent probes, TRITC-phalloiciin (0.7 FM), that stains actin
filaments, and SuVml DNase 1 fluorescein that stains G-actin monomers. Cells were
incubated with the above mentioned reagents for 60 minutes, in the dark, at room
temperature. Merthe ha1 wash, cells were mounted in anti-fading Dako medium for
mounting fluorescent probes and visuaiized using fkst a fluorescent microscope, followed
by the confocal microscope to provide information on the changes in microfilament
distribution on a subcellular level. The distribution of F-actin and G-actin was haged in
both control cells and in cells that have been stimulated with PMA with or without
cytochalasin D pretreatment, as well as in SLO permeabilized cells, with or without
thymosin treatment as mentioned in chapter 3.2.6. Using the confocal microscope
(Zeiss LSM 4- 10 Inverted Microscope, Gemany) and the imaging processing software
(Zeiss LSM program 3.80), the relative arnounts of F-actin and G-actin in certain parts of
the ce11 cmbe quantified and followed as a function of the. For example, during surfactant secretion, only some of the regions of the celi could be affécted (being those cortical, cytoplasmic, and/or perinuclear), while others would not. This rnethod allowed us to quanti@ the amount of F-ach and G-actin by the intensity of fluorescence in each region, enabling detemination of the precise subcellular localization of the changes in distribution of actin. Images of cells visualized using the confocal microscope were standardized and saved by maintainhg the sarne contrast and bnghtness for each and every sample and ce11 treatment. The confocai microscope had an Omnipron Series 43
Laser with 568 and 647 wavelengths. A 63d1.40 Oil resolution Plan-Apochromat objective was used. For the red channel, the laser excitation wavelength was 568 nm, while the long pass filter was 590 nm. For the green channel, the laser excitation wavelength was 488 nm, while the band pass filter was set at 5 15-540 nm. The confocal scanning microscope pemiits visualization of fluorescent molecules in a single plane of
focus, thereby creating a vastly sharper image than the classical fluorescence rnicroscopy.
At any instant during the coafocal irnaging, oaiy a single mal1 part of the sample is
illuminated with exciting light frorn a focused laser beam, which rapidly moves to different spots in the sample focal plane. Images from these areas are recorded by a detector and stored in a computer, a.the composite image is displayed on the computer
screen. By means of a rehement known as optical sectioning, the computer records
serial sections - fluorescent images of planes at different depths of the sample, for each
fluorophore, and can combine the two images into one containing both fluorophores, by
way of the " overlay" command. To quantify the amount of F- and G-actin as indicated
by the two fluorophores, Z scans were performed at different levels of the monolayer and at different angles. For each condition, five Z scans were saved. The Z scans were analyzed using Adobe Photoshop 5.0 and for each colour, red (F-actin) and green (G- actin), histograms were achieved. Means for each histogram were saved, representing the average brightness for the two colours, red and green, for each particular Z scan
(appendices A-D). Values were assigned using an arbitrary bnghtness scale.
3.2.9. Statistical Metbods of Analysis
Andysis of variance (ANOVA) for repeated masures with correction for multiple cornparisons (tests of least-significant difference, Tukey, or Scheffé) was used for statistical analysis. Pc0.05 were considered statisticdly significant. Chapter 4
Resdts and Discussion The goal of this project was to examine the bilateral relationships between surfactant secretion and depolymerization of cortical actin. This proj ect demonstrates that surfiactant secretagogues invoke changes in the actin cytoskeleton including depolymerization of cortical actin and changes in the intracellular distribution of F- and
G-actin. Modulation of the actin cytoskeleton results in changes in active and passive secretion of surfactant. Disruption of the actin cytoskeleton appears to be both an obligatory first step in exocytosis as well as an important trigger of surfactant secretion.
We poshilated that that the actin network regulates secretion of surfactant and this process takes place as early as after 30 seconds of exposure to diflerent secretagogues or actin filament disnipting agents.
To address this hypothesis, we used isolated rat alveolar type II cells in primary culture. Cells were exposed for bnef periods to either secretagogues (e.g. terbutaline or
PMA), or cytoskeletal dimpting agents (actin monomer-binding proteins - thymosin P,, or actin severing proteins - cytochalasin D). Their effects on type II ce11 secretion were assessed by using a lipid extraction assay. The role of the above-mentioned agents on surfactant uptake was estimated by using radioactively labeled surfactant-like liposomes.
We have found an association between PMA stimulation of type II cells and changes in actin. Furthermore, we found that brief exposure of type II cells to cytochalasin D or thymosin f3, significantly increased surfactant secretion. Treatment of type II cells with both secretagogues and cytoskeletal disnipting agents (cytochalasin D or thymosin BJ dramatically modulated the relative content of F- and G-actin in type II cells at early tune points after exposure. These agents had no identifiable effect on cellular uptake of liposomal dipalmitoylphosphatidylcholineover these time hes.
These &ta suggest that suffactant secretion and uptake may be regulated by independent
mechanism.
4.1. Influence of Secretagogues on Surfactant
Secretion: Early Tirne Points
In vitro shidies of isolated primary cultures of type II cells have helped to elucidate the various signaling pathways of secreti~n.~'Evidence supports the
involvement of CAMP-, PKC-, and calmodulin kinase-dependent pathw ay s in agonist stunulated surfactant secretion'". Phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, increases both membrane-associated PKC and surfactant PC secretion. '" Accordingly, PMA was used as a positive stùnulus for the experiments described in this section. in control cells, unstimulated secretion proceeds linearly with tirne: (figure 1). As illustrated in figure 2, exposure to PMA resulted in a two-phase response. PMA stimulated surfactant secretion in type II cells within the first five minutes of exposure. Secretion was standardized to control rates and is illustrated in figure 3. The effects of PMA were most prominent in the first five minutes after stimulation, with a maximal effect seen at 3 minutes. At 30-minute incubation, a statistically insignifiant trend towards an inhibitory effect on secretion was noted in severai (of 13) experirnents. To monitor for cellular cytotoxicity, samples of cells and supernatant £kom each experimental and control condition were monitored as rnentioned in chapter 3.2.7. Cells were cultured with [3~]cholinechloride and treated with PMA, after which lactate dehydrogenase activity in the cells and medium was assayed by rneasuring disappearance of NADH at 340 m. None of the experiments resulted in statistically significant release of lactate dehydrogenase above control levels, which were within 1-2% of total cellular lactate dehydrogenase released du~ga 3-hour incubation per-iod.
tlme in minutes
Figure 1. Standardized surfactant secretion in control type II cek. Mer isolation, ceils were seeded on plastic tissue culture treated plates, labeled with 'H-choline chloride and incubated at 37OC, 5% CO2 overnight. The next day, the amount of 'H-labeled phosphatidylcholine was deterxnined in both the supematant and celi fiactions at different time points. Percent secretion of PC was measured as disintegrations per minute mediaf(disintegrati011~per minute cek + disinteptions per minute media). Amaunt of secretion fiom each sample was compared relative to the controI for each time point and smdardized. Values represent the mean f standard error of the mean (S.E.M.) for a representative experiment done in triplicate. Vertical bars indicate standard error of the mean. , Tirne in minutes and ceil treabnent
Figure 2. Role of PMA on surfactant secretion in type II ceb. After isolation, cells were seeded on plastic tissue culture tceated plates, labeled with 'H-choline chloride and incubated at 37OC, 5% COovernight. The next day, cclls were exposed to 10-'M PMA for difierat tirne periods, as mentioned in the " methods" section. Further, the amount of 'H-labeled phosphatidylcholine was detennined in both the supernatant and ceU fractions, and percent secretion was calculated as desmibed. Percent secretion of PC was measured as disintegrations per minute media/(disintegrationsper minute ceils + disintegrations per minute media). Amount of secretion fiom each sample was compared relative to the control for each time point and standardized. Values represent the mean t S.E.M. for 13 separate experiments done in triplicate. Vertical bars indicate standard error of the mean. Asterisk indicates pC0.05 with respect to the conuoi after normalization for interexperiment variability, detennined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD).
These shidies demonstrate that there is an early maximal response to PMA stimulation at 3 minutes, with continued secretion of surfactant for at least three hours.
Possible explanations for this observation include counterbdanced stimulatory and inhibitory effects of the phorbol ester, stimulation of a fast and a slow stimulatory pathway, fatigue or rehctoriness of second messagers, experimental error, or release of sUTfactant hmtwo or more compartments with differing tune constants.
In a number of ce11 types, chronic treatment with phorbol esters reduces PKC activity in a time- and concentration-dependent manner, and reduces the cellular response to a subsequent challenge with phorbol e~ters.~~*~~~Some loss in PKC enzyme activity is possibly due to proteolytic breakdown of the enzyme2a or to posttranslational changes in the enzyme.246The results of Chander et al.247showed that treatment of type II cells with phorbol esters caused a loss of PKC activity. The underlying mechanism for loss in activity was not clear, but was moa likely not related to proteolytic degradation because they noted a significant loss of activity at as early as 15 minutes. Chander et al. suggested the possibility that phorbol pre-treabnent leads to activation of an endogenous
PKC inhibitor, previously demonstrated to be present in neutrophils, lung, and brain.'".
249.250 They observed that phorbol-ester pretreated cells did not show stimulated secretion with a second challenge with phorbol ester. PKC desensitization-dependent down- regulation of secretion was stimulus specific: terbutdine stimulated secretion was unaltered.
Given that the in vitro assay used to measure surfactant secretion examines the bulk transport of 3H-choline labeled phospholipid, it is also possible that the apparent two-phase response to PMA is explained by the stimulation of a concomitant surfactant uptake pathway. This possibility was examined merin experiments descnbed in section 4.3. One explanation for these findings is the existence of an early pool released hm a cortical location, and a slowly released pool that requires intracellular transport prior to exocytosis. There is sorne evidence for the existence of the two pools of ~UTfactant."~The first ("ready-releasable pool") is relatively mail, tums over rapidly, and is under control of the sympathetic nervous system. The second, " late-releasable pool", is larger and is released in response to an increase in tidal volume. Power et al. 252 have isolated two subfiactions of the lamellar bodies, each of which contains the full spectrum of surfactant phospholipids. There is evidence that the two fractions can be released differentially.33
There is also support for the notion of two phases of stimulated secretion in pancreatic f3-cells. In this system, two stages of insulin release in response to glucose concentrations have been identified. Malaisse et al. suggested that the " early component" corresponds to the mobilization of secretory granules already entrapped in the thick cortical actin rim. When depolymerization of this shell occurs, secretory vesicles are released outside the cell. The " late component" of stimulated insulin secretion may correspond to the exocytosis of granules, that require transport through the cotical actin shell, and is under control of the rnicrotubular apparatus, which is sensitive to colchicine. Experiments that examine the impact of PMA stimulation on the cortical actin shell of alveolar type II cells are described in section 4.2. U O O O
Time in minutes and cell treaEment
Figure 3. Role of PMA on surfactant secretion in type II celis. After isolation, cells were seeded on plastic tissue culture treated plates, labeled with 'H-choline chioride and incubated at 37"C,5% CO, ovemight. The next &y, celis were exposed to 10-'M PMA for different time periods, as mentioned in the
" methods" section. Further, the amount of jH-labeled phosphatidylcholine was determined in both the supematant and ceil fractions, and percent secretion was cdculated as descnied. A significant uicrease in secretion was noted at 3-minute incubation with PMA, witb no evident effects thereafter. The values represent the mean i S.E.M. of 13 separate experiments done in tripkate. Vertical bars indicate standard error of the mean. Asterisk indicates p<0.05 with respect to the control after normalkation for interexperirnent variability, detennined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD). 4.2. Effect of Secretagogues on Cytoskeletal Actin
Based on the observation that in other systems- LCL DL Lncyto~keletalevents and regulated exocytosis are interconnected and the recent findings of Bhandari et al?, who found an increase in the G-actin hction concomitant with a decrease in the cytoskeletal F-actin fiaction, within 1 minute of exposure to terbutaline, we wished to examine such linkages in alveolar type 11 cells. Since cytoskeletal changes appear early during secretagogue stimulation in adrend chromaffin, mast, parotid and pancreatic B-cellsP7*
=, the present experiments were designed to investigate the early effects of PMA on surfactant secretion and cytoskeletal elements.
Two approaches were used to quanti@ F-actin in type II cells: a fluorescent assay for F-actin in cell monolayers using binding of NBD-phallacidin and a £iuoroscopic method using probes for both F- and G-actin to detect changes in both actin pools.
The first approach attempted to detexmine if there were changes in the amount of
F-actin and the relationship (if any) to stimulated surfactant secretion in monolayers of cultured alveolar type II cells. As shown in figure 4, however, this technique did not reveal changes in the amount of F-actin related fluorescence, even at time points during which there were significant changes in surfactant secretion. Similarly, no changes were seen during a 3-hour stimulation with phorbol esters (data not shown). Figure 4. Time-dependent variations in the amount of F-actin with PMA treatments. Cells in primary culture for < 24 h were stimdated with IO-^ M PMA for the periods of time indicated, after which they were fixed in 3.7% paraformaldehyde for 15 minutes and then labeted with NBD-phallacidin, which was extracted in methanol and fluorescence readings at 522 nm detennined after exciting samples at 488 m. Results were expressed as a ratio of F-actin content of stimulated cells to unstimulated cells. The values represent the mean f S.E.M. of 12 separate experiaients done in duplicate. Vertical bars indicate standard error of the mean.
The NBD-phallacidin binding technique detects F-ach in the entire type II ce11
monolayer. Since phallatoxins have similar *ty for both large and small actin
filaments (but do not bind monomeric G-a~tin)~'and bind in a stoichiometric ratio of
about one phallatoxin per actin subunit, possible interpretations for this negative
experiment include:
a. there was no change in F-actin levels under these conditions; there were changes in F-actin, but below the sensitivity of the
technique
there are compensatory increases and decreases in F-actin, that are
averaged throughout the monolayer
there is no change in total F-actin, but merely a redistribution, that
would not be detected by this technique
since the NBD-phallacidin binds large and mail F-actin filaments,
there are changes in the ratio of large to smaîi filaments, but no
changes in total F-actin.
We used confocal scanning microscopy to address some of these issues. Figure 5. Variations in the amount of F-ac~with time in control and PMA treated alveolar type II ceus. Actin filaments were stained with TRITC-phdoidin, whereas G-actin monomers were Iabeled with DNase I fluorescein. Cells were visualized under the confocal microscope and images saved by maintaining the same contrast and brightness for each and every sampIe and ceii treaûnent. To quant@ the amount of F- and G-actin as indicated by the two fluorophores, Z scans were performed at different levek of the monoIayer and at different angIes. For each condition, five separate Z scans were saved. The Z scans were aAerwards opened in Adobe Photoshop 5.0 and for each colour, red (F-ac~)and green (G-actin), bistograms were achieved. Means for each histogram were saved, representing the average brightness for the two colours, red and green, for each partidar Z scan. Values assigned for brightness of each colour are arbitrary. The values represent the mean f S.E.M. of five separate readings. Vertical bars hdicate standard error of the mean. (*) represents p < 0.05, whde (**) represent p < 0.01 with respect to contr01 after nomiaiization for interexperiment variabiiity, detdedby analysis of variance for repeated rneasures with correction for multiple cornparisons (LSD). Tirne in seconûs and celi trWnmt
Figure 6. Variations in the amount of G-actin with time in control and PMA stimulated alveolar type II cells. Ce& originate fiom the same isolation of rat alveolar type iI cek as in figure 5. Actin fdaments were stained with TRITC-phailoiclin, whereas G-actin monomers were labeled with DNase 1 fluorescein. Cells were visualized under the confocal microscope and images saved by maintaining the same contrast and brightness for each and every sample and cell -&arment TO quantify the amount of F- and G-actin as indicated by the two fluorophores, Z scans were performed at different levels of the monoIayer and at ciiffereut angles. For each condition, five separate Z scans were saved. The Z scans were aflerwards opened in Adobe Photoshop 5.0 and for each coiour, red (F-actin) and green (G-actin), histograms were achieved. Means for each histogram were saved, representing the average bnghtness for the two colours, red and green, for each particular Z scan. Values assigned for brightness of each colour are arbitrary. The values represent the mean f S.E.M. of five separate readings. Vertical bars indicate standard error of the mean. (*) represents p < 0.05, wbile (**) represent p < 0.01 with respect to control after nonnaiization for interexperiment variabiiity, determined by analysis of variance for repeated measures with correctior. for multiple cornparisons (LSD). Figures 5 and 6 illustrate the measurement of F and G actin, respectively. The
amounts of F- and G-actin in control cells did not vary under conditions of these
experiments. By contrast, within minutes after phorbol stimulation, increases in both F-
and G-actin were noted. PMA treated cells showed an increase in the amount of F-actin
within the first three minutes. G-actin also increased with time, as early as after one-
minute stimulation with PMA, continukg at three-minute and five-minute incubation.
These changes are contemporaneous with the early thecourse of surfactant secretion
described in section 4.1 (figure 3), which was also maximal at three minutes. The time
courses obsented, in which changes in levels of G-actin precede the increase in secretion
seen at 3 minutes, suggest the possibility that stimulated surfactant secretion requires
antecedent cytoskeletal depolymerization.
The ability to simultaneously and cleariy visualize multiple i.ntrace1lula.r
fluorophores represents a great advantage of confocal microscopy over conventional microscopy techniques. The confocal imaging technique we have used was previously employed in type II cellsx6, as well as in other ce11 However, to the best of our knowledge, these experiments are the first application of double labeling method for cytoskeletal characterization in type II cells, (appendix A). In conml cells, the distribution of F-actin filaments is characterized by a thick F- actin rim at the periphery of the cell, with no change during the five-minute observation period. By contrast, G-actin, identified by means of the DNase 1 fluorescein green dye, is scattered throughout the cell. Following PMA stimulation, there is an obvious decrease in cortical F-actin, at three-minute incubation and an increase in the amount of G-actin trends. Although this needs fùture experimental investigation, the disparity seen betw een the confocal image (appendix A: figure 10) and the biochemical determination of F- and
G-actin (figures 5 and 6) rnay be explained by depolymerization of cortical F-actin with possible cytoplasmic reorganization, correlated with accumulation of the globular form.
2-scans detect total cellular F- and G-actin content, but are not designated to detect regional distribution. 2-scans represent one of the 3-D features of confocal microscopy. These are fiontal sections performed at different levels throughout the monolayer of the ce11 culture. For each ce11 treatment, five Z-scans were saved and analyzed for content of each colour (red, which represents the amount of F-actin as evidenced by staining with TRITC-phalloidin; and green, that represents the arnount of
G-actin, labelled with DNase 1fluorescein) by means of Adobe Photoshop 5.0. For each colour, average brightness units were assigned.
Phalloidin staining to visualize assembled actin filaments and fluorescein stainuig of G-actin show that assembled actin is concentrated at the ce11 periphery (appendix A: figures 1,5,9, 13) and, af€er PMA stimulation, staining is lost around the whole cell, or in patches that may coincide with the sites of exocytosis (see Appendix A, figure 10). Bhandari et al.* recently described an increase in the amount of G-actin,
(maximal at 60 seconds) following exposure of cultureci type II cells to terbutfie, a
CAMPdependent protein kinase signal. They noted a decrease in F-actin and increase in
G-actin with terbutaline stimulation concluding that terbutalhe causes depolymerization of cytoskeletal F-actin in type II cells.
A relationship between secretory activity and the mimtubular-microfilamentous system has been previously noted in parotid and pancreatic acinar cells. 2hE9 The stimulation of depolymerization by agents which activate protein kinase A (terbutaline) and protein kinase C (PMA) supports our hypothesis that actin depolymerization may be a necessary antecedent to stirnulated phosphatidylcholine secretion by type II cells.
Following stimulation of alveolar type II cells in culture, one of the subsequent early events appears to be disassembly of the cortical actin network. It seems plausible that the subplasmalemmal actin network restricts the access of secretory granules to the plasma membrane. Visualization of this network allowed the demonstration that it is rapidly disassembled following stimulation and this may, therefore, be an important aspect of the control of exocytosis by agonists. 4.3. Role of Secretagogues in Surfactant
One possible explmation for the apparent early reduction in mrfactant secretion seen in experiments outhed in section 4.1 ., since these measurements reflect net transport of labeled phospholipid, and given considerable evidence suggesting coordinate regulation of surfactant secretion and uptakeI8,could be the eEect of early uptake. We examined the uptake of surfactant-like liposomes to study the effects of secretagogues and/or cytochalasin D on surfactant secretion.
The mechanism by which type II cells intemalize liposomal DPPC is incompletely understood. Endocytosis has been reported to be the major route of uptake of negatively charged liposomes in other ce11 typeslMand there is evidence that this is the mechanism by which different proteins are intemalized by type II ~ells.'~'The data of
Chander et al?' and Rice et al.'" suggest that lipid exchange is an unlikely mechanism of liposome uptake in type II cells. However, fusion of the plasma membrane cannot be excluded. The uptake and metabolic degradation of liposornal phosphatidylcholine by granular pneumocytes is of considerable importance for precise evaluation of surfactant synthesis, secretion, and clearance.
Experiments described in this section used liposomes prepared nom a mixture of [3~-dipaimitoylphosphatidyicholine-eggphosphatidy lcholine-phosphatidyl gl ycerol- cholesterol(10:5:2:3), as describeci in section 3.2.4. This composition of Liposomes approximates the Lipid composition of pulmonary surfactant.'" Phosphatidylglycerol was used for preparing liposomes, due to enhancement of lipid uptake in the presence of a negatively charged lipid. An effect of sdace charge on uptake of liposomes has previously been suggested for rat hepatocytes2%nd adip~cytes~~'.
Initial studies examined the performance of the system for up to four hours, at
37OC and at 4°C. At 37°C uptake was linear during a four-hour incubation (Figure 7). It was intereshg to note that at 4°C- there was also a time dependent increase in liposome uptake, although, as expected, the absolute amount and slope of uptake were less than at
37°C. CPM - spccific uptake
Time in hours and qmimental temperature
Figure 7. Uptake of liposomes prepared fiom synîhetic phospholipids (['q-DPPC) during incubation with isolated granuiar pneumocytes afler 24 h in primary culture, as a function of tirne and temperature. Cells were innibated with liposomes (50 pM [3H-dipalmitoylphospahtidyIcholine)at 37OC and at 4OC. For each experimenf celis were incubated with liposomes for 1,2, 3, or 4 h and uptake was calcuiated as radioactivities recovered fiom the non-trypsinizabte fraction.
We exarnined the percentage of radioactivity that was trypsin-resistant. Ce11 surface proteins, as indicated by the ability of trypsin to release a fiaction of the cell- associated radioactivity, may mediate the lipid association with granular pneumocytes.
We considered that the trypsin-releasable fiaction represented surface-bound lipids, whereas the trypsin-resistant component represented intemalized lipids, as previously described. ~2.2~~'~'The decreasing fraction of trypsin-releasable radioactivity with tirne of incubation observed in the present study suggests a process of surface binding and intemalization, with little surface adsorption of liposomes. In distinct contrast to our initial hypothesis, there was no effect of PMA stimulation on uptake of labelleci liposomes under the thecoune and experimental conditions examined in the course of these experiments (figure 8). PMA has been previously reported to stimulate uptake of phospholipids in isolated, pefised lungs. ='*= Although previously published studies described increases in uptake of surfactant-like liposorne~,~~these investigaton did not describe results of experiments at these early (40minutes) time points after stimulation.
Tirne in mlnutes and cell beatment g&ular pneumocytes, as a hiaction of tirne. Ceils wkincubated for the times shown and at indicated phosphatidylcholine concentrations. Uptake, as calculated fiom the trypsin-resistant cornponent, is shown as a net increase over the. Results are means + S.E.M. of six separate experiments done in triplicate for each point.
One explmation for the disparity between the present study and previous work may be differences in the experimental preparation. The present study used prirnary cultures of alveolar type II cells to examine uptake of surfactant like liposomes. Other experimental strategies include the isolated - perfbed lung preparation noted above, in vivo shidiesIo, and examination of er vivo lung slices? Geiger used aerosolized tritium- labeled DPPC to study the uptake in rats in vivo1? Appleton oze en-section autoradiographs showed greater than four times background radioactivity in approximately 30% of the alveoli at 1 minute and 2 hours deraerosol administration.
As tritium content in the lung decreased, it increased in liver, spleen, kidney, blood, and urine.
The use of cultures of type II cells implies absence of infiuences of other ce11 types, neural, or humoral factors. Disadvantages include the use of proteolytic treatment in the isolation method, which rnay adversely affect some differentiated cellular char acte ris tic^.^^^ The substrate on which type II cells are cuitured apparently influence the rate of surfactant lipid and protein ~~take.~~'The present experiments do not address the possibility that there may be influences of secretagogues at later thepoints.
The present study found no evidence of coordinate regulation of liposome uptake and surfactant secretion. This contrasts with a significant amount of published data that demonstrate coordinate regulation in other systems at later time points. For example,
Fisha et al."'. 272, 273 found that surfactant s~retzigOgUeSSUC~ as isoproterenol, terbutdine, and others enhanced the clearance of radiolabelled phosphatidylcholine fkom the alveolar cornpartment of an isolated perfùsed rat lung model. Petenazzo and colleag~es~~~reported that the B-agonist metaproterenol enhanced the alveolar clearance in a whole animal model. hcreases in ventilatory rate enhance both secretion and clearance-274The observation that SP-A enhances lipid uptakeaand inhibits lipid secretionln has lead to the speculation that SP-A may be involved in feedback regdation of surfactant pool size.
4.4. Effects of Cytoskeletal Disruption on Surfactant
Uptake
Experiments in this section were designed to test the possibility that modulation of the actin cytoskeleton might influence uptake of surfactant-like liposomes in monolayer cultures of aiveolar type II cells. We used surfactant-like liposomes to study the effects of secretagogues anaor cytochalasin D on surfactant secretion.
The granular pneumocytes used in this study showed thedependent uptake of liposomal dipalmitoyl phosphatidylcholine. Other cells in culture have also been shown to accumulate liposomal phospholipids. The experirnental outhe and methods of procedure were similar to those used in section 4.3. SPE331 tg$ =.Es 55s ËEE EEE
O
Time in minutes
Figure 9. Standardized specific uptake of iiposomal phosphatidylcholine by granuiar pneumocytes, as a fllnction of time and differeot ceU treatments. Ceiis were incubated for the amount of time shown and at indicated phosphatidykholine concentrations. Uptake, as calculated fiom the trypsin-cesistant componenc is shown as a net inmase over tixne. Results are means f SDV of a nuniber of observations shown for each point.
To determine if the actin cytoskeieton was involved in the early uptake process, we examined the effect of cytochalasin D at concentrations known to stimulate secretion
(0.2 PM). As shown in figure 9, cytochalasin D reduced uptake of liposomes DPPC,but this effect was noted at 2 30-minute incubation. Maximum inhibition, however, was only about 25%, demonstrated at 30-minute incubation. Cytochalasin D did not show a statistically significant effect on uptake. When both CD and PMA were added to the system, there were no significant effects on uptake during the fïrst 30 minutes of treatment.
Griese et al." have show that cytochalasin B inhibited uptake of liposomal dipalmitoylphosphatidy lcholine in rat alveolar type II cells in a concentration-dependent mamer- They also noted a maximum inhibition of about 25 %. Cytochalasin D aiso had a small inhibitory effect. However, their experimental outline differed fiom ours, in that they preincubated the cells with cytochdasins, after which they added the liposomes and assessed uptake only der two hours. Therefore, they could not detect the early (if any) effects of cytoskeletd disniption on the uptake of liposomes. Kotsif& et al." investigated the liposome-ce11 association with the aim of inserting methotrexate into the cytoplasm of intact cells. Methotrexate-containing liposome entrapment was dependent on theand concentration. They remarked that approximately 90% of the uptake was inhibited by cytochalasin D, consistent with the uptake being the result of endocytosis. They concluded that the remaining uptake was the result of adhesion of liposomes to the ce11 membrane.
Using an in vitro system composed of liposomes and actin, St-Onge and
Gicqua~d~'~showed that actin may interact directly with membrane p hospholipids. Actin deposited at the surface of the liposome is organized in two regular patterns: a paracristalline sheet of parallel filaments in register, or a netlike organization. These results suggest that the interaction of the cytoskeleton with the membranes involve a direct association of actin with phospholipids.
It is possible that the rate-limiting step in liposome uptake is adhesion of liposomes to the ce11 membrane. In such case, the rate of uptake rnight be independent of the influence of cytoskeletal disrupting agents, such as cytochalasin. The involvement of the actin cytoskeleton in uptake might, however, be apparent at later tune points. The finding that cytochalasins slightly inhibited clearance of surfactant suggests that actin filaments do not play a major role in liposome uptake by type II cells, and do not seem to play any role in the early uptake processes, at Ieast as revealed by methods of the present investigation. We did not, however, investigate the effects of higher concentrations of cytochalasin D, other methods of liposome preparation, nor extended time courses. 4.5. Influence of Actin Cytoskeleton Disrupting Agents
on Early Surfactant Secretion
The ahof these experiments was to examine the relationship between disruption of the cortical actin cytoskeleton and surfactant secretion. In chapter 4.1 we have shown an early increase in surfactant secretion with PMA stimulation. In chapter 4.2, we demonstrated that increased surfactant secretion was preceded and associated with changes in the amount of F- and G-actin. It has been shown previously that lamellar bodies inside type II cells, especially those located close to the surface of the cell, and those caught in the process of exocytosis, are closely associated with actin filaments.'76
Membrane-cytoskeleton interactions and regulated exocytosis appear to be tightly connected.
Two approaches were used to examine the effect of F-actin depolymerization on secretion. In the first senes of experiments, a well-charactenzed cytoskeletal disnipting agent, cytochalasin D, was used. A second series of experiments utilized a highly specific actin monomer-binding protein, Pd-thymosin, and permeabilized alveolar type II cells to investigate their role in surfactant secretion.
Williams and Tsilibary demonstrated a decrease in the number of larnellar bodies per ce11 profile as compared to untreated controls afier cytoskeletal disruption."l In adrenal chromah cells, agonist stimulation is associated with transient depolymerization of F-actin, with generation of G-actin, and these changes are concomitant with increased exocytosisaP7Depolymerization of F-actin has been suggested to promote granule fusion. Based on these data, we hypothesized that early exposure to cytochalasin D of type II cells in primary culture, wouid result in a brisk release of lamellar bodies constrained by the subcortical actin shell, located underneath the plasma membrane (" ready releasable pool").
We found that cytochalasin D enhanced the release of L3w-pC fYom cdtured type
II cells (figure 10). Reqonse was timedependent; cytochalasin was most effective at one-minute incubation, when secretion was increased twofold. This was due probably to local actin depolymerization of filaments surrounding vesicles found very close to the plasma membrane. Tirne in m[nubes and œil ûwtmnt
Figure 10. Effects of CD on surfactant secretion, Day- 1 adult aiveolar type II celk in primary culture were exposed to 0.2 pM CD and ['Hl-PC were extracted fiom both cells and supematants after a modified method of Bligh and Dyer, as descriied in " materials and methods" . Percent secretion of PC was measured as disintegrations per minute media/ (disintegrations per minute ceils + disintegrations per minute media). Amount of secretion fiom each sample was compared relative to the control for each time point and standardized. VaIues represent the mean i S.E.M.of 8 separate experiments done in triplkate. Vertical bars indicate standard error of the mean. (a) represents p < 0.05 with respect to control after noRnalization for interexperiment variability, determined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD).
The maximal response was seen at 1 minute. Similar responses were seen at 3 and 5 minutes, @ut were of lesser magnitude and did not achieve statistical significance).
No major changes in secretion were seen after a five-minute exposure, and secretion decreased following a 30-minute incubation with cytochalasin D. Interestingly, secretion measured at 30 minutes der CD application was less than that noted at five minutes, without any significant changes in the intracellular LDH content, as compared to control.
Under conditions of these experiments no Merincrease in secretion was noted when cells were exposed to a combination of both cytochalasin D and PMA (figure 1 1). By contrast, we observed a Merdecrement in secretion when combined the two agonists over a 30-minute incubation.
ïïme in minutes and cell treatment
Figure II. Effects of PMA and CD on surfactant secretion. Day-l adult alveolar type II cek in primary culture were exposed to 0.2 pM CD and/or 10" PMA and ['HI-PC were extracted fiom both cells and supernatana afir a modified rnethod of Bligh and Dyer, as described in " materials and methods". Percent secretion of PC was measured as disintegrations per minute media/ (disintegrations per minute cells + disintegrations per minute media). Amount of smetion fiom each sample was compared relative to the control for each the point and standardized Values represent the mean t S.E.M. of 8 separate experiments done in triplkate. Vertical bars indicate standard mor of the mean. (*) represents p < 0.05 with respect to control after normaiization for interexpriment variability, determined by analysis of variance for repeated meanves with correction for multiple cornparisons (LSD). Rice et aP5studied the effects of cytochalasin on cell shape. microfilament organization and phosphatidyl ['Hl choline ([)Hl-PC) release in rat alveolar type II cells to test the hypothesis that alteration in microfilaments was associated with surfactant release. They demonstrated a dose dependent uicrease in secretion of [3Hl-PC and disruption of micro filaments following cytochalasin treatment . They obsewed a significant effect of cytochalasin D-induced sdactant release at one hour, which continued over a 3-hour incubation.
Our results are consistent with those of Rice et al.'7s, who found that cytochalasin
D, which disrupts actin filaments, enhanced surfactant release fkom type II cells by more than threefold. To the best of our howledge, Rice and other researchers interested in this field have only studied the effects of cytochalasins on surfactant secretion only after 30 to
60 minute exposure to cytochalasin. However, the actin cytoskeleton is an extremely malleable system, changes in its structure and content occurring within seconds, if not f?actions of a second. Therefore, in the curent shidy we hypothesized that the effects of cytochalasin exposure on type II ce11 secretion could be quantified deras early as 30 seconds.
At concentrations of 2 4 FM, Rice noted that cytochalasin D had an inhibitory effect on P-agonist induced release of surfactant fiom isolated type II cells? Biphasic dose response curves have also been observed for cytochalasins treatment of lymphocytes, which transport amino acids'" and for steroid output from adrenocortical tumour ~ells.~"Although the etiology of this biphasic response is not clear, it is possible that at low concentrations of cytochalasin D in type II cells, microfilaments are disnipted, cells in monolayer culture, it was essentid to perform these experiments in permeabilized cells. The role of actin depolyrnerization in regulated exocytosis under close to physiological conditions was explored b y using a streptolysin O (SLO)penneabilization protocol that rnaintained the structural and functional polarity of cells and preserved their agonist-responsiveness.
We compared two published techniques for ce11 penneabilization. We used a recently published approach, using beta-escin to fom pores in the Under these conditions, the cells released approximately 30-40% lactic acid dehydrogenase into the medium, although maintaining secretory capacity and agonist responsiveness.
Phosphatidylcholine release derone hour in these cells was four times higher than in control cells (data not shown). These values suggested a search for an alternative protocol, since it was anticipated that huge effect of penneabilization alone would overshadow the experirnental response to cytoskeletal modulation by TB4.
SLO permeabilized cells rnaintained their secretory capacity, as indicated by the ahost equal arnounts of 3H-phosphatidylcholinerecovered fiom both control and SLO treated cells. SLO efficiency in penneabilizing cells was assessed by using the trypan blue exclusion assay, which showed that der 10-minute treatment, at 37OC, over 90% of cells were permeabilized. The rate of lactate dehydrogenase release into the medium was also detemiined as descnbed in section 3.2.7. Under conditions of these experiments, there was a 10- 15% release of lactate dehydrogenase into the medium.
Permeabilization by bacterial pore-forming toxins, a-toxin, and streptolysin O
(SLO)is now a widely accepted approach in the functional analysis of the intracellular organelles. The native forms of the toxins assemble as amphiphillic hexamers hto the target lipid bilayer, where they generate mail stable transmembrane pores. SLO pores are heterogeneous with a larger liam me ter.^^' The large pores generated by SLO oiigomers also let proteins pas. To avoid damage to intracelldar membranes, cells are incubated for a very short penod of tirne or at O°C. h our experiments, cells were incubated with
0.4 dm1 SLO for 10 minutes at 3 7OC. Under the later conditions, the SLO monomers just bind to the plasma membrane. Pore formation is initiated by warming up the temperature. SLO treated type II cells were 90% permeable after this ce11 treatment, as shown by trypan blue, a vi ta1 dye exclusion assay.
In îhis study, we opcimized the SLO permeabilization protocol to preseme agonist signaling cornpetence and its coupling to the exocytotic machinery. Cells permeabilized in the absence of actin depolymerizing proteins retained an intact exocytotic apparatus.
Quantitated exocytosis was measured by determining ['Hl-phosphatidylcholine secretion after fusion of lamellar bodies with the plasma membranes, using the lipid extraction assay, modified der Bligh and Dyer, as described in " Matenals and Methods" .
Secretion experiments in pemeabilized type II cells were perfomed in a medium that matches the iniracellular ionic concentration of cells, as mentioned in chapter 3.2.6.
Thymosin binding of actin monomers increased basal and stimulated secretion of type II cells in culture. 1 pM TP4 stimulated PC release f5om SLO permeabilized cells, in the absence of agonists. PC was secreted more rapidly than after stimulation with optimal concentrations of PMA (10-'M). As anticipated, TP4 did not significantly stimulate PC release fkom non-permeabilized cells, suggesting that its site of action be inside the cell.
To evaluate if TP4 stimulated exocytosis by the sarne mechanisms as phorbol agonists, permeabilized cells treated with 0.4 uniWml SLO were challenged with PMA.
IO-' M PMAdid not enhance secretion above the level observed with TP4 alone (figure
12). Sirnilar results were seen with terbutaline at 10 ph4 concentration (figure 12).
Figure I2. Stimulation of exocytosis with PMA and terbutaiine at dehed [Ca27. Cells were permeabilized and diluted into media with defined [Ca2+]as descnied in Materials and Methods. Twwas added during the cold incubation. One hour after starting the experiments, samples were removed to measure ['H+PC. Data show the mean * S.E.M. of three separate experiments performed in trïplicate. Vertical bars indicate standard enor of the mean. (*) represents p < 0.05 with respect to control after norrnaiization for interexperiment variability, deterrnined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD). SLO permeabilized cells mauitained their secretory capacity, as demotlsfrated by the sirnilar arnounts of 'H-phosphatidylcholine recovered fiom both control and SLO treated cells. Thymosin, a ce11 impermeable agent, did not significantly influence secretion. The effect of PMA (IO-' M) or terbutabne (10 CIM), as secretagogues, der 1 hour of exposure are lower than those reported in the literature for responses of intact cells (secretion increases 2-3 times after PMA stimulation for one hour, when compared to control).'" We attribute this difference to the functionai integrity and longevity of permeabilized cells, and to some of the harsh experirnental conditions. For example, cells were kept on ice for 8 minutes, to allow closure of pores. Our secretion experiments show an increase of about 2.5 times in PMA stimulated cells, compared to controls (data not show).
Thymosin did not modulate secretion induced by PMA or in non-permeabilized cells treated, confirming the requirement of an intracellular site of action of thymosin.
An unexpected finding was the inhibition of secretion observed by the addition of thymosin to SLO permeabilized cells stimulated with either secretagogue. The explanation for this hding is not clear. SLO permeabilized chromaffin cells secrete catecholamines by an exocytotic mechanism in response to micromolar concentration of cal~iurn.'~Above a certain concentration (HOpM), calcium inhibits stimulated secretion in SLO permeabilized cells. Since PMA is known to stimulate intracellular calcium release in type II cells, the observed inhibition of secretion may be due this secondary effect. TB4 inhibited secretion in response to PMA. This inhibition was not due to nonspecific ce11 damage; the lactate dehydrogenase assays showing about the same values as for those expressed by control SLO permeabilized type II cells. Since the only known intracellular function of TP4 is to sequester actin monomers, the rnost straightforward interpretation of our result is that TP4 stimulated exocytosis by depolymerizing actin
filaments. It, therefore, follows that other actin depolymerizing proteins, which are srna11 enough to penetrate the SLO-penneabilized membrane pores, should also induce secretion. Tp 10, a stnicturally sirnilar thymosin, showed a comparable effect on secretion in pancreatic achar ~ells.~'~
The SLO-permeabilized ce11 system used in the present study retained the major
features of the exocytotic machinery and its regulation. Because of the preservation of the well-characterized stimulus-secretion coupling responses, the SLO-permeabilized type II cells permitted a careful dissection of the role of the actin cytoskeleton on secretion in vitro. Our results show that actin filaments have a direct facilitatory role in regulated exocytosis: limited depolymerization of actin triggers exocytosis.
4.6. Surfactant Secretion Induced by Cytoskeletal
Disrupting Agents Modulates the Distribution of F-
and Gactin in Alveolar Type II Cell
The goal of this study was to determine the presence and distribution and to
quanti& the amount of filamentous actin and changes that occur in response to the cytoskeletal modulating agents utilized in section 4.5. The experiments were performed in paralle1 to those described in section 4.2. Cytochalasins interact with actin filaments and impede their elongation; this eventually resuits in disruption of the filaments.
Nonetheless, the disintegration of filaments and networks of actin could result in replacement of the orderly microfilamentous meshwork.
To examine changes in F- and G-acth under conditions in which the actin cytoskeleton had been modulated, actin filaments were stained with TNTC-phalloidin, whereas G-actin monomers were labeled with DNase 1 fluorescein. Cells were visuaiized using the confocal microscope and images saved by maintaining the sarne contrast and brightness for each and every sarnple and ce11 treatment, as described in chapter 4.2.
Changes were observed in the distribution of both F- and G-actin. In control cells, there was no change in disposition of actin filaments throughout the cell. A thick actin rim at the penphery of the ce11 and a fainter staùiing inside the cytoplasm were observed consistently (appendix B: figures 1, 5,9, 13 and appendix C figures 1, 5,9, 13).
Globular actin was present throughout the cytoplasm, as well as at the ce11 periphery
(Appendices B and C: figures 1,5,9, 13).
Treatment of type II cells with cytochalasin D for 30 seconds resulted in disruption of the cortical actin shell and an increase in the amount of G-actin (appendix
B: figure 2). The change temporally preceded the increase in surfactant secretion noted in section 4.1. A similar pattern was observed during the first 5 minutes of incubation with
CD (appendix B: figures 2,6, 10). Comparable results were obtained when both PMA and CD were added to cells at the above mentioned concentrations (appendix C: figures 2 and 10). However, the distribution of the two actin pools in PMA treated cells (figures 5,
6 - current chapter) was different than the one dernonstrated in CD treated ceils (figures
13, 14 current chapter). in PMA treated cells, both F- and G-actin fiactions increased with stimulation, whereas, in CD treated cells, there was an evident increase in the amount of G-actin, accompanied by an abrupt decrease in F-actin. This leads us to speculate that there are two different mechanisms involved in early secretory stimulation, one detexmined by cytoskeletal involvement, and the other probably employing signaling mechanisms, such as a protein kinase C dependent pathway. Tirne in seconds and ceil treaanerit
Figure 13. Changes in the arnount of F-actin with CD f PMA treatment. Confocal images were use& as previously mentioned, and to quantify the arnount of F-actin as indicated by TRITC-phalIoidin, Z scans were performed at different levels of the monolayer and at different angles. For each condition, five separate Z scans were saved. The Z scans were afterwards opened in Adobe Photoshop 5.0 and for the red colour (F-actin) histograms were achieved. Means for each histogram were saved, representing the average brightness for red for each particuiar Z scan, The values represent the mean f S.E.M. of five separate readings. Vertical bars hdicate standard error of the mean. (*) represents p < 0.05, whiie (**) represent p < 0.01 with respect to control after normalization for interexperiment variability, detennined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD). Figure Id. Changes in the amount of G-actin with CDeh4.A treatment. Confocal images were used, as previously mentioned, and to quant@ the amount of G-actin as iadicated by DN-ase I fluorescein, Z scans were perfonned at different levels of the monoiayer and at Merent angles. For each condition, five separate Z scans were saved. The Z scans were aflerwards opened in Adobe Photoshop 5.0 and for the green colour (G-actin) histograms were achieved. Means for each histogram were saved, cepcesenthg the average bnghtness for green for each particular Z scan. The values represent the mean f S.E.M. of five separate readings. Vertical bars indicate standard error of the mean. (*) represents p < 0.05, while (**) represent p < 0.01 with respect to control after nonnalization for interexperiment variability, detennined by anaîysis of variance for repeated measures with correction for multiple cornparisons (LSD).
There was no change in the pattern of actin distribution either under control conditions, or after exposure of non-permeabilized cells to thymosin (10 PM). When thymosin was added to SLO pemeabilized type II cells, at five minute incubation with thymosin there was marked decrease in the amount of F-actin, whereas G-actin was at the highest level at 3 minute incubation, indicating a rapid effect of the acth monomer- binding protein (figures 15, 16; appendix D: figures 3,9, 15,21).
Time in seconds and cell treabnent
Figure 15. Effects of thymosin on F-actin. Confocal images were use& as previously mentioned, and to quant@ the amount of F-actin as indicated by TRITC-phalIoidin, Z scans were performed at different Ievels of the monolayer and at different angles. For each condition, five separate Z scans were saved. The Z scam were afterwards opened in Adobe Photoshop 5.0 and for the red colour (F-actin) histograms were achieved. Means for each histogram were saved, representing the average brightness for red for each particuiar Z scan- The values represent the mean f S.E.M. of five separate readings. Vertical bars indicate standard error of the mean. (*) represents p < 0.05, whiie (**) represent p < 0.01 with respect to control after nonnaiization for interexperiment variability, determined by analysis of variance for repeated measures with correction for multiple cornparisons (LSD). Time in seconds and dltreabnerrt
Figure I6. Effects of thymosin on G-actin content. Confocal images were used, as previously mentioned, and to quant@ the amount of G-actin as uidicated by DN-ase 1 fluorescein, Z scans were performed at Merent levels of the monoiayer and at different angles, For each condition, five separate Z scans were saved. The Z scans were afterwards opened in Adobe Photoshop 5.0 and for the green colour (G-actin) histograms were achieved. Means for each histogram were saved, representing the average brightness for green for each particdar Z scan. The values represent the mean L S.E.M. of five separate readings. Vertical bars uidicate standard error of the mean. (*) represents p < 0.05, while (**) represent p c 0.01 with respect to control after normalization for interexperiment variabiiity, detemiined by analysis of variance for repeated measures with correction for multiple cornparisons (Scheffé).
The present experirnents demonstrate a time-dependent effect of CD and thymosin
P, on both actin depolymerization and PC secretion. These data suggest a key role for disassembly of the actin shell in the modulation of surfactant secretion in alveolar type II cells. The stimulation of depolymenzation by agents which activate protein kinase A
(terbutaline) and protein kinase C (PMA) Mersupports our hypothesis that actin depolymerization may be a key step in the stimulateci secretion of phosphatidylcholine by type II cells. Although ~XOC~~O~Srequires actin depolymerization, it cannot occur without a minimal actin structure. The comprehensive inhibition of exocytosis suggests that actin filaments are required for completion of a step close to or downstream of grande docking to the plasma membrane. The use of highiy specific actin modulatory proteins pemiits the molecular dissection of the membrane-cytoskeletaI luikages between the very early and late events in regdated exocytosis. Chapter 5
Summary and Significance SUTfactant secretion by alveolar type II celis is regulated by complex mechanisms that respond to a wide variety of stimuli. Both physical and chernical stimuli affect stdactant secretion. Cytokines and biologically active chernicals that are surfactant secretagogues may act through at least three different intracellular signalling systems
(e.g. increasing calcium and CAMPand activating PKC). The sequence of chemîcal and physical events that link intracellular signaling and exocytosis of suffactant larnellar bodies remain unclear. This project focused on the relationship between secretory stimuli and cytoskeletal changes that accompany surfactant secretion. Unlike previously published studies, the present study examined cellular and cytoskeletai events that occur within the first 30 minutes after stimulation with conventional secretagogues. We investigated the modulation of surfactant secretion and uptake in the early time penod after stimulation, and examined corresponding changes in the actin cytoskeleton. We found that stimulation of type II cells with PMA for three minutes resulted in increased surfactant secretion and that this was associated with dramatic changes in the amount and distribution of F- and G-actin. Disruption of the actin cytoskeleton with cytochalasin D caused an early increase in secretion. We found neither an effect of phorbol stimulation, nor actin depolymerization on uptake of surfactant like liposomes at these time points.
Using a highly specific actin monomer-binding protein, thymosin, these shidies confirm that modulation of the subcortical actin cytoskeleton is associated with Surfactant secretion. Targeted inhibition of actin polymerization caused an uicrease in surfactant secretion in permeabilized alveolar type II cells. The results of these studies document the existence of a subcortical actin shell in adult dveolar type II cells. They suggest that actin depolymerization per se cm cause exocytosis in the alveolar type II cells. Further, they suggest that grande docking, fusion, and content release are Limited in resting cells by the subcortical actin shell. It Unplies that substantial pomons of the granule and plasma membrane are fusion competent, provided that the actin barrier is removed. This is consistent with the hypothesis that the cells have a constitutive fusion rnachinery that is clamped to prevent fusion, until an appropriate activation signal is received.la 14% ln In pancreatic acinar cells, for example, the actin network at the site of exocytosis acts as a dominant negative clamp for regulated exocyt~sis.~~
The actin clamp is likely to exist in other ce11 types. However, since cytochalasin, which depo lymerizes actin filaments, does not trigger exocytosis in neutrophils and neuroendocrine cell~,'~~~'"other regulatory mechanisms likely exist. Previous studies in other ce11 types have suggested that intact actin filaments are required for earlier steps such as granule transport to the site of exocyt~sis.~~~*n9*290 Inhibition of exocytosis by extensive acM depolymerization was also observed dertreatment with supramaximal agonist concentration^^^' or cytochalasin D."'
The direct relation between actin depolymerization and exocytosis found in type
II cells makes them a particularly attractive mode1 to study how agonists induce actin depolymerization to elicit exocytosis and why a minimal actin structure is required for regulated exocytosis. The use of highly specific actin-modulatory proteins pemits the molecular dissection of the membrane-cytoskeletd linkages between the very early and late events in regulated exocytosis. Given the potential for locai/topical dmg delivery in pulmonary systems, it is conceivable that cytoskeleton-modulating agents may prove usefûl in modulating surfactant secretion in vivo. Chapter 6
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Figure 1. Control cells - 30 seconds Figure 2. PMA treated cells - 30 seconds
Figure 3. Conuol cells 30 seconds - z scan Figure 4. PMA treüted cells - 30 seconds - z scan Appendix A - page 2
Figure 5. Control cells - 60 seconds Figure 6. PMA treated cells - 60 seconds
Figure 7. Control cells - 60 seconds - z scan Figure 8. PMA treated cells - 60 seconds - z scan Appendix A - page 3
Figure 9. Control cells - L80 seconds Figure 10. PMA ueated cells - 180 seconds
Figure 1 1. Control cells 180 seconds - Figure 12. PMA treated cells - 180 seconds - Z scan z scan Appendix A - page 4
Figure 13. Control cells - 300 seconds Figure 14. PMA treated cells - 300 seconds
Figure 15. Control cells 300 seconds - Figure 16. PMA treated cells - 300 z scan seconds - z scan Appendix B - page 1
Figure 1. Control cells - 30 seconds Figure 2. CD ueated celis -30 seconds
Figure 3. Control cells 30 seconds - Figure 4. CD treated celis - 30 seconds - z Scan z scan Appendix B - page 2
Figure 5. Control cells - 60 seconds Figure 6. CD treated ceUs - 60 seconds
Figure 7. Control cells 60 seconds - Figure 8. CD ueated ceiis - 60 seconds - z scan Z scan Appendix B - page 3
Figure 9. Control cells - 180 seconds Figure IO. CD treated ceUs - 180 seconds
Figure 1 1. Control cells 180 seconds - Figure 12. CD ~eatedceiis - 180 seconds - z Zan z scan Appendix B - page 4
Figure 13. Control cells - 300 seconds Figure 14. CD veated ceils - 300 seconds
Figure 15. Conuol cells 300 seconds - Figure 16. CD treated celis - 300 seconds - z scan z scan Appendix C - page 1
Figure 1. Control cells - 30 seconds Figure 2. PMA + CD treated çells - 30 seconds
Figure 3. Control cells 30 seconds - z scan Figure 4. PMA + CD treated cells - 30 seconds - z scan Appendix C - page 2
Figure 5. Control cells - 60 seconds Figure 6. PMA + CD treated cells - 60 seconds
Figure 7. Control cells 60 seconds - z scan Figure 8. PMA + CD treated cells - 60 seconds - z scan Appendix C - page 3
Figure 9. Control cells - 180 seconds Figure 10. PMA + CD treated cells - 180 seconds
Figure 1 1. Control cells 180 seconds - Figure 12. PMA + CD treated cells - 180 z xan seconds - z scan Appendix C - page 4
Figure 13. Control cells - 300 seconds Figure 14. PMA + CD treated cells - 300 seconds
Figure 15. Control cells 300 seconds - Figure 16. PMA + CD ueated cells - 3ûû z xan seconds - z scan Appendix D - page 1
Figure 4. Conuol cells - 30 seconds z scan
Figure 1. Control celis - 30 seconds
Figure 5. Thymosin ueated cells 30 seconds - z scan Figure 2. Thymosin ueated cells - 30 seconds
Figure 6. SLO permeabilized cells ueated with thymosin - 30 seconds Figure 3. SLO permeabilized cells z scan treated with thymosin - 30 seconds dix D - page 2
Figure 10. Control cells 60 seconds - z scan
Figure 7. Control cells - 60 second:
Figure I 1. Thymosin treated cells 60 seconds - z scan Figure 8. Thymosin treated cells 60 seconds
Figure 12. SLO permeabilized cells treated with thymosin - 60 seconds Figure 9. SLO permeabilized celis z scan treated with thymosin - 60 second! Appendix D - page 3
Figure 16. Control cells 180 seconds - z scan
Figure 13. Conuol cells - 180 seconds
Figure 17. Thymosin treated cells 180 seconds - z Kan Figure 14. Thymosin treated cells 180 seconds
Figure 18. SLO permeabilized cells treated with thymosin - 180 seconds z scan Figure 15. SLO permeabilized cell!E treated with thymosin 180 seconds Appendix D - page 4
Figure 22. Control cells 300 seconds - z scan Figure 19. Control cens - 300 seconds
Figure 23. Thymosin treated cells 300 seconds - z scan Figure 20. Thymosin treated cells 300 seconds
Figure 24. SLO permeabilized cells ueated with thymosin - 300 seconds z scan Figure 2 1. SLO permeabilized ce1 ueaied with thymosin - 30seconc IMAGE EVALUATION TEST TARGET (QA-3)
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