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Phospholipids as adjuncts for chromaffin granule release Nayar, Rajiv 1981

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PHOSPHOLIPIDS AS ADJUNCTS FOR CHROMAFFIN GRANULE RELEASE by RAJIV NAYAR BSc, The University of British Colunbia, 1979 THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( Department of Biochemistry ) We accept this thesis as confirming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1981 (c) Rajiv Nayar, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 DE-6 (2/79) i i ABSTRACT The chromaffin granules of adrenal medulla ce l l s are membrane bound entities which act as storage vesicles for catecholamines, ATP, and protein. Release of contents to the extracellular medium appears to occur via calcium-stimulated exocytosis which involves fusion with the plasma mem-brane. The molecular mechanism of exocytosis was approached from two points of view. 31 Fi r s t , i t was shown by P-NMR techniques that the endogenous phos-pholipids assume a liquid-crystalline configuration at physiological temperature both i n the intact membrane and in model systems composed of the extracted l i p i d . This is consistent with a structural role of phospholipids in vivo, serving to maintain membrane integrity. Addition of calcium to these systems resulted i n l i t t l e change in the biological membrane spectra and in the appearance of a relatively small component (<10 %) , possibly arising from phospholipid in the hexagonal H^ .^ phase in the model systems composed of the isolated total l i p i d s . Second, incubation of intact granules in the presence of upto 10 mM calcium did not cause significant release of contents. Similarily, incub-ation in the presence of exogenous l i p i d vesicles alone/did" not induce release. However, incubation with phospholipid systems, which have the a b i l i t y to undergo structural transitions in the presence of calcium, followed by the introduction of calcium caused immediate and total release of granule contents. This behaviour is attributed to disruption i i i of membrane integrity arising from calcium induced fusion of the phos-pholipid vesicles with the granules. In contrast, incubation with phos-pholipid systems which do not undergo structural transitions in the pre-sence of calcium is quite ineffective. On the basis of this information, a mechanism of calcium-induced exocytotic release of catecholamines in  vivo is proposed. iv CONTENTS Page 1. INTRODUCTION 1.1 Definition of Membrane Fusion and Exocytosis 1 1.2 Structure of Membranes 3 1.3 Phase transitions and f l u i d i t y characteristics of li p i d s A 1.3.1 Gel-liquid crystalline phase transitions 5 1.3.2 Fluidity of Membranes 5 1.3.3 Polymorphic phase transition 7 1.3.4 Asymmetric nature of biological membranes 8 1.4 The Chromaffin Granule: Structure and Function 10 1.4.1 Historical Perspective 10 1.4.2 Composition of the chromaffin granule 10 1.4.3 Dynamic role of the chromaffin granule 13 1.5 Approach to the problem of elucidating the molecular 16 mechanism of exocytosis 31 1.6 Use of P-NMR in determining membrane structure 17 2. MATERIALS AND METHODS 2.1 Isolation of chromaffin granules 21 2.1.1 Preparation of the Large Granule Fraction 21 2.1.2 Preparation of highly purified chromaffin 22 granules 2.2 Isolation of the chromaffin granule membrane 24 2.3 Determination of mitochondrial contamination in the 24 Large Granule Fraction 2.4 Lowry Protein Assay 25 2.5 Lipid Isolation and Purification 27 2.5.1 Lipid extraction from chromaffin granule membranes 27 2.5.2 Isolation of erythrocyte membrane phospholipids 27 V Pag_e 2.5.3 Isolation and purification of phosphatidyl- 28 choline from egg yolk and soya beans 2.5.4 Preparation of Phospholipase D 30 2.5.5 Preparation of phosphatidylserine by base- 31 -exchange reaction 2.5.6 Preparation of soya phosphatidylethanolamine 33 2.5.7 Purification of egg and soya phosphatidyl- a 33 ethanolamine 2.6 Thin Layer Chromatography (TLC) 34 2.6.1 Micro-slide TLC 35 2.6.2 Two-dimensional TLC 35 2.6.3 Phospholipid spray reagents for identification 36 2.7 Phosphorus Assay 38 2.8 Preparation of phospholipid vesicles 40 31 2.9 P-NMR experiments 40 2.9.1 Bilayer to hexagonal '.transition temperature 41 2.9.2 Polymorphic behaviour of the chromaffin granule 41 membrane and extracted l i p i d liposomes 2.9.3 Quantification of the amount of chromaffin 44 granule membrane phospholipids contributing to the *' P-NMR signal 2.9.4 Influence of exogenous li p i d s on the chromaffin 45 granule 2.10 Spectrophotometric Release Assay 46 2.11 Assaying for the release products of the chromaffin 47 granules 3. RESULTS 3.1 Structural organization of the chromaffin granule membrane 49 3.1.1 Chromaffin granule membrane and total extracted 49 l i p i d 31 3.1.2 Contribution of endogenous phospholipid to P- 51 -NMR signal v i Page 3.2 Influence of exogenous li p i d s 53 31 3.2.1 P-NMR experiments 53 3.2.2 Spectrophotometry release assay: Soya PE-PS 54 3.2.3 Egg PE-PS vesicles 59 3.2.4 Pure PS vesicles and pure CL vesicles 61 3.2.5 PC-PS vesicles „ 61 3.2.6 Erythrocyte l i p i d systems 63 3.2.7 Calcium Titration 65 4. DISCUSSION 4.1 Models of Membrane Fusion 68 4.1.1 Lysolecithin and Membrane Fusion 68 4.1.2 Fluidity and Membrane Fusion 70 4.1.3 Role of Calcium 70 4.1.4 Crystallization Model of Fusion 71 4.1.5 Polymorphic Model of Fusion 72 4.2 Chromaffin Granule Membrane 74 2+ 4.3 Phospholipids as adjuncts for iJa -stimulated release 74 of chromaffin granules 4.4 Possible Implications for the Mechanism of Exocytosis 77 5. BIBLIOGRAPHY 79 v i i LIST OF TABLES Pase Table I Composition of bovine adrenal chromaffin granules 11 Table II Phospholipid composition of the chromaffin granule 12 membrane v i i i TABLE OF FIGURES Page Figure 1 Membrane Fusion 1 Figure 2 Subcellular dynamics of the chromaffin c e l l 15 31 Figure 3 P-NMR spectra of phospholipid phases 20 Figure 4 Isolation of chromaffin granules 23 Figure 5 Protein standard curve 26 Figure 6 Two-dimensional TLC of chromaffin granule membrane 37 phospholipids Figure 7 Phosphorus standard curve 39 Figure 8 Bilayer to hexagonal transition of soya phosphatid- 42 ylethanolamine Figure 9 Bilayer to hexagonal transition of egg phosphatid- 43 ylethanolamine Figure 10 Correlation of chromaffin granule release products 48 31 Figure 11 P-NMR spectra of chromaffin granule membrane and 50 chromaffin granule membrane l i p i d systems Figure 12 Quantitative calibration of chromaffin granule membrane 52 phospholipids detected by 3 1 P-NMR 31 Figure 13 P-NMR spectra of chromaffin granules in the presence 55 of calcium and in the presence of exogenous soya PE-PS vesicles and calcium 31 Figure 14 P-NMR spectra of soya PE-PS vesicles 56 Figure 15 Release of chromaffin granule contents in the presence 58 of exogenous soya PE-PS vesicles and calcium Figure 16 Release of chromaffin granule contents in the presence 60 of soya and egg PE-PS vesicles and calcium at 20° and 35° C Figure 17 Release of chromaffin granule contents in the presence 62 of various exogenous l i p i d vesicles in the presence of Ca r + ix Page Figure 18 Release of chromaffin granule contents after incubation 64 with erythrocyte l i p i d vesicles and calcium Figure 19 Effect of calcium concentration on the release of chro- 66 maffin granule contents in the presence of excess soya PE-PS vesicles Figure 20 Proposed mechanism of exocytotic release of chromaffin 78 granule contents ±n vivo X ABBREVIATIONS ATP Adenosine-5'-triphosphate BSA Bovine serum albumin EDTA Diaminoethantetra-acetic acid g Centrifugal force HEPES 4-(2-Hydroxyethyl)-l-pipperazine ethanesulphonic acid NAD+ Nicotinamide adenine dinucleotide, oxidized NADH Nicotinamide adenine dinucleotide, reduced 31 P-NMR Phosphorus nuclear magnetic resonance ACKNOWLEDGEMENTS I am indebted to Dr. P. R. Cullis for allowing me the opporunity to develop my interests in membrane biology and for his continual support and enthusiasm in this project. I would also like to thank Dr. M. J. Hope for many helpful discussions and for providing information on var-ious techniques in lipidology. Finally, I would like to acknowledge Dr. C. P. S. Tilcock for critically reviewing this thesis. x i i To Anuradha / 1. Chapter I; INTRODUCTION Membranes have been extensively studied over the past thirty years due to the awareness that many important biological processes in animal and plant c e l l s and in microorganisms are mediated by membranes. This has led to various proposed models of membrane structure and also to the revelation of the enormous diversity of membrane-mediated functions, such as, intracellular compartmentalization of specific organelles, regulation of enzyme ac t i v i t i e s , f a c i l i t a t e d transport, c e l l fusion, endo- and exocytosis, and c e l l - c e l l interactions. The purpose of this thesis i s to gain some insight into the molecular mechanism of one of the above processes, namely exocytosis. 1.1 Definition of Membrane Fusion and Exocytosis Membrane fusion i s defined as the process by which two separate membranes join or unite to form a single membrane. This i s illustrated in Figure 1, where two membrane bound vesicles fuse to form a single vesicle. Figure 1. Membrane Fusion 2 . It is obvious that prior to the fusion event, close apposition of the two membranes is necessary in order to allow some kind of interaction between the two fusing membranes which would promote fusion. After fusion, the contents of the two vesicles and their respective membranes become confluent. Exocytosis is a specific membrane fusion event between the secre-tory vesicles of a cell and the cell plasma membrane. This fusion process results in the release of the vesicle products directly into the extracellular space. Exocytosis is basic to the processes of cell excretion and secretion and is involved in the release of a wide variety of enzymes, hormones, and neurotransmitter substances from such cells as the newly fertilized egg, blood platelets, leukocytes, mast cells, nerve cells, cells participating in the formation of k&nins, angiotensin, and erythropoietin, and hormone-producing cells in the adrenal medulla, neurohypophysis, anterior pituitary, thyroid, and pancreas (reviews; 1,2,. . 3) . That the release of these secretory products is essential for homeo-stasis is self-evident. The localization of the secretory products in membrane-bound vesicles and their export from the cell by exocytosis offers several advantages. The products are protected against degrad-ation from the cytoplasmic enzymes and can therefore be transported over fairly long distances, as in the case of nerve axons. The products can be released in quantal amounts in response to a direct physiolog-ical stimulus for release on the plasma membrane. For nerves, the stimulus is an electrical depolarization of the presynaptic terminal 3. (4) whereas for hormone secretion, the stimulus is usually a chemical which induces membrane depolarization (2), but in a l l the exocytotic processes investigated so far, calcium ions have been found to be vital for the operation of the secretion mechanism. Therefore, exocytosis is a specific membrane fusion event which is calcium dependent, however, the exact role of calcium is unclear. Membrane fusion cannot be considered in isolation from the other aspects of membrane activity and analysis of the mechanism of membrane fusion must take into account the basic properties of the membranes. 1.2 Structure of Membranes Membranes are composed predominantly of two major types of mole-cules: lipids and proteins. The lipids are thought to provide the fundamental building blocks of the membranes by forming a bilayer with their polar groups at the intracellular and extracellular surfaces and their hydrophobic fatty-acyl chains stacked roughly perpendicular to the plane of the membrane. This bilayer structure was first proposed theo-retically by Gorter and Grendel (5) in 1925 and later confirmed through experimental evidence by Danielli and Davson (6) in 1935. It has since then been extensively supported by numerous independent techniques (e.g. X-ray diffraction (7), electron microscopy (8), freeze-fracture (9), and nuclear magnetic resonance (10) ). The bilayer model was extended by Singer and Nicolson in 1972 into a Fluid Mosaic model of biological membranes (11). The fluidity was 4. suggested to be due to the rotational and translational motion of the phospholipids arranged in a bilayer matrix, while the mosaic nature included integral proteins that penetrated into or through the membrane, and also peripheral proteins that were "attached" onto the membrane surface. The integral proteins have been shown to contain a higher percentage of non-polar amino acids which are presumably involved in hydrophobic interactions with the phospholipid fatty-acyl chains. The peripheral proteins are relatively polar and can easily be removed from the membranes by changes in ionic strength as they are largely attached by weak electrostatic bonds. The fluid mosaic model has been widely accepted due to its dynamic nature with respect to protein function. It predicts that the motion of proteins may be modified by the fluidity of the lipid bilayer. 1.3 Phase transitions and fluidity characteristics of lipids A typical biological membrane contains over a hundred different species of lipids, predominantly phospholipids. A typical eukaryotic membrane, for example, may contain phosphatidylcholine (PC), sphingo-myelin (Sph), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), and cholesterol or other sterols. Each of the phospholipid species have associated with them a range of fatty acids of differing chain length and degree of saturation. Therefore, in order to unravel any functional role of these phospholipids, model systems using synthetic phospholipid of well defined composition were developed. 1.3.1 Gel-Liquid Crystalline Phase Transition The thermotropic behaviour of a number of pure lipids was invest-igated by Chapman and coworkers (12) using the techniques of different-i a l thermal analysis and differential scanning calorimetry. Pure phos-pholipids in aqueous dispersions were found to undergo a phase transition from a rigid gel-state to a fluid liquid-crystalline state. This transition temperature (T ) is dependent upon the lipid class, acyl-chain length, degree of acyl-chain unsaturation, hydration, and ionic environment (13). In most well defined lipid systems, the transi-tion is highly co-operative. Since a biological membrane consists of a large diversity of phospholipid species, the gel to liquid-cryst-alline transition is not so abrupt. Although gel-liquid crystalline transitions have been observed in pure lipid systems , they have been observed in a few biological membranes, such as in Acholeplasma  laidlawii, relevance of this type of transition is s t i l l in doubt for in vivo situations. 1.3.2 Fluidity of Membranes Fluidity of a biological membrane refledts the degree of order or disorder in the phospholipid acyl-chains. It can be measured by spectroscopic techniques, such as, electron spin resonance (14), nuclear magnetic resonance (15), or fluorescent polarization (16), and by differ-6 . ential scanning calorimetry techniques (13). The f l u i d i t y i s related to the gel-liquid crystalline phase transition of the component individual l i p i d s , that i s , membranes containing a large percentage of unsaturated li p i d s w i l l be more f l u i d than those containing a larger percentage of saturated l i p i d s . Temperature, presence of cholesterol or protein in the bilayer also modify membrane f l u i d i t y . Cholesterol i s the predominant neutral l i p i d found in biological membranes and has dramatic effects on membrane f l u i d i t y . In a liq u i d -crystalline medium, cholesterol has a "condensation effect" by increas-ing the degree of order in the fatty-acyl chains and thereby decreasing f l u i d i t y . In a gel-state medium, i t has a "liquifying effect" by pre-venting crystallization of the phospholipids (18). Therefore, the pre-sence of cholesterol in a biological membrane has been implicated in maintaining the f l u i d i t y of the bilayer. The biological significance of f l u i d i t y in a biomembrane is as yet unclear. It has been suggested to be of importance to the permeability of membranes, for example, in trans-membrane ion fluxes, or in controll*-ing enzyme activity. Evidence for the former has resulted from permea-b i l i t y studies correlating the effect of fatty-acyl chain composition of phosphatidylcholine membranes to the permeability to glycerol or eryth-r i t o l (19), and evidence for the latter has resulted from observations of discontinuities in Arrhenius plots of membrane-bound enzyme a c t i v i t -ies which have been attributed to the "melting" of the annular 7. lipid shell around the enzyme (20). 1.3.3 Polymorphic Phase Transition Apart from the gel-liquid crystalline phase transition, phospholipids also experience a polymorphic phase transition from the liquid-crystal-line bilayer structure to a hexagonal configuration or another non-bilayer structure (21). This polymorphic phase transition occurs o within 10 C of the gel-liquid crystalline transition and is also sensi-tive to temperature, lipid composition, pH, and divalent cations (22). Studies on isolated pure phospholipid species have revealed that a significant proportion of the phospholipids in a biological membrane would preferentially adopt in isolation a hexagonal configuration at physiological temperatures. Important examples of such lipids include unsaturated phosphatidylethanolamines (21,22,23), monoglucosyldiglycer-Ide (24), as well as phosphatidic acid (25) and cardiolipin (25) in the presence of calcium. In addition, lipids such as cholesterol (21,22,27) and unsaturated fatty acid (28) have been shown to induce the formation of hexagonal H.^  phases from bilayer systems. The effect of pH in inducing the hexagonal H has also been demonstrated in the case of phosphatidyl serine, where the phase was induced by lowering the pH to 3.0 (29) . The polymorphic behaviour of phospholipids and their possible functional roles in biological membranes has been recently reviewed by Cullis and De Kruijff (30). They have suggested non-bilayer structures 8. such as the hexagonal H.^  and inverted lipidic micellular structures occur as possible intermediates in membrane-mediated processes in-volving membrane fusion and transbilayer transport of lipids (flip--f lop). 1.3.4 Asymmetric nature of biological membranes Most of the membranes studied so far have been shown to have asymmetry with respect to their lipid, carbohydrate, and protein envir-onment in the two bilayer leaflets. The best documented evidence comes from the erythrocyte cell membrane. Protein labelling studies, phospholipid modification and phospholipase treatment studies, and lectin binding experiments (31) have indicated that the outer leaflet is composed of oligosaccharides containing terminal glucose or mannose sugar residues, contains two major proteins (acetylcholinesterase and 5'-nucleotidase), and is predominantly composed of phosphatidylcholine and sphingomyelin phospholipids. The inner cytoplasmic leaflet is assoc-iated with most of the proteins (spectrin, myosin and actin-like proteins, and protein kinase) and is predominantly composed of aminophospholipids such as phosphatidylethanolamine and phosphatidylserine (32). The presence of asymmetry can be rationalized by the fact that the reactions occurring on the inner and outer leaflets of a biological membrane are quite distinct and therefore would require different en-vironments. There is now good evidence for the specific role of carbo-hydrates in processes involving cell adhesion and recognition (33), 9. and the activities of the proteins on the inner leaflet has been assoc-iated with various cellular reactions. However, the reason whereby phospho-lipid asymmetry is maintained is unknown. As mentioned earlier,membrane fusion occurs ubiquitously in nature. The fusion event has been shown to involve the interactions between the two phospholipid monolayers of the opposing membranes. For example, in exocytosis, the fusion event involves direct interaction between the outer monolayer of the secretory vesicle and the inner monolayer of the plasma membrane. This has been implied from electron microscopic studies (34). Furthermore, freeze-fracture studies of the exocytotic event in chromaffin cells (35) and mast cells (36) have demonstrated that the membrane proteins segregate away from the fusion site and are therefore probably not directly involved during the fusion reaction. The proteins might possibly be involved in bringing about close appos-ition of the two membranes and in the recognition of the fusion site on the plasma membrane, but the fusion event itself clearly would in-volve some sort of structural transition of the phospholipids. Hence, phase transitions, fluidity, and asymmetric distribution of phospholipids could be important factors in allowing fusion to occur. This thesis investigates the possible role of phospholipid speci-ficity in the process of membrane fusion. The fusion system studied is the chromaffin granule which specifically fuses with the chromaffin cell plasma membrane by the process of exocytosis. The ease of isolation, 10. low cost > and the extensive characterization of the chromaffin granules make i t a convenient system for studying membrane fusion. 1.4 The Chromaffin Granule: Structure and Function 1.4.1 Historical Perspective The first indication that the hormones of the adrenal medulla, noradrenalin and adrenaline, were stored in a subcellular particle was obtained by Blashko and Welch (37) and by Hillarp and Hokfelt (38) in 1953. They showed that the bulk of the catecholamines resided in the fraction obtained by high speed centrifugation after removing the cell debris by a preceeding low speed centrifugation step. Electron micro-graphs of the adrenal medulla by Lever (39) in 1955 showed membrane bound vesicles (200nm. dia.) which were smaller than the mitochondria and he suggested that these stored the hormones of the adrenal medulla. Sjostrand and Wetzstein (40) in 1956 obtained similar micrographs and introduced the term, chromaffin granules. Morphological and biochemical characterization was subsequently correlated by Hagen and Barrnett (41) and it was concluded that the chromaffin granules were distinct organelles which were the site of storage of the catecholamines in the adrenal medulla. 1.4.2 Composition of the Chromaffin Granule The two components of the chromaffin granule,, water-soluble (i.e. 11. granule contents) and water-insoluble (i.e. granule membrane) fractions can easily be separated by lysis of the chromaffin granules in a hypo-osmotic buffer solution. The composition of the contents and of the membrane are listed in Table I. (42) Table I: Composition of the bovine adrenal chromaffin granule Constituent Amount, % total dry wt. soluble content catecholamines 20.5 adenine nucleotides 15 protein 27 calcium 0.1 magnesium 0.02 membrane phospholipid 17 cholesterol 5 protein 8 calcium 0.06 magnesium 0.02 The soluble contents are rich in catecholamines (0.71 M), adenine nucleotides (0.13 M ATP), and proteins (210 mg/ml). The soluble proteins are termed chromagranins. There are at. least twelve different chroma-granins present but their functions are s t i l l unknown. The only protein with known function is dopamine-j&-hydroxylase, which is predominantly found in the membrane. The water-insoluble proteins (presumably membrane proteins) are moi 2 | termed chromembrins and include dopamine--hydroxylase, Mg -activated ATPase, NADH oxidoreductase, phosphatidylinositol kinase, and cytochrome k 559. Spectroscopic analysis have also found a flavoprotein. Charact-erization and topography of the glycoproteins by Winkler et al. (43) showed that the carbohydrate portions of the glycoproteins faced the luminalgide of the granule.. Recently, Abbs and Phillips (44) invest-igated the organization of the chromamembrins and found that most of the proteins were accessible on the cytoplasmic side of the granules with atleast two proteins spanning the membrane. The chromaffin granules are relatively rich in lipids with a protein to lipid ratio of 0.45 (w/w) in the membrane. The lipids are chiefly cholesterol and phospholipids with a characteristic relatively high concentration of lysolecithin (about 15 %) . Table II lists the average phospholipid composition of the chromaffin granule membrane. Table II: Phospholipid composition of the Chromaffin  Granule Membrane. Phospholipid Percentage Tompo s it ion* Phosphatidylcholine 27 Phosphatidylethanolamine 34 Phosphatidyl serine ^ Phosphatidy1inosito1 Sphingomyelin 13 Lysophosphatidylcholine 15 Phosphatidic acid 1 Cardiolipin Average of a l l the literature values including the author's (45). So far, it has been difficult to elucidate the phospholipid asymmetry of the membrane because about 50 % of the total phospholipids have been found to be resistant to phospholipases (45,46). Although the 13. nature of this protection is unknown, phospholipase degradation of a l l the phospholipids after heat-treatment of the granules suggests that i t could be due to binding with the membrane proteins (45). 1.4.2 Dynamic Role of the Chromaffin Granule The chromaffin granule does not function as an inert reserve of • catecholamines in the adrenal medulla, but also stores several other secretory products and is directly involved in the biosynthesis and secretion of catecholamines. Figure 2 illustrates some of the dynamic aspects of the chromaffin granule inside the chromaffin c e l l . Storage Apart from the significant store of catecholamines (0.71 M), the contents also include large quantities of adenine nucleotides (0.13 M ATP) and proteins (210 mg/ml) (47) . The total amount of stored subst-ances account for about 63 % of the dry weight of the chromaffin granule. The high concentration of ATP has been suggested to be involved in maintaining high concentrations of catecholamine by formation of high molecular weight complexes (48). Synthesis The chromaffin granule contains the enzyme dopamine-j8-hydroxylase which plays a vital role in the biosynthesis of catecholamines. Since the cytosol of the chromaffin cell has potent inhibitors of this enzyme, the conversion of dopamine to noradrenalin occurs inside the chromaffin 14. granule. This has been supported by H-tyrosine pulse-labelling ex-periments and also by the finding that reserpine , which inhibits active uptake of dopamine into the chromaffin granule, inhibits synthesis of noradrenalin in slices of adrenal medulla (49). Secretion The ultimate role of the chromaffin granule is in the release of the secretory products into the blood, thus fulf i l l i n g the physiolo-gical function of the adrenal medulla. The release is accomplished by exocytosis which involves specific fusion of the granule membrane with the chromaffin cell plasma membrane. Jacobj (50) in 1892 demon-strated that electrical stimulation of the splanchnic nerve or direct stimulation of the adrenal gland resulted in secretion of biologically active substances. Feldberg and coworkers (51) later showed that \ acetylcholine was the physiological stimulus for secretion and found both nicotinic and muscarinic receptors on the chromaffin cells. Finally, extensive studies by Douglas and coworkers (52) found calcium to be the universal and necessary step in this stimulus-secretion coupling event. The molecular mechanism of release has yet to be elucidated. 15. Figure 2. Subcellular dynamics of the chromaffin cell. Release of chromaffin granule (CG) contents occurs by exocytosis. The empty membrane may pinch off to give rise to several small coated vesicles (CV). The CV lose their coating and are either degraded by lysosomal enzymes in the multivesicular bodies (MVB) or may regenerate back into a chromaffin granule. CP, coated pit; Mito, mitochondria. 16. 1.5 Approach to the problem of elucidating the molecular  mechanism of exocytosis. The objective of this thesis was to gain some insight into the molecular mechanism of exocytosis. Since this membrane fusion event involves direct interaction between the phospholipids of the chromaffin granule membrane and the plasma membrane, the problem was approached from two points of view. First, the dynamic organization of the chroma-ffin granule membrane and isolated lipids was investigated by phosphorus 31 nuclear magnetic resonance ( P-NMR) techniques. This was to ascertain if the specialized chromaffin granule membrane was different from other biological membranes and whether any differences observed could be correlated to functions such as fusion. In particular, it was import-ant to characterize the bilayer and non-bilayer preferences of the endogenous lipid and the influence of calcium, given recent suggestions that non-bilayer lipid configurations occur as intermediates in fusion of model (53) and biological (28) membrane systems, and that calcium can tagger formation of such structures in certain lipid systems (26,54). Secondly, from the point of view of efficient extracellular release of catecholamines, i t is obviously advantageous if granule--plasma membrane as opposed to granule-granule fusion is preferentially stimulated by the presence of calcium. Therefore, the influence of specific exogenous lipid model membranes, which may approximate the inner monolayer of the chromaffin cell plasma membrane or prefer either bilayer or non-bilayer configurations in the presence of calcium, on 17. calcium-stimulated release of chromaffin granule contents was invest-igated. Hence, specificity of phospholipids in promoting release of chromaffin granule contents was determined. 31 1.6 Use of P-NMR in determining membrane structure Since biological membrane lipids are predominantly phospholipids, 31 P-NMR is an attractive non-perturbing tool to investigate the motion and average orientation of the phosphate groups. The detection of 31 phospholipid polymorphism by P-NMR techniques rests on three factors (30). First, a large chemical anisotropy is exhibited by the phosphorus nuclei, which for large liquid-crystalline bilayer systems (> 2000 A) is only partially averaged by the restricted modes of motion available. These consist primarily of rapid rotation of the molecules along their long axis. Under proton decoupling conditions, it is possible to remove the P-H dipolar interactions and this results in a characteristic broad spectrum with a low field shoulder and a high field peak separated by approximately 40 ppm. for large lamellar organizations. A typical bilayer spectrum is illustrated in figure 3a. Second, a l l glycerol-based phospholipids (PC,PE, PS, PG, PI), except PA, and sphingomyelin have a similar lineshape when in the liquid--crystalline bilayer. Therefore, in a mixed lipid system, a l l the endogenous phospholipids that are in a bilayer contribute to a composite lineshape. 18. Finally, in a bilayer system the third factor contributing to 31 the P-NMR spectrum is the lateral diffusion of the phospholipids. In large bilayer structures, such as liposomes and biological membranes, the process of lateral diffusion is not fast enough on the NMR time-scale (10 ^  seconds) to produce an effective motional averaging mech-anism. In contrast, in small sonicated vesicles or other small struc-tures, the ability of phospholipids to diffuse laterally around the vesicle and vesicle tumbling produce line-narrowing effects. These isotropic motional averaging effects are observed for lipids in invert-ed micellular configurations and in other small structures, such as the cubic or rhombic phases. A narrow isotropic signal is therefore 31 difficult to interprete - by P-NMR techniques alone. The other non-bilayer structure of interest is the hexagonal phase. As shown in figure 3b, the hexagonal configuration consists of long cylinders of phospholipids whose polar headgroups are oriented towards small (20 A diameter) aqueous channels. Such structures exper-ience additional motional averaging as compared to the large bilayer structures due to lateral diffusion around the aqueous channels. Hence, 31 the hexagonal H phase exhibits a characteristic P-NMR lineshape which has the reverse asymmetry compared to the bilayer lineshape and is narrower by a factor of two. A summary of a l l three lineshapes are illustrated in figure 3. Close correlation between the polymorphic phase behaviour of phosph-31 olipids as detected by P-NMR, freeze-fracture, and X-ray studies (26) 19. confirms the validity of the P-NMR technique for determining membrane structure. Corresponding 3 1 P N M R spectra Phospholipid phases Bilayer . . . . . „ . . . ; . : . < : ^ j Hexagonal (H, ) Phases where isotropic motion occurs a, Cubic b, Rhombic c, Micellar, inverted micellar J, Vesicles -50 pnm.— H-Figur,e 3. P-NMR spectra of phospholipid phases. 21. Chapter II: MATERIALS AND METHODS 2.1 Isolation of Chromaffin Granules A modification of the methods of Smith and Winkler (55) and of Helle et al. (56) was used to isolate the chromaffin granules from bovine adrenal medulla. Between twenty and thirty fresh bovine adrenal glands were obtained from a local abattoir, where they were placed on ice until ready for isolation. A l l subsequent steps were carried out at 4°C 2.1.1 Preparation of the Large Granule Fraction The adrenal glands were defatted and dissected free of cortical tissue. The adrenal medullae were placed in ice-cold .3 M sucrose solution containing 10 mM HEPES and 1 mM EDTA at pH 7.0 (this solution is referred to as "buffered sucrose"). Homogenization of the adrenal medullae was more conveniently carried out employing 40-100 mesh sand prepared by the method of Guena (57). The homogenate was filtered through glass wool and centrifuged at 755 g. for 10 min. The pellet (cell debris) was discarded and the low speed supernatant was centrifuged at 17,000 g. for 10 min. The upper fluffy brown layer on the pellet (mitochondria and lysosomes mainly) was carefully decanted and the pink pellet was washed with buffered sucrose. The high speed centri-fuge step was repeated twice after resuspending the pink pellet contain-ing the chromaffin granules in 40 ml. of buffered sucrose. The final 22. pellets obtained after the high speed centrifugations were pooled and resuspended i n approximately 10 ml. of buffered sucrose to give a protein concentration of around 30 mg/ml. This fraction contained partially purified chromaffin granules and was referred to as the "Large Granule Fraction" (LGF). The large granule fraction was- stable in an isoosmotic medium and had a small amount of mitochondrial contamination (see purity section 2.3). In the majority of the release experiments, the large granule fraction rather than the highly purified granules was employed because of the osmotic f r a g i l i t y of the pure preparations. 2.1.2 Preparation of the highly purified chromaffin granules Due to the high density of the chromaffin granules relative to that of the mitochondria and lysosomes, the chromaffin granules can be highly purified by centrifugation through a hyperosmotic sucrose med ium About 2 ml of the large granule fraction was layered onto 30 ml. of 1.6 M sucrose and centrifuged at 80,790 g for 1 hr. Several different layers resulted after the step-gradient centrifugation. These are illustrated and numbered in figure 4. Analysis of the fractions by Smith and Winkler (55) have shown that the interface layers 2 and 2' contain most of the mitochondrial and lysosomal activity. The pink sediment, 5, corresponded to the highly purified chromaffin granules 23. Bovine Adrenal Medullae (1) Homogenized i n buffered sucrose (0.3 M sucrose, 10 mM Hepes, 1 mK EDTA, pH 7.0) Homogenate (2) 750 g, 10 min. Pe l l e t ( c e l l debris, nuclei) Low Speed Supernatant (3) 17,000 g, 10 min. High Speed P e l l e t resuspended i n buffered sucrose Supernatant (microsomes, c e l l sap) F l u f f y layer 4)-Large Granule Fraction (resnspended i n buffered sucrose) 17,000 g, 10 min. Supernatant (microsomes, c e l l sap) (5) 2 ml layered on 30 ml 1.6 M sucrose -2 ml of the large granule f r a c t i o 1.6 M sucrose (6) 80,790 g, 60 min. P u r i f i e d chromaffin granules 0.3 M sucrose 1.6 M sucrose 1 y — 1 (clear solution) 2 (opaque, dark brown) 2' (dark brown) •3 (opaque, l i g h t brown) •I* ( l i g h t brown) -5 (pink p u r i f i e d chromaffin granules) Tig. 1. Summary of the i s o l a t i o n procedure for chromaffin granules of bovine adrenal medulla. Step (i) was repeated twice. 24. i. and were used for l i p i d extraction and determination of the phospho-l i p i d composition of the chromaffin granule membrane. The scheme of isolation i s illustrated in figure 4. 2.2 Isolation of the chromaffin granule membrane The chromaffin granule membranes were prepared by ly s i s of the chromaffin granules in a hypoosmotic medium (5 mM HEPES, pH 7.0), follow-ed by a freeze-thaw cycle, and subsequent centrifugation at 27,000 g for 30 min. This was repeated four or five times u n t i l the absorbance of the supernatant at 265 nm. after centrifugation was less than 5 % of the i n i t i a l value. The brown pellet obtained after the centrifugation step corresponded to the chromaffin granule membrane fraction. 2.3 Determination of mitochondrial contamination in the  large granule fraction Since the major contaminant of the chromaffin granule preparation was mitochondria, i t was of interest to determine the extent of impurity in the large granule fraction. The enzyme, malate dehydrogenase (MDH), was used as a mitochondrial marker enzyme. MDH catalyzes the follow-ing reaction in the mitochondrial matrix: Oxaloacetate + NADH + H + > L-Malate + NAD+ r!' • NADH absorbs at 340 nm , therefore tbe decrease in absorbance over time was taken as corresponding to MDH activity. The A^g/min/nig protein was taken as the specific activity of MDH, which was proportional to 25. the amount of mitochondria in the sample. Fraction 2 in the ultra-centrifugation step of the preparation was assumed to correspond to the pure mitochondrial fraction. The stock solutions needed for the assay were 0.1 M phosphate buffer, pH 7.5, 15 mM oxaloacetate solution (2 mg/ml. in phosphate buffer), and 12 mM NADH (10 mg/ml-). The reaction was preformed in a 5 ml cuvette by adding the ingredients in the following order: 2.83 ml phosphate buffer, 0.10 ml oxalaacetate solution, 0.05 ml NADH solution, giving a final concentration of 95 mM, 0.5 mM, and 0.2 mM respectively. The reaction was started by addition of 0.02 ml. of the sample. The decrease in absorbance was recorded for about a minute at 340 nm. The reference cell had a l l the ingredients except the NADH solution. The specific activity of MDH in the large granule fraction and the highly purified chromaffin granules was approximately 15 % and 5 % of the pure mitochondrial fraction respectively. 2.4 Lowry Protein Assay Protein was measured according to the procedure of Lowry et al. (58), using crystalline bovine serum albumin as a standard. Stock solutions required for the assay were 2 % Potassium Tartate, 1 % Copper Sulfate, and 2 % Sodium Carbonate dissolved in 0.1 N Sodium Hydroxide. Solution I was freshly prepared by mixing 98.0 ml of Sodium Carbonate with 1.0 ml. of Potassium Tartate and 1.0 ml. of Copper Sulfate. Solution 0.5 h 50 100 150 200 250 Protein (ug.) Figure 5 . Protein standard curve 27. II was prepared by diluting 2 N Folin's Reagent to 1 N. The assay was carried out by adding 5.0 ml. of solution I to 1.0 ml of sample and water. After 10 min, 0.5 ml. of solution II was added followed by immediate vortexingy; Absorbance at 550nm. was recorded after 30 min. A typical Lowry standard curve i s shown in figure 5. 2.5 Lipid Isolation and Purification 2.5.1 Lipid Extraction from Chromaffin Granule Membranes Lipids were extracted by the procedure of Bligh and Dyer (59). Briefly, a sample of chromaffin granule membranes was diluted to 5 ml with water. Another 2.1 volumes of methanol and 1.0 volume of chloro-form was added and the one-phase solution was stirred for 15 min. To extract the l i p i d s , the solution was made into two-phases by the addit-ion of 1.0 volume of chloroform and 1.0 volume of water. The cloudy solution was centrifuged on a bench-top centrifuge at approximately 3,000 rpm. for 5 min. The top water-soluble fraction (containing proteins, ions, etc. ) was aspirated off and the remaining chloroform phase contain-ing the l i p i d s was flash-evaporated in a round bottom flask under vac-uum. The dry l i p i d was redissolved in some chloroform and stored at -20^C under nitrogen. 2.5.2 Isolation of erythrocyte membrane phospholipids The erythrocyte membrane phospholipids were a g i f t from Dr. M.J. 28. Hope. They had been isolated from human erythrocytes and purified using low pressure liquid chromatography on s i l i c i c acid and carboxy-methyl cellulose columns. The l i p i d s were eluted by mixtures of chloro-form and methanol, and were > 99 % pure with respect to phosphorus (60). Chloroform mixtures of the outer erythrocyte monolayer consisted of 44 mol % PC, 44 mol % Sph, and 12 mol % PE, with an equimolar amount of cholesterol. The inner monolayer mixture consisted of 47 mol % PE, 28 mol % PS, 15 mol % PC, and 10 mol % Sph, with an equimolar amount of cholesterol (61). 2.5.3 Isolation and purification of Phosphatidylcholine from egg yolk  and soya beans. Crude soya PC was purchased from Sigma while crude egg PC was isolated from egg yolk as follows. Thirty egg yorks were stirred intensively in 1.25 L. of acetone in order to precipitate the li p i d s which were subsequently fi l t e r e d through a course G3 glass f i l t e r . The precipitate was washed with 2 L. of acetone and the l i p i d s were extracted three times with 500, 400, and 300 ml. of chloroform/methanol (1:1) by stirring for 5 min. and subsequent filtration. The combined filtrate was flash-evaporated under vacuum and residual l i p i d s were dissolved in about 100 ml, of chloroform. To obtain a partially purified preparation of PC, the crude l i p i d solution (about 100g.) was purified by Aluminum Oxide chromatography. One kilogram of Aluminum Oxide (Al 90 q) was suspended in chloroform/ methanol (1:1) and fi l t e r e d over a coarse glass f i l t e r in order to remove most of the fines. The washed A l ^ was then suspended in chloroform and packed into a 200 X 5 cm glass column. The column was washed with 1.5 L of chloroform and the crude PC l i p i d solution was loaded onto the column. Neutral l i p i d s (triglycerides and cholesterol) were eluted with 1.0 L of 95 % chloroform-methanol at a flow rate of 6 ml/min and the partially purified PC fractions were quickly collect-ed by washing the column with 50 % chloroform-methanol at maximum flow rate in order to prevent PC degradation to lysoPC on the column. A l l fractions containing PC and no lysoPC were pooled and dried down by f l a sh-evaporat ion. The par t i a l l y purified PC fraction was then highly purified by High Pressure Liquid Chromatography (HPLC) on the Waters Prep LC-500 liquid chromatography system as described by Patel and Sparrow (62). Briefly, the compressed s i l i c a gel column was washed with 2.2 L of chloroform-methanol-water (60/40/10) and re-equilibrated with chloro-form-methanol-water (60/30/4). About twenty grams of partially puri-fied PC dissolved in chloroform (1 gm/mL) was applied to the column and eluted at a flow-rate of 100 ml/min. One hundred m i l l i l i t e r fractions were collected and the phospholipid elution profile was followed by thin layer chromatography on glass slides. Fractions containing absol-utely pure PC were combined and dried down by flash-evaporation ar r i v -ing at white compounds which were ^99 % pure PC. 30. The pure soya PC was used to synthesize its respective PE and PS by employing the base-exchange capacity of phospholipase D. Egg PS was also synthesized similarily while egg PE was purified from total egg yolk lipids by HPLC. 2.5.4 Preparation of Phospholipase D Phospholipase D, which hydrolyzes the headgroups of phospholipids, is very useful for the synthesis of less abundant phospholipids, such as phosphatidylserine. A study of phospholipase D activity in various plant tissues by Davidson and Long (63) found that Savoy cabb-ages was the richest source of the enzyme. Hence, a partially puri-fied preparation of phospholipase D from fresh Savoy cabbages accord-ing to their procedures was utilized to synthesize PS and PE. Phospholipase D purification was done as follows. The inner light--green leaves of Savoy cabbages (4 Kg) were homogenized in a Waring blender at maximum speed in 3 L.of ice-cold water for 3 min intervals. The homogenate was freed from fiber by squeezing through four layers of cheesecloth and then centrifuged at 13,000 g. for 30 min. The pH of the supernatant was adjusted to 5.5 with 4 N. HC1 and 250 ml fractions were quickly heated to 55°C in a boiling water-bath and then immediately cooled to 0°C. The heat-treated filtrate was spun again at 13,000 g. for 30 min and added to 2 volumes of ice-cold acetone with continual stirring in order to precipitate the * proteins. The acetone preci-31. pitate was allowed to stand for 2 hr. Subsequently, most of the yell-owish supernatant was aspirated off and the precipitate was transferred into metal GSA tubes and spun iat 1,000 g for 5 min. The white pellet was lyophilized in order to remove a l l the residual acetone in the preparation. The dry powder was resuspended in 75 ml of 0.2 M sodium acetate buff er, pH 5. 6, containing 0.04 M calcium chloride and continually stirred for 30 min in an ice-cold water bath. The solution was then transferred to an SS-34 centrifuge tube and spun at 17,000 g for 10 min. The brown supernatant, corresponding to the partially purified phospholipase D fraction, was carefully decanted into six test-tubes o and' stored at -20 C until used. 2.5.5 Preparation of Phosphatidylserine by Base-Exchange reaction Both egg and soya PS were synthesized from their respective PC by employing the base-exchange capacity of phospholipase D according to the procedures of Comfurius and Zwaal (64). The transphosphatidylation reaction catalyzed by phospholipase D was carried out in the presence of excess L-serine either with egg PC or soya PC. The phosphatidylcholines were dissolved in anhydrous ethyl-ether at a concentration of 20 mg/ml. L-serine was first lyophilized to o remove traces of methanol and subsequently dissolved at 45 C up to saturation (46 % w/w) in 100 itiM Acetate buffer (pH 5.6) containing 100 mM CaCl^. One tube of partially purified phospholipase D was added 32. to the serine solution and an equal volume of the phosphatidylcholine solution in ether was also added. The incubation flask was immediately closed and shaken continuously in a water-bath at 35 C. Incubation was stopped after 35 min and pressure in the nalgene incubation flask was relieved by cooling i t under a stream of cold water. The mixture was transferred to a glass centrifuge bottle and spun at 1,000 g for 10 min. The top ether phase was carefully transferred into a round bottom flask and the phospholipids were extracted once more from the incubation mixture with about 50 ml of ether. The combined ether fractions were flash-evaporated and the phospholipid was washed as follows in order to remove any water soluble components. The phospholipid was dissolved in 25 ml of chloroform-methanol (2:1), followed by addition of 6 ml of water. The two phases were mixed with a pasteur pipette and then centrifuged at 1,000 g for 5 min. The top aqueous layer, containing the water-soluble components was aspirated off and the chloroform lipid phase was dried down under nitrogen. Analysis of the total phospholipids showed that atleast 20-30 % of the phosphatidylcholine had been converted to phos-phatidylserine. Phosphatidylserine was purified from the reaction mixture by carboxymethyl-cellulose (CM-cellulose) chromatography as described by Comifurius and Zwaal (64), except that the elution of the phospholipids was done by a continous chloroform-methanol gradient rather than by a step-gradient. The pure PS fractions were pooled together and converted 33. to the sodium salt by dissolving the dry lipid in an acidic Bligh and Dyer monophase (chloroform/methanol/0.4 M HC1) which was subsequently titrated to pH 7.5 with a Bligh and Dyer monophase where the aqueous component was 0.5 M NaCl and 1.0 M NaOH. Addition of 0.4 volumes of water and chloroform to the total titrated volume resulted in a two-phase system. The chloroform phase containing the sodium salt of phos-phatidyl serine was dried down under nitrogen. The phosphatidylserine was shown to be > 99 % pure by two-dimensional thin layer chromato-; graphy. 2.5.6 Preparation of Soya Phosphatidylethanolamine Soya phosphatidylethanolamine was also prepared by the base-extfh-age capacity of phospholipase D. The procedure was identical to the one described for the preparation of phosphatidylserine except that the reaction mixture contained 15 % (w/v) of ethanolamine instead of L-serine. The ethanolamine was neutralized prior to incubation by — the addition of concentrated HC1. The transphosphatidylation reaction" in this case was much more complete with approximately 80 % of the phosphatidylcholine being converted to phosphatidylethanolamine. The purification of the phosphatidylethanolamine was done by HPLC as des-cribed below. 2.5.7 Purification of Egg and Soya Phosphatidylethanolamine Both egg and soya phosphatidylethanolamines were purified by HPLC 34. techniques as described by Patel and Sparrow (62). Egg PE was purified from a partially pure fraction obtained during the purification of egg PC by HPLC, while the soya PE was purified from the products of the transphosphatidylation reaction catalyzed by phospholipase D as described in section 2.5.6. The compressed silica gel column of the HPLC was washed with 2.2 L of chloroform-methanol-water (60/40/10) and 1.0 L of chloroform-methanol--water (60/30/2). The column was then equilibrated with 2.0 L of chloroform-methanol-water (60/30/2). Between 5-10 gm of partially purified PE dissolved in chloroform at a concentration of 1 gm/ml. was applied to the column and the phospholipids were eluted at a flow-rate of 100 ml/min. into 150 ml fractions. The pure PE fractions were pooled and flash-evaporated arriving at white compounds which were > 99 % pure. Apart from the TLC analysis of PE, the bilayer to hexa-gonal HJJ transition temperature is very critical to the purity of PE. a For example, the transition temperature of soya PE is 15 C and that o of egg PE is 25 C, and an impurity of upto 1 % can shift this transition temperature of PE by 10°C. Therefore, determination of the character-istic transition temperature of PE was also essential in accessing the purity of egg or soya PE. The procedure is described in section 2.9.1 2.6 Thin Layer Chromatography (TLC) TLC is considered to be the most effective and versatile technique in the separation and identification of lipids. Silicic acid is used 35. as the adsorbent on a glass plate or on a glass slide and offers the advantage of simplicity, ease of manipulation, sensitivity, rapidity, and high resolving power. For following elution profiles, microscope slides were utilized while for separationof the chromaffin granule membrane lipids and for quantitatively determining the purity of phospholipids, the high resdlution . two-dimensional TLC technique was used. 2.6.1 Micro-slide TLC Materials: Adsorbent; silica gel G suspended in chloroform at a con-centration of 50 g/100 ml. Plain microscope slides (2.5 7.5 cm.) TLC microslides were prepared by dipping the microscope slides into a slurry of silica gel G. and allowing the thin film of silica to air dry. Prior to runs, the silica was heat activated for approximately 30 sec. over a hot plate and the lipid sample was applied with a capillary tube. The TLC plate was run either in an acid or a base solvent. The acid solvent consisted of chloroform-methanol-glacial acetic acid-water system (25/15/4/2) and the base system consisted of a chloroform-methanol--ammonia-water mixture (90/54/5.7/5.4). After each run, the solvent was evaporated by gently heating the slide over a hot-plate and sub-sequently sprayed with the appropriate stain for detecting specific phospholipids (discussed in section 2.6.3). 2.6.2 Two-Dimensional Thin Layer Chromatography (2D-TLC) In order to separate the complex mixture of phospholipids in the 36. chromaffin granule membrane or to determine the purity of the phospho-lipids quantitatively, use was made of 2D-TLC according to the method of Broekhuyse (65). Pre-coated TLC plates (Silica Gel 60, 0.25 mm. thickness, 20 *}C 20 cm. plates) were used. They were activated at o 120 C in the oven for one hour before use. After sample application, the plate was run vertically in the base solvent system, dried and sub-sequently run horizontally in the acid solvent system. The composition of the solvent systems are described in section 2.6.1. After the runs, the plate was dried and exposed to iodine vapour for visualization of the phospholipids. The amounts of specific phospholipids components was determined by scraping off the silica corresponding to the specific phospholipid species and determining the amount of phosphorus by the method of Fiske and Subarrow (66). Figure 6 shows the separation of the chromaffin granule membrane lipids acheived by 2D-TLC. 2.6.3 Phospholipid Spray Reagents for Identification  Phosphorus reagent The specific reagent for detection of phospholipids is the molybdenum blue reagent. Reagent I was made up by adding 40.11 g. of MoO^  to 1 L. of 25 N rL^ SO^ , and boiling gently until the molybdic an-hydride was dissolved. Reagent II was prepared by adding 1.78 g. of powdered molybdenum to 500 ml. of Reagent I and boiling gently for 15 min. The solution was cooled and decanted. The specific phospholipid reagent was prepared by mixing equal volumes of Reagent I and Reagent 37. _H> ^ N . r O Cholesterol Acid « — — Base ' / V E P I ' ^ LPC Origin Figure 6. Chromaffin Granule Membrane Phospholipids separated by by two-dimensional thin layer chromatography. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatid-ylcholine; SPH, sphingomyelin. 38. II and diluting the solution with 2 volumes of water. This final reagent, molybdenum blue reagent, has a greenish yellow colour and is stable for months. Ninhydrin Reagent The ninhydrin reagent was used to detect phospholipids containing free amino groups (e.g. phosphatidylethanolamine, phospha-tidylserine, and their lyso derivatives). The reagent consisted of 0.2 % ninhydrin in butanol saturated with water. When sprayed with ninhydrin, lipids with a free amino group showed up as red-violet spots. 2.7 Phosphorus Assay The amount of phospholipids was determined according to the Fiske and Subbarow method (66). Reagent I was 70 % Perchloric Acid, Reagent II contained 0.22 % ammonium molybdate made up in 2 % (v/v) concentrated I^SO^, and Reagent III was made up by dissolving 30 g- of Sodium Bisul-fite, 1 g of sodium sulfite, and 0.5 g. l-amino-2-napthol-4-sulfuric acid at 40 C and was filtered after storing it overnight in the dark in order to remove the crystals. The procedure for detecting the amount of phosphorus was as follows. Between 0.1 and 0.5 umol of phospholipid sample was hydrolyzed by add-ition of 0.5 ml. of perchloric acid and one hour digestion at 196 C. In order to prevent evaporation, the tubes were covered with glass marbles. When the hydrolysis was complete (i.e solution was clear), 0.9 h 0.1 0.2 0.3 0.4 0.5 Phosphorus (umol) Figure 7. Phosphorus standard curve 40. the tubes were cooled and 14 ml. of ammonium molybdate reagent was add-ed followed by 0.6 ml of the Fiske-Subarrow reagent and immediate mix-ing. Colour was allowed to develop by heating the tubes in a boiling water-bath for 15 min. The tubes were cooled and absorbance at 830 nm. was recorded. Figure 7 show the standard phosphorus curve (0-0.5 umol P) . 2.8 Preparation of Phospholipid vesicles The phospholipid model systems were obtained by mixing appropriate quantities of lipid in chloroform and then evaporating the chloroform under a stream of nitrogen. The thin film of phospholipids was then stored under vacuum for 2 hr in order to remove any residual solvent. The lipid was subsequently hydrated in the buffered sucrose solution and sonicated intermittently (30 sec intervals) in an ice-water bath employing a tip sonicator. Sonication was continued until the disper-sion became optically clear (approximately 5 min). 31 2.9 P-NMR Experiments 31 All P-NMR experiments were performed on a Bruker WP 200 NMR Spectrometer, operating at 81 MHz for phosphorus, which was equipped with proton decoupling and temperature control. Accumulated free in-duction decays were obtained for up to 2,000 transients employing a 11 j*sec pulse and 0.8 sec interpulse time. 41. 2.9.1 Bilayer to Hexagonal H^ . Transition Temperature In order to determine the bilayer to hexagonal transition temp-31 erature of phosphatidylethanolamines, P-NMR spectra were accumulated over the appropriate temperature range by employing the VART (varia-tion temperature) program. Briefly, the phospholipid sample (50 /imol) was transferred to an NMR tube and the chloroform was evaporated under a stream of nitrogen. Subsequently, the tube was stored under vacuum for 2 hr and the lipid was hydrated in 1.0 ml of NMR buffer (100 mM NaCl/10 mM HEPES/1 mM EDTA/10 % D20, pH 7.4) by vortexing. 31P-NMR spectra at various temperatures was accumulated and the temperature of the bilayer to hexagonal transition was determined. Figure 8 and 9 show the spectra obtained for the bilayer to hexagonal H^ transition for soya and egg PE respectively. Their transition temperatures were 15 and 3 0 C respectively. 2.9.2 Polymorphic Behaviour of the Chromaffin Granule Membrane  and Extracted Lipid Liposomes The chromaffin granule membranes and their total lipids were iso-31 lated as described in sections 2.2 and 2.5.1 respectively. The P-NMR experiments on the chromaffin granule membrane were performed by pack-ing the membranes into an NMR tube containing buffered sucrose with 10 % D2O. The extracted membrane phospholipids were viewed after evaporating the chloroform under a stream of nitrogen, storage under 44. vacuum for 2 hr. , and subsequent hydration with 1.0 ml. of NMR buffer. Addition of calcium from a stock 1 M or 0.1 M solution of CaC^ was immediately followed by vortexing. 2.9.3 Quantification of the amount of Chromaffin Granule Membrane i 31 Phospholipids contributing to the P-NMR signal In order to quantify the amount of chromaffin granule membrane 31 phospholipids contributing to the P-NMR signal observed, two types of calibration experiments were performed. Initially, the ratios, CG Rpe, of the integrated signal intensities (recorded sequentially under exactly similar experimental conditions) from a standard sample of egg yolk phosphatidylcholine and the chromaffin granule membrane-sample were obtained. This was subsequently Compared f i r s t , to the ratio obtained from phospholipid phosphorus assays of the standard egg PC / CG sample and thejgranule membrane sample. Secondly, Rp^ was compared to the ratios of the egg PC standard and the chromaffin granule membrane 31 P-NMR signal intensities after the addition of 0.4 ml. Triton X-100 to both samples. This was sufficient detergent to solubilize both membrane systems, giving rise to translucent dispersions and a narrow, 31 symmetric P-NMR spectrum. In the case of the ratios obtained from phospholipid phosphorus assays and after solubilization with detergent, it may be presumed that a l l the chromaffin granule phospholipids contri-CG bute to the ratio R^ obtained. Therefore, comparison of the ratios obtained employing the intact granule membrane to those obtained via 45. phospholipid phosphorus assay and after detergent treatment gave a measure of the amount of phospholipid not detected in the intact granule 31 membrane by P-NMR. 2.9.4 Influence of Exogenous Lipids on the Chromaffin Granule -r 31 P-NMR techniques were employed to investigate the influence of exogenous lipids on the chromaffin granules in the presence and absence 2+ of Ca . The following protocol was followed. Intact chromaffin granul o (large granule fraction) were incubated at 37 C for 15 min. either in the presence or absence of exogenous PE-PS (3:1) vesicles with and without the addition of calcium. These incubated preparations were 31 subsequently concentrated for P-NMR studies by centrifugation ajtg: 17,000 g for 15 min. The packed pellet was washed with buffered suc-rose by gently swirling and then transferred into a NMR tube for signal accumulation. Since the chromaffin granules contain high concentrations 31 of ATP, the dominant features of the P-NMR spectra were the three peaks corresponding to the cL , fl , and K phosphates of ATP and . one peak corresponding to inorganic phosphate. Thus, release of the chromaffin granule contents was monitored by the presence or absence of the ATP peaks. The major disadvantage of this assay was the large amount of chromaffin granules required for each experiment (approxi-mately 100 mg. protein). Therefore, a much more economical spectro-photometric assay was employed during majority of the experiments. 4 6 . 2.10 Spectrophotometrlc Release Assay The spectrophotometrlc release assay varied from the ^ P^-NMR experiments in that the contents released by the chromaffin granules were measured rather than the contents remaining inside the granules. Typically, chromaffin granules (0.8-1.2 mg. protein, 40-50 of the large granule fraction) in buffered sucrose were incubated in the pre-o sence of sonicated vesicles, CaC^, or NaCl at 25 C for 15 min in a total volume of 1.0 ml. Where CaC^ was not added, a corresponding milliosmols of NaCl was added in order to minimize release due to osmo-tic differences. The reaction was stopped by addition of 4.0 ml ice-cold buffered sucrose and the chromaffin granules were pelleted at 17,000 g for 10 min. The supernatant was assayed for the release of the chromaffin granule contents, namely protein, ATP, and catecholamines. A reproducible level of about 25 % background release was observed in a l l controls in agreement with the observations of Hillarp et al (67). Release of contents was hence expressed as a percentage after subtracting the background. A measure of the total release of the chromaffin granule contents was taken as the amount release after lysing the chromaffin granules in 5.0 ml. of 5 mM HEPES, pH 7.0, follow-ed by a freeze-thaw cycle. 2.11 Assaying for the release products of the Chromaffin Granules  Protein The amount of protein released in the supernatant was measured 47. by the procedure of Lowry et a l (58). The determination was made in 1.0 ml of the supernatant and the blank contained 1.0 ml of buffered sucrose. Catecholamines The amount of catecholamines was measured according to the procedure of Von Euler and Hamberg (68). Briefly, to 1.0 ml. of the supernatant, 1.0 ml of 1 M Acetate buffer (pH 6.0), 50 u l of 10 % SDS, and 0.2 ml of 1 N iodine^ solution was added. After exactly 10 min , 0.2 ml, of 0.5 M sodium thiosulf ate; was added to bleach the colour of the oxidized catecholamines and absorbance at 530 nm was read immed-iately. Total Contents Since the contents of the chromaffin granules absorb quite strongly at 265 nm, , a very convenient assay f o r monitoring release was simply to determine the absorbance of the supernatant at 265 nm. It was found that the results obtained by this assay correlated very favourably to those obtained by the protein and catecholamine deter-minations (see figure 10). Therefore, in the majority of the release experiments, the A^fiC; assay was employed. 4 8 . Figure 10. Correlation of chromaffin granule  release products. The amount of release of chromaffin granule contents in the presence of exogenous PE-PS (3:1) ve s i c l e s with sub-sequent addition of CaC^ as determined by absorbance at 265 nm. ( /\> ) , amount of protein ( O ) » a n c* catecholamine content ( D ) i n the supernatant. 49. Chapter III; RESULTS 3.1 Structural Organization of the Chromaffin Granule Membrane The first priority in approaching the exocytosis process invol-ving the chromaffin granule and the chromaffin cell plasma membrane was to establish the dynamic structural organization of the endogen--ous lipids of the chromaffin granule membrane. As indicated in the 31 introduction, P-NMR is a useful technique for determining the poly-morphic preferences of phospholipids in model and biological membranes. 3.1.1 Chromaffin Granule Membrane and Total Extracted Lipid 31 The P-NMR spectra arising from the isolated chromaffin granule membranes, as well as model liposomal systems consisting of hydrated preparations of the total extracted lipids are indicated in figure 11. o Two features are apparent. First at 37 C, a large majority of the 31 endogenous granule membrane lipids exhibit a P-NMR lineshape charact-eristic of the bilayer phase. This is with the exception of a small component ( 5 % of the total phospholipid) which gives a signal of isotropic motional averaging. This may arise from small membrane frag-ments or lipids in other structures which undergo isotropic averag-ing (see figure 3). Secondly, the model membrane liposomal system of the total extracted chromaffin granule membrane lipids (figure 11c) exhibits a very similar bilayer spectrum. This observation is consis-tent with the structural role of phospholipids in an intact membrane, 50. " ^ 4 0 ' 0 ' 4 0 ~ - 4 0 ' 6" ' ~£o~ p p m p p m 31 Figure 11. P-NMR spectra of chromaffin granule membrane and 31 chromaffin granule membrane systems. 81.0 MHz P-NMR spectra at 37 C arising from (a) isolated chromaffin granule membranes; (b) isolated chromaffin granule mem-brane in the presence of 10 mM-CaC^ ; (c) liposomes com-posed of lipids extracted from chromaffin granule membrane and (d) as (c) in the presence of 10 mM CaC^. All preparations contained 10 mM Tris-HAc (pH = 7.2) and, where CaC^ was not present, 2 mM EDTA. The solutions used for the biological membrane contained 10 % D2O, whereas that of the liposomes contained 90 % D2O. 0 ppm. refers to the resonance position of sonicated phosphatidyl-choline vesicles. 51. that is maintenance of the bilayer i n t e g r i t y . Since calcium ions have been found to be essential for the exo-cytosis process, i t was of interest to study its effect on the membrane and the membrane model systems of the chromaffin granule. Figure 11 shows that the addition of 10 mM CaC^ results in l i t t l e change in the biological membrane spectrum (figure lib) and in the appearance of a relatively small component ( ^  10 % of that total phospholipid), possibly arising from phospholipids in the hexagonal phase in the model system composed of the isolated total lipids (figure l i d ) . 31 3.1.2 Contribution of Endogenous Phospholipid to P-NMR Signal In order to show what fraction of the endogenous chromaffin jjpanule membrane phospholipids was actually detected, the observed intensity 31 of the P-NMR signal for the granule membranes was quantitatively calibrated against a known amount of egg PC liposomes as described 31 in the methods section. Figure 12 shows the P-NMR spectra of the egg PC liposomes and chromaffin granule membrane taken under similar. parameters and figure 12b shows the spectra taken after solubilization of the phospholipids with Triton X-100 detergent. The ratios of the chromaffin granule membrane phospholipid signal intensity to that of CG egg PC standard, Rp^  , were 0.35 for the intact systems, 0.36 as deter-mined by phospholipid phosphorus assays, and 0.37 as determined after solubilization with Triton X-100. These results indicate that more 52. (a) Soya PC Soya PC Figure 12. Quantitative c a l i b r a t i o n of chromaffin granule membrane phospholipids detected by 5'P-NMR. The amount of chromaffin granule membrane phospholipids detected by 3' p-NMR signal was determined as described in the methods section. Spectra (a) are the signals obser-ved for the standard Soya PC sample and the chromaffin granule membrane obtained under similar conditions. Spectra (b) are the signals observed after the mem-branes were s o l u b i l i z e d by Triton X-100. It was assum-ed that a l l the phosphlipids contributed to the 31p_NMR signal upon s o l u b i l i z a t i o n . 53. than 90 % of the endogenous chromaffin granule phospholipids contri-31 bute to the observed P-NMR signal. 3.2 Influence of Exogenous Lipids The second stage of the research was to investigate the influence of exogenous lipid and calcium on chromaffin granule release. This 31 was first approached by P-NMR techniques and subsequently by spec-trophotometry techniques. 31 3.2.1 P-NMR Experiments As indicated in the methods section, intact chromaffin granules (large granule fraction) were incubated in the presence or absence of 2+ exogenous lipid vesicles with the subsequent addition of Ca and 31 then concentrated by centrifugation for P-NMR studies. Figure 13 shows the spectra obtained after incubation of intact chromaffin gran-ules at 37°C in the absence (fig. 13a) and in the presence (fig. 13b) 2+ 31 of 10 mM Ca . In both cases, the dominant features of P-NMR spectra arise from the three phosphate groups of ATP and that of inorganic phosphate. These spectra confirmed previously published spectra of intact chromaffin granules (69). The presence of ATP in the spectrum of fig. 13b indicated that the concentration of calcium of upto 10 mM did not induce significant release of the granules contents. This observation was in agreement with similar electron-microscopic studies 54. of intact qhromaffin granules done by Edwards et al(70) . Against this background, the results were quite dramatic when the granules were incubated in the presence of varying amounts of PE-PS (3:1) vesicles (10 min.,37 C) to which calcium was subsequently added and incubation continued for an additional 5 min. These results are illustrated in figure 13c-e. Defining R as the molar ratio of added exogenous phospholipid vesicles to endogenous chromaffin gran-ule lipid, i t is clear that for R=6 (fig. 13e) complete release of ATP has occurred as revealed by the absence of ATP in the chromaffin granules. In addition, a new spectral component is apparent in the region of -7 ppm. which coincides with the position of the low field peak arising from phospholipids in the hexagonal H^ j. phase (53) . The appearance of the hexagonal phase phospholipid was not unexpected on the basis of the behaviour of the PE-PS (3:1) in the presence of calcium (fig.1 14) . The addition of calcium to PE-PS ves-icles induces immediate precipitation of the vesicle suspension and triggers the formation of the hexagonal H^ .^ phase as indicated by the 31 characteristic P-NMR lineshape in figure 14b. This result was con-sistent with previous studies done on bilayer liposomal PE-PS systems (29) where the presence of calcium also triggered precipitation and hexagonal H T T phase formation. 3.2.2 Spectrophotometry Release Assays: Soya PE-PS In order to further characterize the ability of exogenous lipid 55. -20 0 20 -20 0 20 Ppm ppm Figure 13. P-NMR spectra of chromaffin granules in the presence of calcium and in the presence of exogenous soya PE-PS (3:1) vesicles and calcium. 81.0 MHz ^ P-NMR spectra at 37°C obtained from (a) intact o chromaffin granules; (b) granules incubated at 37 C for 15 min in the presence of 10 mM CaC^; (c) same as (a); (d) chromaffin granules in-o cubated in the presence of PE-PS vesicles (R = 4) for 10 min. at 37 C, followed by introduction of CaC^ to a concentration of 10 mM, after which the incubation at 37° C was continued for an additional 5 min; (e) same as (d) with the exception that R = 6. R is defined as the molar ratio of exogenous (vesicular) PE-PS phospholipid to endogenous (chromaffin granule) phospholipid. The buffered sucrose medium (see methods) was employed throughout. For other details see Methods section. (a) 1 1 1 1 1 r — — — — — ! — — — — — T -20 0 20 ppm 31 Fi g u r e 14. P-NMR s p e c t r a of Soya PE-PS (3:1) v e s i c l e s . 31 81.0 MHz P-NMR s p e c t r a at 25 C a r i s i n g from ( a ) s o n i -cated PE-PS v e s i c l e s ; (b) so n i c a t e d PE-PS v e s i c l e s to which 5 mM C a C l 2 was added. The b u f f e r e d sucrose med-ium c o n t a i n i n g 10 % D,0 was employed. 57. 2+ to act as adjuncts for Ca -stimulated release of granule contents, an independent and a more economical spectrophotometry assay was 31 performed as summarized in the methods section. The results of P-NMR experiments using the PE-PS model systems were confirmed by the spectrophotometric assay. Figure 15 illustrates the percentage of chromaffin granule contents released in the presence of varying amounts 2+ of exogenous PE-PS vesicles with subsequent addition of 5 mM Ca At a concentration corresponding to a model membrane phospholipid to chromaffin granule membrane phospholipid ratio, R, of 5 (mol/mol) 2+ there was complete release of chromaffin granule contents when Ca was present. This contrasted strongly with the results obtained when PE-PS vesicles alone were added as well as the situation when Ca alone was present, in which cases no release above the control level was observed. Also, no significant refease for calcium concentrations as high as 10 mM was observed. Therefore, these results clearly estab-2+ lish a requirement for both PE-PS vesicles and Ca as indicated by 31 P-NMR results. Furthermore, the amount of release was also sensitive to the order in which these agents were added to the chromaffin granules 2+ prior to incubation. In figure 15 when Ca was present prior to the addition of PE-PS vesicles, the release was significantly lower. This 2+ behaviour was interpreted as arising from Ca induced precipitation of the vesicles to form HJJ phase aggregates before the exogenous vesicles could interact with the chromaffin granules. 58. 1 2 3 A 5 6 R Figure 15. Release of chromaffin granule contents in the 2+ presence of Ca and exogenous Soya PE-PS (3:1) ves-i c l e s as assayed by spectrophotometrlc techniques: ( £ ) incubation in the presence of PE-PS vesicl e s (15 min.) where 5 mM CaC^ was added after 10 min. incubation; ( | ) incubation in the presence of PE-PS vesicle s where 5 mM CaC^ was introduced prior to -. v e s i c l e addition. R i s the molar r a t i o of exogenous l i p i d to chromaffin granule phospholipid. 59. The mechanism by which PE-PS vesicles act as adjuncts for Ca^'-stimulated release of chromaffin granule contents is of particular 2+ interest. It may imply that the release resulted from Ca induced fusion of the vesicles with the granule membrane followed by lysis as a result of the fusion event itself, or by the presence of non-bilayer lipid in the granule membrane which would no longer support the bilayer structure. As indicated by Tilcock and Cullis (29) the 2+ addition of Ca to PE-PS systems results in a structural segregation of the PS component into crystalline (presumably "cochleate") regions, allowing the PE to revert to the phase i t prefers in isolation. 2+ Questions then arise whether i t was the ability of Ca to induce cryst-alline cochleate structures or the hexagonal phase organization (or both) which was related to the lytic event. These questions were approached by testing the ability of vesicles composed of specific phospholipids which would preferentially adopt a bilayer or a non-bilayer conformation under the incubation conditions. The phospholipid systems utilized were egg PE-PS, pure PS, and pure cardiolipin (CL). 3.2.3 Egg PE-PS vesicles As mentioned previously, the bilayer to hexagonal H.^  transition temperature of pure egg PE was 25°C. Therefore the pure egg PE would adopt a bilayer conformation at 20°C and a hexagonal H I T conformation 1 2 3 A 5 R Figure 16. Release of chromaffin granule contents in the presence of soya and egg PE-PS (3:1) vesicles at 20*0 and 35°C: (• ) incub-ation in the presence of soya PE-PS vesicles at 20°C and 35°C with subsequent addition of 5 mM CaCl,,; incubation in the presence of egg PE-PS vesicles and 5 mM CaCl 0 at 20°C CO ) and at 35°C ( A ) . 61. at 35"C. The results obtained after incubating ;egg PE-PS (3:1) mixtures at 20*C and 35°C are shown in figure 16. It is evident that signifi-cant release of the chromaffin granule contents occurred when the egg o PE-PS vesicles were incubated at 35 C. As a control incubations with soya PE-PS vesicles resulted in significant release at both temperatures. This experiment strongly suggests a possible role for the occurrence 2+ of non-bilayer hexagonal H.^  phases in Ca -stimulated release of the chromaffin granules as one of the parameters that was influenced at the two temperatures was the bilayer to hexagonal transition. 3.2.4 Pure PS vesicles and pure CL vesicles Calcium induces formation of crystalline cochleate structures for PS dispersions (71,72) while the addition of calcium to cardiolipin model systems triggers formation of the hexagonal phase (26) . Since i t was of interest to ascertain which of the two phases promoted release of the chromaffin granule contents, pure PS and pure CL ves-2+ icles were tested as adjuncts for Ca -stimulated release. As shown 2+ in figure 17 both PS and CL vesicles were effective adjuncts for Ca stimulated release of chromaffin granule contents. These results there-fore did' not clearly distinguish the preferred conformations necessary for release. 3.2.5 PC-PS vesicles Phosphatidylcholine is a bilayer lipid and therefore does not Figure 17. Release of chromaffin granule contents in the presence 2+ of various exogenous lipid vesicles in the presence of Ca : ( O ) incubation in the presence of pure soya PS vesicles; ( • ) incubat-ion in the presence of pure CL vesicles; ( A. ) incubation in the presence of soya PC-PS (3:1) vesicles. R is the molar ratio of exogenous lipid to chromaffin granule phospholipid. 63. undergo bilayer to nonbilayer phase transitions in isolation or in 2+ the presence of calcium. Addition of Ca to PC-PS will only result 2+ in the interaction of Ca with the acidic PS molecules causing lateral phase separation of the two lipids into separate domains. This obser-vation has recently been observed by freeze-fracture studies of ^ Jacob-son and Papahajoupoulos (73). Therefore^ the interaction of PC-PS vesicles with chromaffin granules was investigated. The results in figure 17 indicate that no significant release of chromaffin granule contents resulted from incubation of PC-PS (3:1) vesicles with the chromaffin granules. Identical results were obtained when equimolar PC-PS (1:1) mixtures were employed. These observations imply that the occurrence of a hexagonal structure may be an tm-2+ portant factor in promoting Ca -stimulated release. 3.2.6 Erythrocyte Lipid Systems Finally, i t was of interest to extend the above observations to model systems which may approximate the composition of the inner mono-layer of the chromaffin cell plasma membrane more closely. One of the best characterized plasma membranes is that of the red blood ce l l . Since its asymmetry with respect to phospholipids, proteins, and carbohyd-rates has been extensively investigated, i t was a convienent model system for study. The polymorphic preferences of the inner and outer erythrocyte monolayers had also been recently investigated by Hope 1 64. Figure 18. Release of chromaffin granule contents after incubation with erythrocyte l i p i d v e s i c l e s : ( CD ) i n -cubation in the presence of outer monolayer l i p i d s (15 min.) where 5 mM CaC^ was introduced after 10 min.; ( O ) incubation in the presence of inner mono-layer l i p i d s (15 min.) where 5 mM CaC^ was introduced after 10 min. R i s the molar r a t i o of exogenous l i p i d to chromaffin granule phospholipid. 65. and Cullis (60). It was shown that the inner monolayer model systems 2+ partially adopted the hexagonal H.^  phase in the presence of Ca , whereas the outer monolayer systems did not. The ability of model systems composed of human erythrocyte phospho-lipids in proportions corresponding to the composition of the inner 2+ and outer monolayers to act as adjuncts for Ca -stimulated release of chromaffin granules was investigated. Figure 18 shows that the inner monolayer model systems acted as effective adjuncts for release of granule contents whereas the outer monolayer model systems did not. Larger ratios of exogenous (vesicular) inner monolayer phospholipid to endogenous (granule) phospholipid were required than for the other adjunct systems discussed before. This situation has been tentatively attributed to the instability of the sonicated inner monolayer systems, which tended to aggregate shortly after sonication, as indicated by an increasingly cloudy lipid dispersion. 3.2.7 Calcium Titration 2+ In a l l the above experiments, excess Ca (5 mM) was used so that the limiting component was the amount of phospholipid vesicles. When 2+ the amount of Ca was made limiting, it was found that atleast 2 mM 2+ Ca was required for causing effective release of the chromaffin gran-ule contents (see fig. 19) in the presence of excess PE-PS vesicles 2+ (R = 4 ). This valueucorresponded with the concentration of Ca req-66. Calcium Cone. (mM) Figure 19. Effect of calcium concentration on the release of chromaffin granule contents in the presence of excess Soya PE-PS (3:1) ve s i c l e s (R = 4) for 15 min. where CaCl 0 was added after 10 min. 67. uired to induce the formation of the hexagonal phase in analogous PE-PS liposomal systems (29). 68. Chapter IV: DISCUSSION 4.1 Models of Membrane Fusion A brief discussion of the models proposed for membrane fusion over the past decade would be useful for illustrating some of the concepts for the membrane fusion event and also for the interpret-ion of the results in this thesis. Four popular models have been proposed, namely, the lysolecithin model, the fluidity model, the crystallization model, and the polymorphic model. 4.1.1 Lysolecithin and Membrane Fusion Rubin (74), Guttler and Clausen (75) suggested the formation of lysophosphatides in promoting membrane fusion and experimental support was presented a few years later by Lucy and coworkers (76) when they fused hen erythrocytes ±n vitro with lysolecithin. They also obser-ved that the cells were very unstable and lysed within a period'of 30 seconds. Studies with a r t i f i c i a l lipid membranes have indicated that the insertion of a wedge-shaped molecule such as lysolecithin could promote substantial perturbation in the packing of the lipid molecules (77) . Also, due to the highly lytic properties of lyso-lecithin, its production during membrane fusion would need to be con-fined to highly localized sites so that the integrity of the rest of the membrane would be maintained. Cells have been found to possess 69. enzymes responsible for the conversion of lysolecithin to non-lytic derivatives (78) but the levels of enzymes reponsible to generate lysolecithin and its removal appear to vary significantly between different cells and even between different membranes of the same cell. It has also been proposed that the high concentrations of lysolecithin found in the chromaffin granules (79) and mast cell granules (80), both of which fuse with the plasma membrane, may be involved in the exocytosis process. Although i t is possible that membrane fusion occurring ^n vivo may involve bilayer to micellar transitions, no experimental support for this concept has been presented. Also, the fact that the turnover of lysolecithin in the adrenal medulla is not altered during secretion (81) argues against the participation of lysolecithin. Hence, the role of lysolecithin in mediating fusion is only supported by evidence of fusion under experimental conditions and similar fusion events have also been demonstrated by other lypophilic and lipolytic agents which create a similar type of disordering in the membrane. Such agents -include retinol, oleic acid, glycerol mono-oleate, propyleneglycol, and phospholipase C. Also, studies of virus-induced cell fusion have failed to provide evidence for the involvement of lysolecithin. There-fore, the role of lysolecithin in membrane fusion is as yet unclear. An important contribution of this hypothesis was that disordering of the fusing membrane seemed to be a common feature of the fusion 70. event. 4.1.2 Fluidity and Membrane Fusion The significance of redistribution of intramembranous particles (presumably representing integral proteins) during virus-induced fusion (82) and in other in vivo fusion events including that of the chroma-ffin granules (35,83) and mast cell granule (36) secretion has led to suggestions that the redistribution and aggregation of intramemb-ranous particles (IMP) in fusing membranes provide areas devoid of proteins (35,36). Therefore, fusion is restricted to areas of part-icle-free membranes and solely involves the interaction between the phospholipids of the two opposing membranes. This hypothesis was ex-tended by Ahkong et al. (84) by the suggestion that the redistribut-ion of the particles would increase membrane fluidity. But, no direct experimental evidence has been presented except from the studies of model membrane systems which indicate that prior to fusion, the phos-pholipids have to be in a fluid liquid-crystalline state (101). Fur-thermore, fusogenic compounds such as lysolecithin and myristic acid have been observed to decrease fluidity while dimethylsulfoxide (DMSO) has been shown to increase fluidity (85). Therefore, the exact role of fluidity in promoting membrane fusion is as yet unclear. 4.1.3 Role of Calcium Calcium has been found to be essential for fusion in a wide variety 71. of fusion events in both natural and model systems (102). In natural 2+ fusion systems, Ca has been demonstrated to induce processes such as exocytosis in the chromaffin cells (52), mast cells (86), and in other neurosecretory cells (87), by either introducing calcium directly into the cell or by calcium inophores A23187 and X537A. Also, the exocytotic process has been inhibited by blocking calcium entry into 2+ 2+ the cell with lanthanum, Mg , Co , and D-600 (2,88). In model systems composed of acidic phospholipids, fusion has been observed only in the presence of calcium. Although the exact mechanism by which calcium promotes fusion is unknown, the strong interaction between calcium and acidic phos-pholipids has led to two recent models of membrane fusion. 4.1.4 Crystallization Model of Fusion The interaction of calcium with phospholipids, particularily acidic phospholipids, has been extensively investigated by Papahad-jopouLos and coworkers (89,101). Calcium has been shown to affect 2+ the permeability properties, form two to one (PS/Ca ) complexes, cause crystallization of the acyl chains of acidic phospholipids and thereby raising their gel-liquid crystalline transition temper* ature, and causing phase separations in mixtures of acidic and neutral phospholipid model systems (PS/PC). On the basis of such observations 2+ Papahadjopoulos has proposed a dual role for Ga in mediating fusion. 72. Firstly, the presence of Ca^T is suggested to promote close apposi-tion of adjacent membranes by enchancing electrostatic interaction between them (90) and by forming a specific intermembrane complex(91). 2+ Secondly, Ca induces destabilization of the apposed membranes by formation of crystalline domains of acidic phospholipids which would represent sites at which fusion would occur (90). Support for such a mechanism of membrane fusion has been shown in the case of pure PS model systems but no evidence has thus far been presented for an ±a vivo situation or in Situations where bio-logical membranes have been employed. 4.1.5 Polymorphic Model of Fusion The observations that specific phospholipids in a biological membrane can adopt a non-bilayer structure in isolation (22) and 2+ that Ca can trigger such structures in mixed model membranes (29) has led to the proposal of a polymorphic model of membrane fusion. The main precept of such a model if that at some stage in the fusion event, whether i t is mediated by protein or lipid, a portion of the lipids must experience a departure from the bilayer structure. Studies with erythrocyte ghost membranes by Hope and Cullis (28), where incorporation of fusogens, oleic acid or glycerol mono-oleate, into the membranes at concentrations sufficient to induce cell fusion have been shown to produce a bilayer to hexagonal H T T phase trans-73. ition in some portion of the membrane phospholipids. Verkleij et al. (53) have also presented freeze-fracture evidence from model membrane systems showing the occurrance of intramembranous lipidic particles. It was suggested that these IMP (presumably inverted lipid ic micelles) were possible intermediaries •• in the bilayer to hexagonal H^ ^ transitions (95). Very recently, Hope and Cullis (28) have shown a positive correlation,between formation of a non-bilayer phase and the extent of cell fusion in human erythrocyte ghosts. On the basis of these observations, a molecular model of membrane fusion was proposed where the intermediate structure consisted of hexagonal Hjj. phase lipid cylinders or inverted micelles (28,30,98). The suggestion of inverted micelles in the fusion process has also been presented by other independent workers. For example, Lau and Chan (92), studying alamethacin induced fusion by proton NMR, Pinto da Silva (93), studying freeze-fracture results of the fusion of peripheral vesicles with plasmalemma of zoospores of the fungus, Phtophthora palmivora, and Gingell and Ginsberg (94), from a theore-tical standpoint, have a l l proposed similar intermediate structures. Although the polymorphic fusion model has been supported by experi-mental evidence from model membrane systems, i t has as yet been dif ficult to provide evidence from an ±a vivo situation due to limit-ations of available biochemical techniques. 74. 4.2 Chromaffin Granule Membrane 31 The P-NMR results obtained from the isolated granule membrane and model systems composed of the extracted lipid clearly established that the large majority (90 % or more) of the endogenous phospholipid experienced the bilayer phase at physiological temperature. This was consistent with the role of the lipid component which is primari-ly structural in nature, that is , serving to maintain granule integ-rity. It should be noted that while this behaviour appeared self--conistent and was similar to results obtained from other biological membranes, such as the erythrocyte ghost membranes (21,30), certain observations for organelle membranes indicate that such behaviour cannot be assumed a priori. In the case of rat liver endoplasmic reticulum, for example, two laboratories have independently reported the occurrance of isotropic motional averaging for endogenous phos-pholipids (96,97). The observation that calcium did not induce major changes in the motional behaviour of isolated granule membrane phos-pholipids was consistent with the observed inability of calcium to increase release of intact granule contents above background levels. 2+ 4.3 Phospholipids as Adjuncts for Ca -Stimulated Release  of Chromaffin Granules The major result, of this thes&s concerns the observation that lipid vesicles which undergo structural transformations in the presence 2+ 2+ of Ca could act as adjuncts for Ca -stimulated release of chromaffin 75. granule contents. It is reasonable to suppose that this ability 2+ is associated with Ca -induced fusion of the vesicles with the granule and that lysis resulted either during the fusion event or v: as a result of the presence of non-bilayer lipid in the granule membrane. In fact, it is difficult to imagine how the vesicles could produce such dramatic effects without interacting directly with the chromaffin granule membrane. The common property of the phospholipid vesicles exhibiting 2+ the lysis ability was that Ca induced aggregation and formation of larger structures for the vesicle system in isolation. In the 2+ case of the PE-PS system, Ca induces lateral phase separation of the PS molecules which results in the fusion between the vesicles aa3 formation of large hexagonal structures by the PE molecules. 2+ In contrast, addition of Ca to the EC-PS systems simply causes lateral phase separation but no fusion due to the retentionuof the bilayer configurations by both lipids. As for pure cardiolipin and 2+ phosphatidylserine systems, addition of Ca results in the formation of large structures of hexagonal cylinders and cochleate forma-tion respectively. The observation that both systems acted as eff-icient adjuncts for release suggested that:the detailed nature of these large structures was not a determining factor. Therefore, the major factor contributing to their ability to act as adjuncts was that both cardiolipin and phosphatidylserine in the presence of 2+ Ca become destabilized and must fuse between themselves or with any membrane that they might be interacting with. Hence, in the process of embedding themselves into the granule membrane, the integrity of the chromaffin granule membrane is disrupted and the granule contents released. 2+ The relationship between the above results and Ca -stimulated release of the chromaffin granule contents ±n vivo is not completely obvious. However, if the inner monolayer lipid composition of the chromaffin cell plasma membrane approximates that of the human eryth-rocyte plasma membrane, i t would imply that the presence of calcium would destabilize the inner monolayer in the sense that a sizable fraction of the phospholipids would prefer the hexagonal H^ phase. As has been indicated elsewhere (99) , formation of the H^ phase from previously bilayer systems appear to proceed as an inter-bilayer event. Thus, lipids in the outer monolayer of the chromaffin granule membranes closely apposed to destabilized regions of the inner mono-layer of the plasma membrane could serve to relieve this instability by forming local short cylinders (H^ -j. structures) or inverted micelles (lipidic particles) by combining with the inner monolayer phospholipids of the chromaffin cell plasma membrane. This type of reasoning seems consistent with the results obtained when the inner and outer erythrocyte model systems were employed as adjuncts. Release of the chromaffin granule contents was only observed in the presence of inner monolayer model systems which are unstable in the presence of calcium. 77. 4.4 Possible Implications for the Mechanism of Exocytosis From the results of this thesis, certain speculations can be made suggesting an _in vivo mechanism of exocytosis. The role of calcium in the fusion event could be f i r s t to aid close apposition between the chro-maffin granule and the plasma membrane, and second to induce lateral phase separation of the bilayer stabilizing phospholipids (particularily acidic phospholipids such as PS) which would result in local domains of non-bilayer lipids forming inter-bilayer lipidic micellar structures. A model depict-ing such a mechanism of exocytosis with the lipidic particle as the inter-mediary step is illustrated in figure 20. As mentioned previously, the inverted micelles have been implicated as 2+ intermediaties in fusion processes occurring in Ca - induced fusion of model lipid systems of PC-CL vesicles (99) and in the temperature induced fusion of PE-PC-cholesterol vesicles (95). The observation that fusion regions in vivo appear to be devoid of membrane particles as detected by freeze-fracture is potentially in disagreement with the intermediary role of lipidic particles in fusion in vitro (35,36). However, recent obser-vations on the mechanism of release from mast cells (100) suggest that intramembrane particles are present at or near the fusion site., 78. Figure 2 0 . Proposed mechanism of exocytotic release of chromaffin granule contents iri vivo. PM refers to the chromaffin c e l l plasma membrane and (XJ denotes the chromaffin granule. 79. BIBLIOGRAPHY 1. C e c c a r e l l i , B., Meldolesi, J., and Clementi, F. (1974) in Advances in Cytopharmacology. Vol. 2, ,Reven Press, N.Y. 2. Douglas, W.W. 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