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Studies on the roles of lipids in membrane structure and function Bally, Marcel B. 1984

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STUDIES ON THE ROLES OF LIPIDS IN MEMBRANE STRUCTURE AND FUNCTION  BY  MARCEL B. BALLY B.Sc, Texas A&M  University, 1977  M.Sc,  University, 1979  Texas A&M  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE  FACULTY OF GRADUATE STUDIES Department of Biochemistry  We accept this thesis as conforming to the required standard  THE  UNIVERSITY OF BRITISH COLUMBIA August 1984 ®Marcel B. Bally, 1984  In 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 of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  my  It is  understood t h a t copying or 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 gain  s h a l l not be allowed without my  permission.  Department of The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  )E-6  (3/81)  written  ABSTRACT  Lipids in membranes satisfy two basic roles. First they provide a matrix with which membrane proteins are associated and second, they provide a structural bilayer for maintaining a permeability barrier. This thesis investigates certain aspects of the structural and permeability barrier properties of lipids employing simple protein free model membrane systems. It is demonstrated, employing P and H NMR 31  formation  of  non-bilayer  structures  2  in  techniques, that cholesterol engendered  multilamellar  phosphatidylethanolamine (PE) and either phosphatidylserine Further  the presence  formation  of cholesterol, in conjunction  of non-bilayer  vesicles  (MLVs)  composed  of  (PS) or phosphatidylcholine (PC).  with  Mg , facilitated 2+  Ca  2+  triggered  organizations in the PS containing systems. It is indicated that in  these complex multicomponent systems where multiple structural phases (ie. bilayer, hexagonal, and "isotropic") coexist, that the phospholipids exhibit ideal mixing behaviour. A  basic consequence of the barrier properties of a lipid  bilayer is an ability to  maintain a membrane potential (Atp ), which is required for a variety of membrane mediated transport processes. To investigate the role of lipids in maintaining of  ATJJ  hip , and the direct effect  on transport functions, a large unilamellar vesicle (LUV) preparation free of impurities  is required. This thesis describes a novel procedure for generating  LUVs, by extrusion of  MLVs through polycarbonate filters (pore size 100 nm). LUVs can thus be obtained from a wide variety of lipid species and mixtures in the absence of lipid solubilizing agents. Vesicles exhibiting  AIJJ  in response to a NaVK  shown that such a K  +  ion gradient (K  +  +  inside) are characterized. It is  diffusion potential can drive the uptake of a variety of biologically  active molecules (eg. local anaesthetics, antineoplastic agents, biogenic amines (dopamine)) which have cationic and lipophilic characteristics. The transport process appears to proceed by a antiport cation/lipophilic cation exchange process that is driven by the transmembrane potential.  ii  TABLE OF CONTENTS  ABSTRACT  i  TABLE OF CONTENTS  iii  LIST OF TABLES  .'  vii  LIST OF FIGURES  viii  ABBREVIATIONS  x  ACKNOWLEDGEMENTS  xii  1 INTRODUCTION 1.1 Membrane Structure: A Historical Perspective  1  1.2 Rational for Investigating the Role of Lipids in Biological Membranes  4  1.3 Role of Lipids Within the Fluid Mosaic Model of Membranes  5  1.4 Model Membrane Systems  .'.  1.5 Chemical and Physical Properties of Phospholipids and Cholesterol  7 9  1.5.1 Phospholipids  10  1.5.2 Cholesterol  12  1.6 Lipid Polymorphism  14  1.6.1 Techniques for evaluating lipid polymorphism  15  1.6.2 Roles postulated for non-lamellar structures  19  1.7 The Shape Concept  21  1.8 Permeability Properties of Lipid Membranes  24  1.8.1 Passive permeability  24  1.8.2 Membrane potential  26  1.9 Summary  28  iii  2 LIPID POLYMORPHISM: INFLUENCE OF CHOLESTEROL AND DIVALENT CATIONS ON THE STRUCTURAL PREFERENCES OF MIXED LIPID MODEL SYSTEMS 2.1 Introduction  29  2.2 Materials and Methods 2.2.1 Preparation of soyabean lipids  30  2.2.2 Synthesis of dioleoyl phospholipids  33  2.2.3 Sample preparation  35  2.2.4 Nuclear magnetic resonance  36  2.2.5 Freeze fracture  36  2.3 Results 2.3.1 Influence of cholestrol on soya PE/PS lipid mixtures 2.3.2 Influence of cholesterol and Mg transition in PE-PS systems  +2  on the Ca  2+  induced bilayer to H  36 n  2.3.3 Influence of cholesterol on the mixing properties of DOPC/DOPE systems 2.4 Discussion  41 45 48  3 PRODUCTION OF LARGE UNILAMELLAR VESICLES BY A RAPID EXTRUSION PROCEDURE: CHARACTERIZATION OF SIZE DISTRIBUTION, TRAPPED VOLUME AND ABILITY TO MAINTAIN A MEMBRANE POTENTIAL 3.1 Introduction  54  3.2 Materials and Methods 3.2.1 Lipid preparation  56  3.2.2 Vesicle preparation  56  3.2.3 Determination of trapped volumes  56  3.2.4 Freeze fracture and negative staining  57  3.2.5 P Nuclear magnetic resonance 31  3.2.6 Membrane potential and permeability studies  58 58  3.3 Results 3.3.1 Characterization of LUVETs  iv  60  3.3.2 Characterization of LUVETs with a membrane potential 3.4 Discussion  68 76  4 UPTAKE OF SAFRANINE AND OTHER LIPOPHILIC CATIONS INTO MODEL MEMBRANE SYSTEMS IN RESPONSE TO A MEMBRANE POTENTIAL 4.1 Introduction  78  4.2 Materials and Methods 4.2.1 Materials  79  4.2.2 Vesicle preparation  80  4.2.3 Generation of a membrane potential  80  4.2.4 Uptake of safranine  82  4.2.5 Determination of membrane potential and K  efflux  +  4.2.6 Uptake of assorted drugs  82 82  4.3 Results 4.3.1 Characterization of safranine accumulation by LUVETs  83  4.3.2 Uptake of methyltriphenylphosphonium (MTPP )  92  4.3.3 Uptake of charged lipophilic drugs  94  +  4.4 Discussion  97  5 UPTAKE OF DOPAMINE AND OTHER BIOGENIC AMINES INTO LARGE UNILAMELLAR VESICLES IN RESPONSE TO A MEMBRANE POTENTIAL: ACTIVE TRANSPORT IN THE ABSENCE OF A CARRIER PROTEIN 5.1 Introduction  105  5.2 Materials and Methods 5.2.1 Materials  107  5.2.2 Preparation of vesicles  107  5.2.3 Determination of membrane potential  109  5.2.4 Uptake assays  110  v  5.3 Results 5.3.1 Accumulation of dopamine in response to a transmembrane electrochemical potential  Ill  5.3.2 Influence of external pH on the uptake of dopamine  115  5.3.3 Influence of trapped ATP on dopamine accumulation  117  5.3.4 Uptake of other biogenic amines 5.4 Discussion 6 SUMMARIZING  ..120 122  DISCUSSION  128  BIBLIOGRAPHY  131  vi  LIST OF  TABLES  1  Commonly occurring fatty acid moieties and phospholipid headgroups  11  2  Physical characteristics of vesicles produced by extrusion  66  vii  LIST OF FIGURES 1  The fluid mosaic model of membrane structure  2  General structure of a phospholipid  11  3  Structure of cholesterol  13  4 5  P-NMR, H-NMR, and freeze-fracture characteristics of phospholipids phases  31  31  7  31  9  2  in various 17  Polymorphic phases and corresponding dynamic molecular shapes  6  8  3  22  P and H NMR spectra of DOPE as a function of temperature  37  2  P-NMR spectra from dispersions of soya PE in the presence of varying amounts of soya PS and cholesterol 39 Freeze-fracture micrographs of soya and absence of equimolar cholesterol  PE-soya  PS  mixtures  in the presence 40  P-NMR spectra from dispersions of soya PS and soya PE in the presence of varying amounts of cholesterol and Ca 42  31  2+  10 P-NMR Mg 31  spectra from dispersions of soya PS and soya PE after dialysis against 43  2+  11 P-NMR spectra from dispersions of soya PS and soya PE after dialysis against Mg and Ca 31  2+  2+  44  12 P-NMR spectra from soya PS and soya PE mixtures in the presence and absence 31  of 1 M NaCl 13  31  46  P and H NMR spectra from of DOPE/DOPC mixtures and cholesterol 2  47  14 Influence of cholesterol on the quadrupolar splitting observed forH-DOPC 49 15 P-NMR signal intensity arising from egg PC vesicles prepared by extrusion of multilamellar vesicles 61 2  31  16 Micrographs of negatively stained egg PC vesicles  62  17 Freeze-fracture micrographs of vesicles of varying lipid composition  63  18 Size distribution of soya PC LUVETs  64  19 Trapping efficiency of LUVETs  68  20 Membrane potential in soya PC LUVETs in the absence of valinomycin  70  21 Membrane potential in soya PC LUVETs in the presence of valinomycin  72  viii  22 Comparison between K distribution  the membrane  potential  determined  by  [ H]-MTPP and 3  73  +  23 Comparison between the membrane potentials obtained for various transmembrane K chemical gradients as detected by [ H]-MTPP and the theoretical potentials 75 +  3  24 Structures of safranine, MTPP, and adriamycin 25 Spectrophotometric  chlorpromazine,  dibucaine,  propranolol, vinblastine, 81  response of safranine  84  26 Influence of increasing safranine concentrations on the safranine response  86  27 Levels of LUVET associated safranine as a function of transmembrane potential  87  16 Magnitude of the safranine response concentration and membrane potential  89  29 Time course for accumulation 30 K  +  as  a  function  of  internal  safranine  of safranine  90  release from LUVETs on addition of safranine  91  31 Influence of lipid composition on the safranine response 32 Accumulation LUVETs  of [ H]-MTPP 3  +  (initial  93  exterior concentration  of 20 x 10' M) by 8  95  33 Accumulation of MTPP  +  (initial exterior concentration of 2 mM) by LUVETs  34 Uptake of chlorpromazine  and dibucaine into LUVETs  96 98  35 Uptake of propranolol by LUVETs  99  36 Uptake of vinblastine and adriamycin into LUVETs  100  37 Model of safranine uptake  102  38 Structures of dopamine, epinephrine, and serotonin  108  39 Accumulation of dopamine by LUVETs experiencing a K  +  diffusion potential  112  40 Accumulation of dopamine by LUVETs experiencing a H  +  diffusion potential  114  41 Influence of external pH on dopamine uptake 42 Comparison between the membrane potentials obtained detected by [ H]-MTPP and the theoretical potentials 3  116 for various pH gradients as 118  43 Effect of trapped ATP on dopamine accumulation  119  44 Accumulation of epinephrine and serotonin by LUVETs  121  45 Postulated mechanism for the uptake of dopamine  124  ix  ABBREVIATIONS USED  A  A  Change in absorbance at 516 nm  J16  A Q  Quadrupolar splitting  Aip  Transmembrane electrochemical potential  ATP  Adenosine triphosphate  A1 0 2  Aluminium oxide  3  BLM  Black lipid membrane  CAPS  3-(cyclohexylamino) propanesulfonic acid  CSA  Chemical shift anisotropy  CCCP  Carbonyl cyanide m-chlorophenylhydrazone  DMSO  Dimethylsulfoxide  EPPS  N- (2- rTydroxyethyl)piperazine- N'- 3- propanesulfonic acid  ESR  Electron spin resonance  GPC  Glyceryl phosphorylcholine  H„  hexagonal  HCIO,  Perchloric acid  HEPES  [4-(2-Hydroxyethyl)]-piperazine ethanesulfonic acid  KGlu  Potassium glutamate  LUVs  Large unilamellar vesicles  LUVETs  Large unilamellar vesicles by extrusion techniques  MES  2-(N-Morpholino)ethanesulfonic  MLVs  Multilamellar vesicles  M0O3  Molybdenum trioxide  MTPP  +  Methyltriphenylphosphonium  NaHSO*  Sodium bisulfite  Na S0  Sodium sulfite  2  NMR  4  Nuclear magnetic resonance  x  acid  Phospholipids 1,2- dioleoyl- sn- glycero- 3- phosphorylcholine  DOPC [ C - H ] DOPC  1.2-(11,11- dideuteriodioleoyl)-5«-glycero- 3-phosphorylcholine  DOPE  1,2- dioleoyl- sn- glycero- 3- phosphorylethanolamine  2  n  2  [ C - H ] DOPE  1,2—(11,11— dideuteriodioleoyl)-s«- glycero- 3-phosphorylethanolamine  E (egg)  Lipids derived from hen egg yolk  PA  Phosphatidic acid  PC  Phosphatidylcholine  2  n  2  PE  Phosphatidylethanolamine  PG  Phosphatidylglycerol  PI  Phosphatidylinositol  PS  Phosphatidylserine  S (soya)  Lipids derived from soya bean extract  psi  Pounds per square inch  SUVs  Small (sonicated) unilamellar vesicles  Tc  Gel to liquid-crystalline transition temperature  TLC  Thin layer chromatography  xi  ACKNOWLEDGEMENTS  Acknowledgement sections are sometimes treated in a lighthearted fashion and therefore maybe taken in a lighthearted manner. For this reason I want to stress the point, clearly and in a manner that will be taken seriously, that this thesis was  by no means an individual  effort The ideas, concepts, and technical expertise from each member of the lab were vital to the completion of this work. Therefore I want to do more than simply acknowledge these individuals for they are all co-authors of this manuscript. These individuals are: Pieter Cullis, Mick  Hope, Colin Tilcock, Tom  Madden, Lawrence Mayer, Cees van Echteld,  Rajiv Nayer, Blake Farren, Helen Loughley, Tom especially like to thank Pieter and Mick, who  Redelmeier, and Eric Sommerman. 1 would  created an environment which made scientific  research a pure joy. Your enthusiasm is something I will always admire. Also, thanks for not making me  Finish the Sendi virus project.  This work was Columbia, Mom  supported financially  by  fellowships from  the University of British  and Dad, and the Forestry Department (You'll have to think about that one).  Finally, I would like to thank my  brothers, Alain and Andy, for not dropping  me  from that second, story hotel window fifteen years ago, I knew if I was given a chance I could accomplish something worthwhile.  xii  To be is to do (Socrates) To do is to be (Jean-Paul Sartre) Do be do be do (Frank Sinatra) and To Christina and Jlonka  xiii  INTRODUCTION  1.1 Membrane Structure: A Historical Perspective The  cytoplasm  of a cell is physically separated from the surrounding environment by  means of a cell membrane. The fact that the cytoplasm is chemically quite different from the external milieu yet can aquire necessary  metabolites from the surroundings  illustrates  prima  facie the role of the membrane in providing a selectively permeable barrier. However, beyond this basic role of separation, biological membranes participate in many complex phenomena ranging  from  roles involving inter-  and intra-cellular  communication  to mediating  cellular  immune responses. Biological membranes are composed primarily of lipids and proteins, with  a small  amount of associated oligosaccharides. To date, models defining the organization of these components in membranes have been based on three basic observations derived from studies which have spanned the last 50 years. The first observation, based on the initial investigations of Gorter and Grendel (1925) and developed  later by Danielli and Davson (1935), was that  the lipid component of membranes was arranged familiar  structure is represented  by  in a bimolecular leaflet organization. This  the phospholipid  bilayer,  which  accommodates the  amphipathic characteristics of the lipids such that the polar headgroups are in direct contact with the aqueous environment while the fatty acid chains are buried in the hydrophobic core of the membrane. The  second observation(s) concerned the location of the protein component in relation  to the lipid bilayer model postulated by Danielli and Davson. Formulation  of these models  relied on information derived from a number of techniques which had been developed for determining  protein structure (x-ray  crystalography, optical  rotary  dispersion, and circular  dichroism) and for visualizing membranes (electron microscopy and freeze fracture technology). Robertson (1957) proposed the unit membrane model, which summarized data derived mostly  1  from x-ray diffraction and  electron microscopic studies of the mylein  sheath, that suggested  that proteins, in extended 8 -form, were spread out as monolayers coating the lipid bilayer. It was  later demonstrated that a substantial fraction of membrane associated protein was  a -helical conformation and  in an  appeared to be maintained within the lipid bilayer as globular  structures. Characterization of these integral  membrane proteins (in contrast to peripheral  membrane proteins which are loosely associated with the surface of membranes) resulted in the  postulation  of  a  variety of  models  for  membrane  structure  suggesting  hydrophobic  protein-lipid interactions where protein molecules were actually intercalated within the lipid bilayer  (for reviews  see  Vanderkooi  and  Green,  1970;  Hendler, 1971;  Stoeckenius  and  Engelman, 1969; Korn, 1966; Bretscher, 1973). The  third observation  was  that the components of a membrane existed in a fluid,  mobile, state. Utilizing the technique of differential scanning calorimetry it was the temperature was undergo  an  demonstrated as  increased through a particular value aqueous dispersions of phospholipids  exothermic  transition  (the  gel  to  liquid-crystalline  phase  transition) which  corresponded to the transition of the acyl chains from a rigid, immobile, state to a fluid state. At physiological temperatures membrane lipids were found to be generally in a fluid state (Chapman, 1966; of lipids and  Steim et al., 1969;  Melchoir, 1970). In addition, the lateral movement  proteins in biological membranes had  been  demonstrated  techniques. This is exemplified by the classic experiment of Frye and  by  a  Edidin (1970) which  demonstrated, using immunospecific labeling, that the surface components of two intermixed  after the  cells were fused. This  spreading  variety of  of components was  cell types  shown to  be  dependent on the external temperature. These observations have been successfully accommodated in the currently accepted "fluid mosaic" model for the structure of biological membranes, which was Nicolson  in  1972.  Briefly,  considerations concerning  the  model  membranes and  (see  Fig. 1)  was  proposed by Singer and  derived  from  thermodynamic  associated components. Hydrophobic and hydrophilic  interactions were maximized so that the lowest free energy state for the functional membrane could  be  attained (for reviews see  Singer, 1977;  2  Tanford,  1980). This  required  that the  Fig.  1. The fluid mosaic model of membrane structure. In this model, developed by Singer and Nicolson (1972), membrane  proteins are shown to be interspersed within regions of a predominantly fluid phospholipid bilayer, forming a mosaic pattern.  Plasma membrane  Cytosol  nonpolar components of membranes, such as the fatty acid chains of the phospholipids  and  the nonpolar amino acid residues of proteins, were in minimum contact with the aqueous environment while  the polar groups, polar amino acids, headgroups of phospholipids,  associated carbohydrate integral  membrane  moieties, were in maximum contact with the aqueous environment  proteins were  depicted  as  being  embedded  in a  phospholipid  and The  matrix,  providing a mosaic structure in which integral proteins were interspersed within sections of membrane bilayer. Further, the model stressed that both the protein and  lipid components  were capable of lateral movement within the membrane. 1.2 Rationale for Investigating the Role of Lipids in Biological Membranes Beyond  the  fundamental  view  of membrane  structure as modeled  by  Nicolson, it is necessary to explain the great diversity in chemical composition membrane. Any  particular biological membrane may  different polar head groups and  Singer  and  of a biological  contain a variety of phospholipids (having  hydrocarbon chains), varying amounts of sterols, and a host  of differing proteins. In addition, within a given membrane these different components are arranged asymmetrically such that the inner surface of a membrane is compositionally different from the outer surface (for reviews see van Deenen, 1981; Etemadi, 1980a; Etemadi, 1980b). This  compositional  diversity  would, at  least  in part, be  determined  by  the functional  requirements of the membrane. In this context, it has been firmly established that the major functional role of lipids in biological membranes involves the formation and maintainence of the  bilayer  structure. This  has  been  demonstrated  through  observations  that phospholipid  preparations normally spontaneously adopt the bilayer structure on hydration (Dervichian, 1964; Bangham  et al:,  (Finean  and  1965)  and -that biological membranes contain regions of bilayer structure  Robertson,  phosphatidylcholine  could  1958). provide  However, the  fluid  a bilayer  single  phospholipid  species  such  as  structure of membranes. Since several  hundred lipid species can be present in a typical mammalian cell membrane, other functional roles for lipids must exist This aspect of lipid diversity, as it relates to membrane structure and function, provides the major focus of this thesis. Specifically, model systems composed of  4  single and mixed lipid components have been utilized to identify potential roles for lipids in biological membranes. The  objectives of this introduction will be to (i) discuss the functional roles of lipids  in terms of the fluid mosaic model of membranes, (ii) discuss the systems utilized to model the structural and functional features of membranes, (iii) review some of the chemical and physical properties of phospholipids and cholesterol, (iv) describe the structural preferences of lipids, introducing the concept of lipid polymorphism and defining the roles which have been postulated permeability  for non-lamellar  structures, (v) discuss  barrier, and (vi) discuss  the concept  the roles that  of lipids  different  lipids  in providing  a  assume different  molecular shapes. Throughout these discussions the scope of this thesis as it relates to each area will be emphasized.  1.3 Role of Lipids Within the Fluid Mosaic Model of Membranes In  general  terms, the fluid  mosaic model  suggested  that the lateral  mobility of  membrane proteins was essential for proper membrane function and was related to a variety of membrane mediated cellular phenomena, including malignant cell transformation (the original example illustrated by Singer and Nicolson; 1972), fertilization (Johnson and Edidin, 1978), cell growth (Collard et al. , 1977), and differentiation (Kawasaki et al., 1978). One rationale for lipid  diversity  was therefore  established on the basis that membrane  function could be  regulated by changes in fluidity within local regions of the membrane. Indeed, changes in lipid microviscosity had been shown to influence the functions of integral membrane proteins with specific carrier, receptor, and enzymatic functions (Warren et al., 1974a; Warren et al., 1974b; Jacobs and Cuatrecasas, 1977; Kimelberg, 1977; Chapman and Correll, 1977). Since it was demonstrated  that the gel to liquid-crystalline phase transition  sensitive to acyl chain  length  and degree of unsaturation  of phospholipids  and the headgroup  was  composition  (Chapman, 1968), as well as the presence of cholesterol (Ladbrook et al., 1968; Hubbel and McConnel, 1971) and integral membrane proteins (Cornell et al., 1978; Silvius, 1982), it was proposed that in vivo regulation of membrane proteins could be "fine tuned" by a variety of  5  factors that are mediated by specific lipids such as cholesterol. Rationalizing lipid diversity in terms of membrane fluidity presents certain difficulties when one considers that gel state lipids do not appear to be present  in most eukaryotic  systems. The unsaturated nature of naturally occurring lipids results in gel to liquid-crystalline phase  transitions well  below  physiological temperatures, as shown  for the lipids of the  erythrocyte (van Dijck, 1976). In addition, several studies demonstrated that incorporation of integral  membrane  proteins  usually  reduces  the  hydrocarbon  transition  temperature  (Papahadjopoulos et al., 1975b; Houslay et al., 1975) and indicated that integral membrane proteins were excluded from regions of gel state lipid (Grant and McConnel, 1974; Kleeman and McConnel, 1976). Presumably these data indicate that gel state lipids are not accessible to membrane proteins and therefore  would not be able to regulate protein function even if  present in the membrane. A  second rationale for lipid diversity was established from data which indicated that  membrane bound  enzymes required  a well define  lipid  annulus for optimal  function (for  review see Devaux and Seigneuret, 1984). The evidence however does not provide a convincing argument for the presence of a defined lipid annulus. It has been demonstrated that a variety of enzymes, such as cytochrome oxidase (Vik and Capaldi, 1977), (Na , K )-ATPase (Hilden +  and  +  Hokin, 1976), C55-isopreniod alcohol phospholinase (Gennis and Strominger, 1976), and  (Ca \ Mg )-ATPase (Hesketh et al., 1976), function well when reconstituted in model systems 2  2+  which contain any one of a variety of lipids. This point is further emphasized by experiments which demonstrated that (Na , K )-ATPase and sarcoplasmic +  +  reticulum ATPase could function  well in a pure detergent environment (Dean and Tanford, 1977). In addition, conclusions derived from ESR (electron spin resonance) data suggesting the presence of "boundary lipid" appear to be only valid on the ESR time scale of 10" -10" 8  motions requiring longer  times to occur). Deuterium  10  sec (ESR is not sensitive to  ( H) NMR 2  (time  scale of 10" sec) 6  studies have not been able to distinguish between the boundary lipid and the bulk lipid phase suggesting  that lipids in the annulus exchange with bulk lipid at a rate of 10" -10" 6  sec. These points are illustrated by studies on cytochrome oxidase and sarcoplasmic  6  7  reticulum  ATPase which show a two  component ESR  component (Hesketh et al., 1976;  spectra indicative of a "mobile" and  Marsh, 1978)  "immobile"  while H-NMR studies showed the existence 2  of only one mobile component (Oldfield et al., 1978; Oldfield et al., 1979).  Related lipid-lipid  to  the  interactions may  previous  discussion,  several  result in the formation  investigations  of compositionally  suggested  that  specific  distinct lipid domains  (for review see Oldfield and Chapman, 1972) that would be functional significant in biological membranes. For example, investigations which demonstrated that Ca , an agent which mediates 2+  in  vivo fusion events, induced the phase segregation  systems (Papahadjopoulos, 1978;  Dluhy  et al., 1983)  of phosphatidylserine  in mixed lipid  supported the concept that fusion of  membranes would require phase separation of phospholipid  components, providing a site for  fusion to occur at the phase boundaries. Data presented in this thesis (see Chapter 2) and elsewhere (Tilcock et al., 1984), however, indicate that in complex multicomponent membrane systems such as the biological membrane that individual lipid species have no  tendency to  segregate into functionally or structurally distinct domains. 1.4 Model Membrane Systems To investigate the basic structural and physical characteristics of biological membranes it is useful to isolate individual components and attempt to model the more complex system by incorporating these components in simple well-defined  systems. Indeed much of the current  concept of membrane structure and function has been derived from investigations which utilize model membrane systems composed of naturally occurring or synthetic lipid species in the presence or absence of an isolated membrane protein. A  discussion of protein reconstitution in  model membrane systems is beyond the scope of this thesis, for those interested there are several reviews of this subject (see Racker, 1979; Eytan, 1982). Hydration which  are  of a dry phospholipid film results in the spontaneous formation of structures  composed  of  numerous  closed  concentric  lipid  bilayers separated  by  aqueous  compartments (Bangham et al., 1965). These multilamellar vesicles (MLVs) are perhaps the  7  most frequently utilized preparation for defining the properties of lipids. This is due in part to the ease of preparation, which involves quite literally addition of a buffer to a dried lipid film followed by a good shake to generate and disperse the large (radius greater than 200 nm) lipid aggregates. Many of the biophysical approaches used for determining the motional and  structural properties of lipids, including the diffraction, spectroscopic, and calorimetric  techniques, utilize MLVs as model systems (for example see Chapter 2). In addition, MLVs have been used to characterize a variety of membrane associated functions, ranging from monovalent and divalent cation permeability to protein-lipid interactions. The  data derived from these investigations are often ambiguous due to the size heterogeneity  and  the numerous  aqueous  compartments  present,  hence  different model  systems were  developed to circumvent these problems. These included procedures for generating unilamellar vesicles (for reviews see Bangham et al., 1974; Pagano and Weinstein, 1978; Szoka and Papahadjopoulos, 1980) and the black lipid membrane (BLM; for review see Fettiplace et al., 1974). The latter preparation is a mechanically lipid-solvent  solution  across  a  hole  in a  supported lipid bilayer produced by spreading a plastic  sheet  which  separates  two aqueous  compartments. This model system has been particularly useful in characterizing the permeability properties of lipids in a bilayer configuration. However, the results from experiments employing BLMs are difficult to interpret since these preparations always contain a certain amount of residual solvent which has been shown to influence the bilayer thickness (Benz et al., 1975) as well as the transport properties of these membranes (Benz et al., 1977). The  applicability of unilamellar vesicles in defining permeability and transport functions  is obvious, however the majority of techniques which have been developed to produce these vesicles also have shortcomings (see Szoka and Papahadjopoulos, 1980). In the case of small (sonicated) unilamellar vesicles, which have a diameter less then 30 nm, artifacts resulting from the high degree of membrane curvature (the inner to outer monolayer phospholipid ratio is typically 1:2) resulting in packing restraints. Other preparations  which produce large (greater  than 100 nm diameter) unilamellar vesicles (LUVs) utilize organic solvents or detergents that are  difficult  to completely  remove  subsequently. For this reason  8  a novel  technique for  producing large unilamellar vesicles (LUVs) has been developed and Chapter 3  of this thesis. Chapter  3 provides an  therefore  further discussion concerning the  provided  here. It is sufficient  to  adequate review of pertinent literature,  utilization and  state that  is described in detail in  properties of LUVs will not  these vesicles provide  an  ideal model for  characterizing ion permeability (see Chapter 3) and transport properties (see Chapters 4 and in systems of defined lipid mixtures and, in addition, may  be  5)  provide a useful model vesicle  system for the reconstitution of membrane proteins (Madden et al., 1984).  1.5  Chemical and  The in  a  Physical Properties of Phospholipids and  fundamental question as to why  biological membrane  still  Cholesterol  there is such a heterogeneous population of lipids  remains.  Clearly  differences  fundamental roles in membrane structure as well as the activity and  membrane mediated events such as fusion. An  and the properties of these lipids in isolation and  in  regulation  lipid  composition  play  of membrane protein  indication of the lipid diversity  in mixtures will be presented. The variety  of lipid species present in a typical eukaryotic membrane include phospholipids and cholesterol, as well as an assortment of other species including glycolipids, neutral lipids, and  free fatty  acids. In addition there are variations in the headgroup composition, represented by different phospholipids  and  gangliosides (glycolipids), in the fatty acyl chain composition of individual  lipid species, and  the number of chains present (e.g. the lysophospholipids have a single acyl  chain). Clearly it would be inappropriate to review the chemical and physical properties of all lipid  species, much of which is detailed in a variety of texts (for excellent reviews see  Zubay, 1983). The  major lipids employed  in this thesis are phospholipids  and  cholesterol,  therefore the more general aspects of these lipids will be summarized here. The  ability of  different lipids to adopt other structural phases in addition to the bilayer configuration, defined as lipid polymorphism, is an  important physical parameter of lipids that will be  separately (see section 1.6).  9  discussed  1.5.1  Phospholipids  In general, most lipid species exhibit amphipathic characteristics. This is represented by the most abundant lipid class, the phospholipids, which contain a strongly hydrophilic head group and  one, or more commonly two, acyl chains which can be 12 to 24 carbons long  with varying degrees of unsaturation. The major classes of phospholipids are shown in Fig. 2, and the more common variations in acyl chains are given in Table 1. The are esterified to glycerophosphate,  forming phosphatide acid (PA), which can condense with a  base to form phosphatidylcholine (PC), phosphatidylethanolamine phosphatidylinositol  fatty acid chains  (PI), or phosphatidylglycerol (PG).  occurring lipids commonly contain an  The  (PE), phosphatidylserine (PS), fatty  acid chains  even number of carbons, with an  of naturally  unsaturated  chain  normally confined to the sril position of the glycerol backbone and a saturated chain, often palmitic acid (16:0), in the snl position. The physical properties of phospholipids are influenced largely by headgroup interactions and acyl chain mobility (for reviews see Chapman, 1975; de  Kruijff, 1979;  Lee, 1975; Seelig, 1978; Cullis and  Boggs, 1980). Some of these properties have been characterized by  the  temperature at which the gel to liquid-crystalline phase transition occurs (Tc). For example, bilayers composed of a synthetic PC, which contain long saturated acyl chains have a higher gel  to liquid-crystalline phase transition temperature compared to PC  species with shorter  saturated acyl chains. The presence of a double bond in the acyl chains (normally cis) results in  a marked  decrease in the Tc  (Chapman, 1975). The  phospholipids are also influenced by headgroup composition difference of 22° C and  dioleoyl PE  thermotropic  phase properties of  (Cameron et al., 1981), where a  has been observed in the Tc of' dioleoyl (18:lc/18:lc) PC  (Tc=  0°C). Several lipid species, including PA,  negatively charged headgroup at physiological pH  and  PS, PG,  and  (Tc=-22°C) PI, have a  the repulsive effects between these  headgroups results in a decrease in the Tc (van Dijck et al., 1978). Clearly the charge of a lipid is not the only factor influencing the Tc. PA, PG, and PS all bear a negative charge at neutral pH  yet the Tc of PA  can be as much as 25° C  10  higher than either PG  or PS  Fig. 2. General structure of a phospholipid showing the more common headgroups.  Table 1 Commonly occurring fatty acid moieties.  Common name  Structure Saturated fatty acids CH (CH ) COOH 3  2  Laurie  10  CH (CH ) COOH  Myristic  CH,(CH ) COOH  Palmitic  CH (CHj) COOH  Stearic  3  2  12  s  M  3  16  CH (CH )„C00H  Arachidic  CH (CH.,) COOH  Lignoceric  3  2  3  22  Unsaturated fatty acids CH (CH ) CH = C H ( C H ) C 0 0 H 3  2  s  2  Palmitoleic  7  CH (CH ) CH = CH(CH ) COOH 3  2  7  2  Oleic  7  CH (CHJ) CH=CHCHJCH=CH(CH,) COOH 4  3  Linoleic  I  C H 3 C H j C H = C H C H , C H = C H C H C H = CH(CH ) COOH  Linolenic  C H ( C H ) ( C H = C H C H ) C H = CH{CH )3C00H  Arachidonic  2  ;]  2  J  2  2  3  2  11  7  with identical acyl chain composition (van Dijck, 1978). In addition to the effects on thermal properties of lipids, the presence of ionizable groups allows for a number of physiologically relevant factors, such as pH, ionic strength, and the presence of divalent cations, to isothermally modulate the behaviour of lipids. The ability of divalent cations such as Ca  2+  to induce phase segregation  of lipids in model membrane  systems has already been discussed. Several investigators have also shown that changes in pH can exert profound effects on the structure and properties of PS containing membranes (Harlos et al., 1979; Hope and Cullis, 1980; Tilcock and Cullis, 1981). 1.5.2 Cholesterol >  The  major neutral lipid component in the plasma membrane of eukaryotic organisms is  cholesterol. The structure  of cholesterol (Fig. 3) bears no resemblance  to phospholipids,  however this steroid ring does possess amphiphathic characteristics due to the presence of the 3 3-hydroxyl group at one end of the molecule. Cholesterol orients itself in a phospholipid bilayer  such  that  this hydroxyl  group  is adjacent  to the fatty  acyl  carbonyls  of the  phospholipid (Huang, 1977), while the rigid steroid nucleus is associated with the acyl chains. A consequence of this association is a reduction in the number of gauche confirmations which occur in the region of the first ten carbons of the acyl chains (Kawato et al., 1978; Ranee et  al., 1982), a point  illustrated  by pressure-area  cholesterol exerts a condensing effect (represented  monolayer curves which  indicate that  by a decrease in the area occupied per  lipid molecule) in a PC monolayer. The presence of cholesterol in PC bilayers results in a decrease in the enthalopy change associated with the transition (Ladbroke et al., 1968). This effect has been attributed to the ability of cholesterol to increase lipid order above the phase transition while decreaseing the order below the transition. On the basis of these data it was suggested that the major role of cholesterol in membranes was to control membrane fluidity. As stated previously, the role of fluidity in the regulation of membrane function is doubtful.  12  Fig. 3. Structure of cholesterol.  13  It has been well established that cholesterol is an essential structural requirement for many biological membranes and influences a variety of membrane mediated functions from the passive permeability of ions to the regulation of enzymatic activities (for reviews see Demel and  de Kruijff, 1976; Block, 1981). The influence that cholesterol has on the behaviouT of  lipids (and proteins) in membranes is still the subject of intense investigations and forms the basis of much  of this thesis. In Chapter  2 the role of cholesterol in modulating the  polymorphic phase behaviour (see section 1.6) of mixed lipid systems is examined. In addition, Chapter 4 presents a preliminary study investigating the effects of cholesterol on membrane mediated transport processes. 1.6 Lipid Polymorphism  As discussed previously, many phospholipids  spontaneously adopt a bilayer organization  upon hydration. In addition to this configuration it has been demonstrated that a variety of lipids can adopt different structural phases, such as the hexagonal (H ) phase characterized by n  cylinders of lipids in an "inverted" (headgroups oriented inwardly  towards aqueous channels)  organization (see Fig. 4). This lipid polymorphism has been recognized  for more than 20  years, when  diffraction, initially  Luzzatti and co-workers, using  documented the occurrence of non-lamellar hydration  the technique  of X-ray  lipid structures depending on the state of lipid  and temperature (Luzzatti and Husson, 1962; Luzzatti et al., 1968; Luzzatti and  Tardieu, 1974). However, the significance of these alternate phases in relation to membrane structure and function was not realized until it was shown that physiologically relevant factors (pH, ionic strength, acyl chain composition and the presence of divalent and/or other lipid species) could isothermally influence the structural preference of naturally occurring lipids (for reviews see Cullis and de Kruijff, 1979; Cullis et al, 1983; de Kruijff et al., 1984). These non-bilayer  phases almost certainly play important roles in defining membrane structure and  function. For this reason, the role of lipids as related to their structural preference will be a central theme for this dissertation. Chapter 2 examines some factors which formation of non-lamellar  regulate the  structures, while the remaining chapters examine the role of bilayer  14  forming  lipids in regulating ion permeability and membrane mediated  transport process. It  would be advantageous at this stage to briefly review the experimental techniques utilized to characterize the macroscopic structures adopted by hydrated model lipid systems and to discuss the roles which have been postulated for these alternate phases.  1.6.1 Techniques  A  for evaluating  lipid  polymorphism  diverse set of techniques have been utilized to investigate the polymorphic phase  behaviour of lipids, including X-ray and neutron diffraction (Luzzatti, 1968; Buldt et al., 1979; et al., 1979; Blaurock, 1982), freeze-fracture  Zaccai  differential  scanning  electron  microscopy  calorimetry (Cullis and de Kruijff, 1978b), and H 2  (Verkliej, 1984), and P nuclear 3I  magnetic resonance (NMR) spectroscopy (Davis, 1983; Seelig, 1977; Seelig, 1978; Cullis and de Kruijff, 1979). NMR  and freeze-fracture techniques were employed in studies presented in this  thesis, therefore the discussion will be confined to these procedures. It should be emphasized, however,  that  diffraction  techniques  are the only  information on the structures adoped by hydrated  procedures  that  provide  unequivocal  lipids. As discussed by Cullis and de  Kruijff (1979), the other techniques are extrapolative in nature, relying on previous X-ray diffraction data. Currently, there ' is a great deal of evidence which demonstrates structural assignments  based  on NMR  data are in agreement with X-ray  that the  diffraction data  (Marsh and Seddon, 1982; Seddon et al., 1983; Tilcock et al., 1984). Nuclear  magnetic  resonance  spectroscopy.  Nuclei such  as P 31  exhibit  a quantum  mechanical quantity known as "spin". For P the spin quantum numbers are +1/2 or -1/2. 31  This imparts to the nuclei characteristics of a small bar magnet when placed in a strong magnetic  field. In particular, nuclei with spin 1/2 can be considered as magnets which are  oriented such that the "north" pole is oriented towards the north pole of the magnet giving rise to the strong magnetic field, whereas for nuclei with spin -1/2 the situation is reversed. Clearly the nuclei with spin -1/2 are then in a "lower energy" situation, and an energy difference then exists between the spin -1/2 and 1/2 states when a strong magnetic field is  15  present  The NMR  experiment involves exciting transitions between these two states (which  requires radiofrequency irradiation) and monitoring the energy absorbed. This gives rise to a resonance at a particular frequency, which is usually characterized in terms of a parameter known as the "chemical shift". This chemical shift is calculated as the frequency separation between the resonance of interest and some standard, divided by the irradiation  frequency  itself. As the irradiation frequency is usually in the MHz range, this chemical shift parameter is usually multiplied by a factor of 10 and expressed as parts per million (ppm). 6  The  magnetic field experienced  phosphodiester  bond found  by the P n  (phosphorus) nuclei associated with the  in most phospholipids is partially shielded by the surrounding  electron cloud. This shielding will differ according to the orientation of the phosphate segment in the magnetic field. As a result a P nuclei will resonate at different frequencies according 31  to the orientation, resulting in a large "chemical shift anisotropy" (CSA) broad  31  P-NMR  and a characteristic  spectrum for non-oriented, dried phospholipid samples. When phospholipids  are hydrated, the motion available to the phospholipid in a bilayer organization influences the 31  P-NMR lineshape. For large (radius greater than 200 nm) liquid-crystalline bilayers (such as  MLVs) this motion is restricted to rapid axial rotation of the lipid. For a non-oriented sample this gives rise to a broad lineshape with a low field shoulder and a high field peak separated by an effective CSA of -40 to -50 ppm (see Fig. 4). Most of the phospholipids examined exhibit similar values of CSA, providing essentially equivalent spectra for these lipids in a liquid-crystalline bilayer organization. This is a useful characteristic if more complex lipid mixtures  (such as a biological membrane) are examined, since all the signals arising from  phospholipids in a bilayer organization combine to produce a composite bilayer lineshape. In situations where lipids can experience other forms of rapid motion, in addition to rotation about the long axis, additional narrowing of the P NMR 31  spectrum is observed. This  is the case for small vesicles, where vesicle tumbling and rapid lateral diffusion of the lipids around  the vesicle  allow  isotropic  motional  averaging  over  all possible orientations.  The  resulting P NMR spectrum is characterized by a narrow lineshape as illustrated in Fig. 4. A 31  variety of other phases, such as the inverted micellar configuration, cubic, or rhombic phases,  16  Fig.  4. " P - N M R ,  2  H-NMR,  and freeze-fracture  characteristics  of phospholipids  in various phases.  kHz  allow isotropic motion (see Fig. 4), hence it is difficult to assign a lipid phase on the basis of this narrow P  NMR  31  techniques such  as  spectrum and conclusions must be corroborated  freeze-fracture, or  X-ray  diffraction.  Lipids  by data from other  in the  H  n  phase also  experience additional motional averaging in comparison to lipids in a large bilayer configuration due  to diffusion of the lipids around the cylinder structures. The  are narrower by a factor of two, with an effective CSA  resulting P 31  of -20  ppm,  NMR  spectra  and have a reversed  asymmetry. The  previously mentioned advantage of P  NMR,  3l  that all P  signals combine to give  3l  rise to a composite spectra reflecting the overall organization of the phospholipids, may be considered times be  a disadvantage. In systems composed of two or more lipid species it would at  useful to determine the phase properties of each individual component (for an  example see Chapter 2). H 2  NMR  provides an excellent technique for determining the phase  properties of a specifically deuterated lipid. The H 2  and  also  nucleus has spin quantum numbers of  -1, which give rise to a quadrupole moment The  2  H  NMR  spectra derived  +1  from a  partially deuterated lipid in a large bilayer organization contains two absorption peaks (see Fig. 4). The  separation  of the peaks, defined as the quadrupole splitting, is related to the  order parameter. Similar to the situation described for P 31  its characteristic quadrupole splitting is dependent on Lipids in the quadrupole  H  n  splitting  phase provide  a spectra  with  in comparison  to  in  lipids  NMR,  the H 2  NMR  2  H  spectrum with  the motional properties of the lipids. essentially a  the  2  fold  bilayer organization.  reduction  in the  When isotropic  motional averaging occurs, as in the case with small vesicles, the quadrupole splitting collapses resulting in a spectrum with a single narrow line. Freeze-fracture membrane  systems  microscopy, and from NMR  electron microscopy. The can  be  observed  local morphology exhibited by lipids in model  utilizing  the  technique  of  freeze-fracture  electron  freeze-fracture data is useful in verifying the structural assignments derived  experiments (see Chapter 2). The  freeze-fracture technique relies on the fact that  in a frozen sample which contains membrane structures a fracture plane will proceed through  18  the hydrophobic interface between the bilayer leaflets (Pinto da  Silva and  Branton, 1970).  Once fractured the sample is coated with platinum and carbon, the sample itself is digested away and the resulting replica is examined using an electron microscope. Micrographs of phospholipids in a bilayer organization show smooth sheets, as the fracture occurs in the center of the bimolecular leaflet, while H striated  patterns created  cylinders  (see  providing  defined  as  Fig. 4). In  indicative of any  the  fracture plane  n  phase structures are seen as  proceeds between  addition, freeze-fracture micrographs  structural information  for lipids  which  the are  exhibit  a  number of phases which allow isotropic motional  hexagonally  packed  sometimes useful in narrow  NMR  spectra  averaging. An  elegant  example of this is given by studies which defined the nature of the lipidic particle (Verkleij, 1984). 7.6.2 Roles postulated for  The  number  non-lamellar  of  structures  functional roles for lipids  in biological  membranes  is increased  substantially when non-bilayer organizations are considered. As indicated earlier, a number of isolated lipids, primarily unsaturated PEs, preferentially adopt the H  u  phase. In general, the  presence of other lipids stablize the bilayer configuration for these lipids (for example see Chapter 2). As previously discussed, a great variety of physiologically relevant factors, including specific ions, other lipids, and proteins, can induce the formation of non-lamellar alternatives in multicomponent systems. A  clear example of this concerns model systems composed of  cardiolipin which assume the bilayer organization until the divalent cation Ca triggering a bilayer to H  n  2+  is added,  phase transition (Cullis et al., 1978b).  Clearly the diversity of lipids in membranes is more complex than required to merely provide a structural matrix  for non-lipid  components and  a semi-permeable barrier. This  function, discussed in greater detail in section 1.7, would be satisfied by the bilayer forming lipids such as PC and sphingomyelin. The presence of non-bilayer forming lipids (PEs) would allow for the formation of non-bilayer structures, a function that could be regulated by the presence of lipids whose phase behaviour  are sensitive to external variables. Numerous roles  19  have been proposed for non-bilayer  organizations  (which most likely precedes via the  in membranes, including membrane fusion  formation of inverted micellar structures; see Verkliej,  1984), exocytosis (Nayar et al., 1982b), transbilayer transport of lipids and ions (Cullis et al. , 1980;  Noordam et al., 1981), intermembrane communication (Cullis et al. ,1983), and  insertion and non-bilayer  transport lipids may  (de  Kruijff et al., 1984). In addition, it has  state, where the  H -phase lipid (Kachar and n  elsewhere (Cullis et al., 1983; The  been suggested that  define the structure of certain regions of biological membranes. For  example it is thought that the tight junction results from two semi-fused  protein  most convincing  inner  lamelli of two  cells are  membranes that exist in a separated  by  a  cylinder of  Reese, 1982). These proposals have been discussed  in detail  de Kruijff et al., 1984). argument for the role of non-lamellar structures in  vivo is in  the case of membrane fusion, a process that mediates a variety of biological phenomena such as fertilization, endocytosis (uptake of extracytoplasmic material), and cellular  material). Since  this area is relevant  supportive evidence will  be  data presented  briefly summarized. It is conceptually  membrane fusion event occurring  (release  in Chapter  difficult  of  2, the  to perceive  a  without some local disruption of the bilayer organization,  hence this process is likely mediated by  the  numerous studies have demonstrated a strong appearance of non-bilayer  to the  exocytosis  formation of an  alternative structure. Indeed,  correlation between membrane fusion and  the  structures. Chemical fusogens, such as glycerolmonooleate, which are  able to induce fusion of both model and  biological membranes, also enhanced the formation  of non-lamellar phases in these systems (Hope and  Cullis, 1981). Structurally similar agents,  e.g. glycerolmonostearate, which were not capable of inducing fusion, do not effect the bilayer organization. In addition, the divalent cation Ca * usually required for in 2  provokes the formation of the H the  acidic phospholipid  PS  n  vivo fusion events,  phase in mixed lipid model systems that contain PE  (Tilcock and  Cullis, 1981), PG  (Farren  and  and  Cullis, 1980), PI  (Nayar et al., 1982a), or cardiolipin (de Kruijff and Cullis, 1980). Addition of Ca  2+  to vesicles  (SUVs) with these lipid compositions induced fusion, accompanied by the formation of lipidic particles (putatively an  inverted micellar structure) localized primarily at the fusion juncture  20  (Hope et al.,  1983). These data are consistent with the proposal that fusion proceeds via  formation of a non-bilayer, inverted micellar, structure. Further studies designed to investigate the mechanism of exocytosis of chromaffin granule contents also suggested that formation of the inverted micelle was  an necessary intermediate for the exocytosis process (Nayar  et al.,  1982b).  1.7 The  Shape Concept  A rationale for lipid diversity and the structural behaviour of lipids in membranes has been based on the proposal that the structures which lipids adopt when hydrated may  be the  result of their dynamic molecular shape (Israelachvili et al., 1977; Cullis and de Kruiff, 1979). This proposal is consistent with the polymorphic phase properties of lipids and lipid mixtures, as well as other properties including the thermotropic behaviour of lipids, the ability of lipids to form intermolecular hydrogen bonds, and the response of lipids to physiologically relevent factors such as divalent cations The  proposed  "shapes" that different lipids may  assume are illustrated in Fig. 5.  Lipids with a relatively small headgroup in relation to the area occupied by the acyl chains have a "cone" shape. The  reverse situation, where the area occupied by the headgroup is  greater than the area occupied by the acyl chains, results in an "inverted cone" shape and if the areas appropriated by the acyl chains and the headgroup are effectively equivalent, then a "cylindrical" shape is assumed. The  latter example would describe the bilayer forming lipids  such as PC, while the "cone" shaped lipids, ie. PE, would be accommodated in the  H  n  phase organization. These effects are illustrated by the properties of synthetic PEs. These lipids undergo a temperature induced bilayer to Hn  phase transition (which occurs above the gel to  liquid-crystalline phase transition) that is acutely sensitive to acyl chain composition. More specifically, this  transition  occurs  at progressively lower  temperatures  in association  with  increases in acyl chain unsaturation (Tilcock and Cullis, 1982). The unsaturated acyl chains can be expected to assume a greater cross-sectional area with respect to the headgroup than the  21  Fig. 5. Polymorphic phases and corresponding dynamic molecular shapes of component lipids.  LIPIDS  SHAPE  ORGANISATION  / / / / / / / / , / / / ,  LYSOPHOSPHOLIPIDS D E T E R G E N T S  INVERTED  C O N E  M I C E L  r  £3 '////////////,  PHOSPHATIDYLCHOLINE S P H I N G O M Y E L I N  /  •/////////////  ^7777777777777777777777777777 BILAYER C Y L I N D R I C A L  /////////////w///////////, P H O S P H A T I D Y L E T H A N O L - A M I N E M O N O  G A L A C T O S Y L DIGLYCERIDE  C H O L E S T E R O L  C O N E  22  H E X A G O N A L  P H A S E  saturated chains. In  addition  to the influence of the acyl chains described above, the phospolipid  headgroup, and phospholipid headgroup interactions, would also have a marked effect on the molecular area occupied by a particular lipid. For instance, it has been suggested that the bulkier choline headgroup of PC prevents close packing of this lipid in comparison to lipids containing the smaller ethanolamine  group (Phillips and Chapman, 1968), providing a rationale  for the lower Tc observed for PC containing membranes in comparison to PE. Similarly, since the area occupied  by a lipid headgroup would be affected by electrostatic repulsion, one  would expect that the presence of negatively charged lipids in membranes would prevent close packing of the lipids, again providing an explanation for the higher Tc observed for these lipids in comparison to neutral lipids. The Ca  2+  induced bilayer to H  n  phase transition in  PE/acidic phospholipid mixtures can also be explained on the basis of molecular shape and the presence of a charged lipid component For example, formation of a cation-PS complex would be expected to decrease the ionic repulsion between headgroups thereby reducing the effective area occupied by the serine headgroup (for discussion see chapter 2). This latter point is further emphasized by data which demonstrated a pH induced bilayer (at pH of 7) to H  n  phase (at pH of 4) transition in pure PS model systems (Hope and Cullis, 1980; the  pK of the PS carboxyl group is approximately 4.0). The shape concept has proven to be of general utility in predicting the properties of lipids under a variety of situation. Taking a building block approach one might expect that appropriate  mixtures  of "cone" and "inverted cone"  shaped  lipids  would  result  in the  formation of a bilayer structure provided ideal mixing occurs. This was shown to be the case for mixtures of PE with a variety of detergents ("inverted cone" shaped lipids), providing the rather  novel concept  that detergents can stabilize  the bilayer  organization of membranes  (Madden and Cullis, 1982). In line with this reasoning, it was suggested  that lipids with  differing "shapes" may be required for optimal packing and sealing around irregularly shaped membrane proteins. Data from model lipid systems containing glycophorin support this notion (Van der Steen et al., 1982). These proposals have been discussed in greater detail elsewhere  23  (Cullis et al., 1983; de Kruijff et al., 1984). 1.8 Permeability Properties of Lipid Membranes  As the previous discussions have indicated, there has been a great deal of effort spent on defining the roles of lipids in regulating membrane "fluidity" and the structural preferences of membranes. Ironically, the role of lipids in regulating the permeability  properties of  membranes is perhaps the least understood role, despite innumerable investigations that have spanned the greater part of three decades. This is due in part to the presence of a great diversity of membrane proteins that are capable of providing selective ion channels or actively generating ion gradients, the roles of which have superseded the passive (or perhaps active) role of lipids in mediating permeability  functions. In addition, studies on the permeability  properties of lipids have been hampered by the lack of an adequate model membrane system which is free of impurities (ie. solvents and/or detergents) and amenable to experimentation. Since this topic is germane to the studies presented in Chapters 3, 4 and 5, a brief summary of the permeability properties of model membrane systems will be provided with an emphasis on passive ion permeability and the role of ion gradients in generating membrane potentials. Due to the quantity of literature available it will be impossible to provide a complete review. This  discussion will  be limited  to formulating  general  concepts defining the permeability  properties of lipids. In addition, it is worthwhile to note that lipids play a role in regulating membrane permeability through specific and non-specific effects on membrane proteins (van Hoogevest et al., 1984a; van Hoogevest  et al., 1984b). These  effects will  also not be  considered here. 1.7.1 Passive  The  permeability  permeability properties of membranes are dramatically illustrated by the properties  of MLVs, in spite of the difficulty in interpreting results from this system (see Section 1.4). MLVs respond to osmotic gradients by shrinking and swelling, behaving as ideal osmometers provided that the lipids are in a liquid-crystalline state. As a result, the permeability of these  24  membrane systems to water can be evaluated by measuring the shrinking and  swelling rates.  Such studies indicate that the permeability of water through membranes increases in association with  increases in acyl chain  "fluidity". Increases in acyl chain unsaturation  results in an  increase in water permeation while introduction of cholesterol reduces membrane permeability above the Tc and increases the rate below the Tc (Blok, 1977). Interestingly, at the point in the transition from the gel to liquid-crystalline phase where the two  phases coexist one  observes a peak in the rate of water permeation. This has been associated with defects  in  the  membrane  at  the  composition  on  water permeability  interface of the establish general  non-electrolytes such as glucose, glycerol, and  two  phases. These  principles. The  packing  effects of lipid  diffusion  properties of  erythritol, show similar characteristics to those  exhibited by water. These molecules permeate more rapidly through bilayers composed of lipids that contain short or unsaturated acyl chains (Demel et al., 1972; Papahadjopoulos, 1973). The  permeability of lipid membranes to a particular molecule can be defined by the  permeability coefficient (P). the  flux  This constant is given in units of velocity (cm/sec) and equates  (change in concentration  with  time  divided  by  the  membrane  area  per  liter;  moles/sec cm )  to the difference in concentration (moles/cm ) of the molecule across the  membrane. The  P  2  3  value for water is between 1 and  100  x 10"  3  cm/sec, a value which  represents a rapid equilibration of this molecule. In order to gain a greater appreciation of this parameter, it can be calculated that for a P value of 10"  5  material from a vesicle with a diameter of 100 nm  cm/sec, half of the entrapped  will be released in less than 1 sec. In  contrast to the rapid permeability of water through lipid membranes, the P {e.g. Na\  K\  CI", (HVOH")) are generally in the range of 10"  10  of lipid composition. material from 100 nm energy necessary kcal/mole;  These P  values correspond  14  cm/sec irregardless  to a half-time for release of entrapped  vesicles of 6.4 hr to approximately  to bring an  to 10"  value of ions  7.3 years. As one might expect the  ion across a hydrophobic (lipid) interface is substantial (40  Parsegian, 1969), therefore this process represents a thermodynamically unfavorable  event Similar to the preceding  examples, the permeability of a given ion appears to be  related to the order or fluidity in the hydrocarbon chain, where a decrease in permeability is  25  observed in membrane systems containing cholesterol or long chain saturated fatty acids. In addition, ions show an increase in permeability under conditions where there is a mixture of gel and liquid-crystalline phases. The charge of a phospholipid headgroup can also strongly influence the permeability of a given ion. For instance, the presence of negatively charged lipids in a membrane will repel anions and attract cations to the lipid water interface, hence these systems are generally more permeable to cations. Under conditions where CI" is present on both sides of a membrane, unusually large P values (10~ ) are obtained for this anion. It 5  has been suggested that this may be due to the formation of HC1 and the subsequent rapid transport of this neutral molecule (Hauser et al., 1973; Nicholls and Miller, 1974). This observation (.P=10"  5  was consistant with  cm/sec) of H  +  data  which  demonstrated  ions in systems containing  rapid transmembrane equilibration  the CI" ion on both sides of the  membrane.  1.7.2 Membrane  A across  potential  transmembrane potential is produced when there is an ion concentration gradient  the membrane. For biological  cytoplasmic membrane  membranes these  gradients  occur  ubiquitously  across  membranes as well as subcellular organelles. It is becoming apparent that the potential has an essential role in a variety of membrane  mediated  processes  including amino acid and metabolite transport (Schuldiner and Kaback, 1975; Fujimura et al., 1983), protein  transport  (Zwizinski  et al., 1983), protein  insertion (Wickner, 1983), lipid  flip-flop (McNamee and McConnell, 1973), and divalent cation transport (Saris and Akerman, 1980). Based on the far reaching implications of these processes in terms of understanding the function of membranes, it is surprising that only a few studies have examined the role of lipids in the development and maintenance of the membrane potential in vesicles. Further, the role of lipids  in relation to these membrane potential dependent events has received no  attention other than the rather preliminary studies presented in this thesis.  26  ':,  The potential generated across a membrane is the result of a number of variables,  including the equilibrium distribution of ions present defining  the surface  and the surface charge (important in  potential; for review see Ohki, 1981). In a situation where an ion  gradient is present, a potential difference across the membrane will be established as the ions diffuse down their concentration  gradient  The resulting diffusion potential can be calculated  employing a derivation of the Nernst equation (Goldman-Hodgkin-Katz equation) that describes the  relation  between  the electrical potential ( A i p ) and the concentrations  (activities) and  permeabilities of the ions present:  RT_ *  A  =  ~ z F  P l n  P  m  [Na ]" + P +  [Na ]' + F +  N a  K  K  [ K ] " + P a [CI-]' + . . . +  [K ]' + P +  C  I  [CI"]"  +  • • •  In descriptive terms this equation indicates that the orientation and magnitude of the diffusion potential is dependent on the mobilities (permeability coefficients, F) of the cations and anions present In addition, the sign of the potential on the side of the membrane where the mobile species is concentrated  will be the opposite sign of this species. It also indicates that the  magnitude of the potential will be greater when there are large differences in cation and anion mobility. As indicated in the previous section, most ions are relatively impermeable therefore it is sometimes necessary to increase the rate at which an ion permeates the membrane (increase the P value) in order to generate a membrane potential. This can be accomplished by the use  of ionophores, a group of lipid  soluble macromolecules that selectively  enhance the  permeability of certain ions (for reviews see Pressman and de Guzman, 1975; Ovshinnikov and Ivanov, 1977; Reed, 1979). The K  +  ionophore valinomycin  is an example of a mobile carrier  which acts by forming a very permeant lipid soluble complex when coupled with the ion species concerned. Other ionophores, e.g. gramicidin, create channels through the membrane allowing certain ions to permeate. It should be stressed at this point that the latter group of ionophores are relatively insensitive to the lipid environment, while the mobile ionophores are sensitive to the lipid composition and function only in membranes where the lipids are in liquid-crystalline state. Using valinomycin  as an example, addition of this ionophore, to vesicles:  27  with a NaVK  +  ion gradient (KC1  inside and  (provided the P value of the CI" and Na of K  ions will eventually  +  be  NaCl outside) establishes a K  ions can be neglected). The  +  restrained by  +  ion gradient  more rapid movement  electrostatic forces, and  the  resulting charge  separation will result in a negative (interior) membrane potential. Additional details concerning the  generation  of the  membrane potential and  methods for evaluating  the  potential are  discussed in Chapters 3 and 4. It must be emphasized again that these studies represent some of the first well characterized investigations of membrane potential in model LUV  systems.  1.9 Summary  The  preceding  review  has  clearly  shown  that  there  is  currently  an  excellent  understanding of the chemical and physical properties of lipids (at least the major classes of eukaryotic lipids) and  how  function of membranes. The  at a molecular level these lipids may  modulate the structure and  fundamental problem of lipid diversity has been reduced to the  question of defining lipid functions. This thesis attempts to elucidate a few of the functional roles of lipids in model membrane systems. Specifically, in Chapter 2 the role of cholesterol in modulating the polymorphic phase behaviour of mixed lipid systems composed of naturally occurring or synthetic PEs and either PC on  the  Ca  2+  induced  bilayer to H  n  or PS is investigated . The phase transition in PS  influence of cholesterol  containing  systems is also  examined. In addition, a basis for examining the role of lipids in regulating ion permeability and  membrane mediated transport function is established (Chapters 3 and  4). It is further  demonstrated that the transport of biologically relevant molecules can proceed in membrane systems in the absence of a carrier protein (Chapters 4 and  5), thereby defining a new  and  potentially exciting membrane dependent function that could be regulated by the presence of specific lipid species.  28  LIPID POLYMORPHISM: INFLUENCE OF CHOLESTEROL AND DIVALENT CATIONS ON THE STRUCTURAL PREFERENCES OF MLXED LIPID MODEL SYSTEMSt  2.1 Introduction  The  functional roles of cholesterol in biological membranes are not well understood. As  emphasized in the preceding chapter (see section 1.5.2), it is generally assumed that cholesterol plays a role in modulating the "fluidity" of the lipid environment, thereby affecting membrane function. However, as described in detail in section 1.3, there is little convincing evidence to support the proposal that fluidity plays an important role in membrane function in vivo. In this context, it is appropriate to examine the influence of cholesterol on physical properties of membrane phospholipids other than fluidity. In previous work (Cullis et al., 1978; Cullis and de  Kruijff,  unsaturated  1979), it was PC  shown  (phosphatidylcholine)  that -  cholesterol exerts PE  a rather  remarkable  effect in  (phosphatidylethanolamine) systems, serving to  destabilize bilayer structure and induce formation  of the hexagonal (H ) phase. In dioleoyl n  (18:lc/18:lc) PE - dioleoyl PC systems, for example, cholesterol induces apparently complete H  n  phase structuring at equimolar proportions with respect to phospholipid (Cullis, 1978). This  result was surprising in view of the ability of cholesterol to "condense" PC monolayers and reduce membrane permeability (Demel and de Kruijff, 1976) and thus in some sense stabilize bilayer structure for PC systems. On the basis of the above, it may also be considered H  u  that cholesterol may facilitate  phase formation. In regard to this, it has been shown that the Ca  2+  concentration needed  to induce bilayer to H n transitions in multilamellar (soya) PE-PS systems and to induce fusion in sonicated  unilamellar vesicles are rather high (2 mM)  systems, particularly in the cytoplasm, where the free Ca  t  2+  in reference to biological  concentrations  are less than 100  This chapter has been based on the references Bally et al., (1983) and Tilcock et al.,  (1982). 29  Vi M.  In this chapter the ability of cholesterol to act as an adjunct to the Ca  examined. Further, the ability of Mg induce bilayer to H  n  to facilitate this process is examined. Mg  2+  effect is  2+  2+  does not  transitions in PE-PS systems but does induce aggregation, a step which  is vital to obtaining the close apposition between bilayers apparently required before H  n  phase  formation can proceed (Cullis et al., 1980). In addition, this chapter also examines in greater detail the influence of cholesterol on the preferences of DOPC-DOPE systems  with the  specific question of whether there is any phase segregation of either phospholipid species in systems exhibiting both bilayer and H  n  phase structures, or whether the lipids exhibit ideal  mixing behaviour. It is demonstrated that cholesterol can induce H PE-PS systems  an order  phase structure in previous bilayer  and that, in combination, the presence of (2 mAf) Mg " and equimolar 2  cholesterol can reduce the Ca than  n  2+  concentrations required to induce such transitions by more  of magnitude. It is also  DOPC-DOPE-cholesterol  systems  demonstrated  exhibiting both  that  bilayer and H  in mixed  liquid-crystalline  phase characteristics, the  n  lipids appear to be equally distributed in both phases. 2.2 Materials and Methods 2.2.1 Preparation  of soyabean  lipids  Soyabean PC was purified from crude (20%, w/w) soya PC purchased from Sigma (SL Louis, MO)  utilizing  two chromatographic  preparation was produced by chromatography Germany; for reference see Sheltawy  procedures in series. A  partially  purified  PC  on aluminium oxide (A1 0 ; E. Merck, Darmstadt, 2  3  and Dawson (1969)). Crude soya PC (75 gm) in  chloroform was loaded onto a 150 X 5 cm column of A1 0 2  3  (previously washed with a 1:1  (v/v) mixture of chloroform/methanol,. and subsequently packed in chloroform) and eluted with a 1:20 (v/v) chloroform/methanol mixture at a flow rate of 6 ml/min. As the PC began to elute  from  the column  the mobile  phase was exchanged  for a 1:1 (v/v) mixture of  chloroform/methanol and the flow rate was increased to 20 ml/min. The partially purified PC  30  (yield of approximately 20 gm) liquid chromatography  was  (PrepLC/System  chloroform/ methanol/water  subsequently purified employing  silica acid preparative  500, Waters Associates; using PrepPak-500/silica) with  (60:30:4, v/v) as the mobile phase (for reference see Patel and  Sparrow, 1978). The resulting lipid was shown to be greater than 99% pure as indicated by iodine vapour stained two-dimensional thin-layer chromatography gel 60 TLC plates (0.25 mm  (TLC; using pre-coated silica  thick; E. Merck, Darmstadt, Germany) run in the first direction  in a base solvent system composed of chloroform/methanol/ammonia/water and  in  the  second  direction  chloroform/methanol/glacial  acetic  with  an  acid  solvent  (90:54:5.7:5.4, v/v)  system  composed  of  acid/water (25:15:4:2, v/v) according to the method of  Broekhuipe (1969)). Soya PE and soya PS were obtained from pure soya PC exchange capacity of phospholipase D gm/  100 ml) was  employing the headgroup  (Comfurius and Zwaal, 1977). Briefly, PC  incubated, with vigorous shaking at 40° C  in ether (5  with 100 ml of a saturated  solution of L-serine (46%, w/v, in 0.1 M acetate buffer (pH 5.6) containing 0.1 M a 15%  ethanolamine solution (w/v, in the acetate/ CaCl  CaCl ) or 2  buffer after netralization of the  2  ethanolamine with concentrated HC1) in the presence of a crude phospholipase D preparation (see  below). The progress of the reaction was  followed on small TLC  plates (2 X  7 cm  plates cut from the large pre-coated plates used for two-dimensional TLC) which were run in the base solvent mixture and subsequently sprayed with a phosphorus staining reagent (a mixture of 2 vol water, 1 vol molybdenum trioxide reagent (40.1 gm concentrated H S0 ), and 1 vol molybdate reagent (1.78 gm 2  which  4  stains phosphorus  containing  compounds  blue  2  4  the reaction was  3  in 1 L of  Na molybdate in 500 ml water)),  upon  heating,  (appearence of black spots) of the lipids and organic compounds due H S0 . Generally, after 30 min  Mo0  followed  by  charring  to the presence of  stopped by cooling the reaction mixture,  followed by low speed centrifugation (500g) for 10 min to facilitate separation of the aqueous and ether phases. The  ether phase was  collected, then dried using rotary evaporation. The  resulting lipid, a crude mixture of unreacted PC, PS or PE, and PA, was washed to remove aqueous contaminants by suspending the lipid in 25 ml of chloroform/methanol (2:1) followed  31  by  addition of 6 ml  phase was  of water to generate a two  isolated and dried down. The  phase system. Subsequently the organic  reaction generally resulted in a 25-50% conversion  for PS and a 50-75% conversion for PE. The  PS  was  purified using conventional  low  pressure column chromatography using  carboxymethylcellulose as a stationary phase (Comifurius and Zwaal, 1977). Briefly, 2-4 crude PS  in chloroform  was  loaded  onto a  5  X  72  cm  CM-52  gm  of  carboxymethylcellulose  (Whatman, England; material was pre-washed in methanol) column packed in chloroform. The lipid was  eluted using a continuous (0 to 50%)  fractions (which eluted at approximately acid  solvent) using  a  ninhydrin  spray  chloroform-methanol gradient PS containing  25% methanol) were identified on TLC reagent  (0.2%, w/v,  ninny drin  plates (run in  in water saturated  butanol), which reacts with free amino groups forming purple-mauve spots on the plate after heating, followed by the phosphorus spray reagent described previously. The pure PS fractions were pooled, dried, and susequently the lipid was  chromatographically  converted to the sodium  salt as described by Hope and Cullis (1980). This procedure involved dissolving the dry lipid in a Bligh and Dyer monophase (chloroform/methanol/water in the ratio 1:2.1:1, v/v) where the aqueous component contained 0.4 M  HC1  and subsequently titrating the mixture to pH  8.0 with a Bligh and Dyer monophase where the aqueous phase contained 0.5 M 0.5 M  NaOH. This was  followed by addition of 0.4 vol of both water and  create a two-phase system. The  =  NaCl and  chloroform to  organic phase containing the sodium salt of PS  was dried  and stored in chloroform under nitrogen at -20°C. The crude soya PE was purified employing silica acid preparative liquid chromatography using a mobile phase composed of chloroform/methanol/water/25% ammonium hydroxide ratio of 60:30:1:1 (v/v). The pure PE fractions were identified on TLC  in a  plates using a similar  procedure described for PS. Pure soya PE was stored in chloroform under nitrogen at -20°C. Both lipids were shown to be greater than 99% pure as indicated by iodine vapour stained two-dimensional TLC chromatography.  32  The phospholipase D used for the transphosphatidylation reactions was partially purified from the inner leaves of savoy cabbage employing the procedure of Kates and Sastry (1969). Briefly, 4-6 kg of the inner yellowish-green leaves of savoy cabbages were homogenized in 3 L of ice-cold water. The resulting homogenate was filtered through cheesecloth and centrifuged 5.5 with AN  at 2,000g for 30 min to remove bulk fiber. The supernatant was adjusted to pH HC1  and fractions were heated rapidly (within 2 min) to 55° C in a boiling water bath to  precipitate  contaminating  subsequently  proteins. The  fractions  centrifuged at 13,000g for 30  min.  were  immediately  The  supernatant  cooled was  to  collected  0°C,  and  and  the  remaining proteins, including phospholipase D, were then precipitated by addition of ice cold acetone (1 vol supernatant: 2 vol acetone) with continuous stirring for 15 min. The precipitate settled out of solution within 2 hr at 4°C and was isolated by centrifugation (lOOOg for 10 min) after the bulk of the unprecipitated material was removed by aspiration. The precipitate was  lyophilized  to remove  residual  conversion reactions, 200-500 mg sodium acetate buffer pH at 0°C  acetone  and  stored at -70° C  of the preparation was  5.6 containing 40 mM  until  used. For  suspended in 20 ml  the  of 0.2 Af  calcium chloride. The suspension was mixed  for 30 min and, subsequently centrifuged at 17,000g for 10 min to remove insoluble  material. The  resulting supernatant was  utilized  for the base-exchange  reactions described  above.  2.2.2 Synthesis  of dioleoyl  phospholipids  Dioleoylphosphatidylcholine was  synthesized by  the procedure  of Warner and Benson  (1977), which involved reacylating glyceryl phosphorylcholine (GPC), derived from egg PC, with the specified fatty acid imidazole. Egg PC  was prepared from hen egg yolks by preparative  liquid chromatography (as described for soya PC) of a chloroform/methanol of  acetone  precipitated lipids (30 egg yolks were mixed with 1.2  L  (1:1, v/v) extract  of acetone and the  resulting precipitate was isolated by filtration, washed 5 additional times with 1 L acetone and subsequently extracted 3X with 500 ml of the chloroform/methanol mixture). GPC from  the pure  egg  PC  by  the procedure  33  of Brockerhoff and  Yarkowiski  was derived (1965), which  involved  the slow  addition of 10 ml tetrabutylammonium  hydroxide  (25% in methanol,  Eastman-Kodak, Rochester, NY) to 10 gm of egg PC dissolved in diethyl ether. After a 1 hr incubation at 20° C the precipitated GPC was isolated by centrifugation at 500g for 10 min.  The GPC was dissolved in methanol and reprecipitated with  contaminating  acyl  chromatographically  chains  or PC. This  procedure  was repeated  ether to remove any 3X  and resulted in  pure GPC which was dissolved in methanol and stored at -20°C until  used. GPC  (5 mmoles), dried and left under high vacuum overnight, was dissolved (aided by  sonication in a bath sonicator) in 120 ml of dimethyl sulfoxide (DMSO). Subsequently, this was  added to a 20 ml solution of DMSO containing 20 mmoles of oleic acid imidazole,  which was prepared by adding 22 mmoles l.r-carbonyldiimidazole (Sigma, SL Louis, MO) to 20 ml of tetrahydrofuran (Aldrich Chemical Co., Milwaukee, WI) containing chromatographically pure oleic acid (Sigma) followed by evaporation of the tetrahydrofuran after the reaction was completed (as the reaction occurs C 0 gas is released as the fatty acid imidazole is produced, 2  indicating the progress of the reaction which was normally complete at 45 min). The acylation reaction was initiated by addition of 72 ml sodium DMSO (prepared by slowly adding 1 gm of sodium metal to 75 ml of DMSO followed by stirring overnight in a flask equipped with a drying tube) and was allowed to procede with vigorous stirring. The reaction progress was followed using small TLC plates run in the base solvent system and was usually complete within 10 to 15 min. The reaction was stopped by cooling the reaction mixture and adjusting the pH to less than 2 with 4N HC1. Immediately following this, 200 ml of water was added and  the mixture was extracted 3X with a half vol of chloroform/methanol (2:1, v/v). The  combined chloroform extracts were washed 5X with a half vol of methanol/water (1:1, v/v) in  order to remove the bulk  of the DMSO. The washed extract was dried (forming a  brownish oil) and the excess DMSO and unreactive fatty acyl imidazole were removed by preparative liquid chromatogrphy on silica using chloroform/methanol/water (60:30:3, v/v) as the  mobile  phase.  In  general  this  was  followed  by  additional  purification  on  carboxymethylcellulose using a step gradient of 1% methanol in chloroform followed by 10%  34  methanol to elute the DOPC. The resulting lipid was greater than 99% pure as determined by two-dimensional TLC. DOPE  was prepared  phospholipase D  from  DOPC  utilizing  the headgroup  as described previously. Deuterated DOPC  exchange  capacity of  and DOPE were synthesized as  described above using 11,11- dideuteriooleic acid which was synthesized by a modification of the procedure of Tucker et al. (1971), based on a Wittig condensation of nonanol-2-d2 with methyl 9-iodononanoate (Farren et al., 1984) and was kindly provided by Dr. S.B. Farren. 2.2.3 Sample  preparation  Samples for NMR (purified 10-mm  studies were prepared from an appropriate mixture of the lipids  as described above, with cholesterol purchased NMR  from  Sigma) in chloroform in a  tube. Chloroform was evaporated under nitrogen and the sample was then kept  under high vacuum for approximately 2 hr. The dry lipid mixtures were hydrated with 0.7 ml of buffer (100 mM NaCl, 10% (v/v) deuterated water, and 10 mM HEPES (pH 7.4)) by vortex  mixing  in the presence  of a  glass  agitator  to facilitate  lipid  dispersal (for  PE-PS-cholesterol mixtures the samples were hydrated at 4°C). In the case of the H-labeled 2  lipids the samples were prepared in deuterium-depleted water (obviously no deuterated water was used in the buffer). Soya PE and PS samples were titrated with calcium by adding aliquots of 100 mM stock solution of the chloride salt To ensure equilibration of the divalent cation with the lipid,  the samples were subjected to three freeze-thaw  Samples membrane  requiring  dialysis  were  sealed  tubing, 12,000-14,000 MW  in 0.6 cm  cutoff;  cycles employing  diameter  Spectrum  Medical  following hydration and equilibrated against the required Ca 4° C for 6 hr. Control experiments employing  45  dialysis  2+  liquid nitrogen.  tubing (Spectrapor  Ind., Los Angeles, CA)  and/or Mg  2+  concentration at  Ca revealed maximal binding of Ca  2+  to soya  PS occurred within 4 hr. A 10-fold molar excess of the divalent cation in the dialysate over PS was utilized in all dialysis experiments. Lipid degradation following the 6 hr of dialysis was found to be less than 1%, as determined by TLC.  35  2.2.4 Nuclear  2  For  31  P  magnetic resonance  H NMR  and "P NMR  spectra were obtained by using a Bruker WP200 spectrometer.  NMR,  free induction  decays were accumulated  from  up to 1000 transients by  employing a 15-ys 90° radio-frequency  pulse, 20-kHz sweep width, and 0.8-s interpulse  delay,  proton  in the presence  corresponding  of broad-band  decoupling.  An  exponential multiplication  to 50-Hz line broadening was applied to the free induction decay prior to  Fourier transformation. For H NMR, 2  free induction decays were accumulated for up to 20,000  transients by employing a 15- ys 90° radio-frequency pulse, 30-kHz sweep width, and 0.04-s interpulse delay. An exponential multiplication corresponding applied  prior to Fourier transformation.  to a 100-Hz line broading  Unless specified, all spectra  were recorded  was at a  temperature of 30° C.  2.2.5 Freeze  fracture  Freeze-fracture  was  performed  on appropriate  samples (described  in section 2.2.3)  containing 25% (v/v) glycerol as a cryoprotectant. The samples were frozen in a liquid freon slush. Freeze-fracture studies were performed on a Balzers BAF 301 apparatus, and replicas were viewed employing a Phillips 400 electron microscope. All freeze-fracture studies were kindly provided by Dr. M.J. Hope. 2.3 Results 2.3.1 Influence  The  of cholesterol on soya PE/PS  use of NMR  lipid  mixtures  techniques to assign phase structures of lipid mixtures is described  in section 1.6.1 and is illustrated by the data in Fig. 6 which shows P and H 31  spectra of DOPE as a function of temperature. The P NMR 31  2  NMR  spectra illustrate a bilayer to  hexagonal (H ) transition occurring as the temperature is raised through 10° C as indicated by n  the change in line shape from one with a low-field shoulder and high-field peak separated  36  -Fig:'- 6. "P and H NMR spectra as a function of temperature of fully hydrated dioleoylphosphatidylethanolamine (DOPE) which is H labeled at the C position of the acyl chains ([C - H ]DOPE). The P NMR spectra were obtained at 81.0 MHz in the presence of proton decoupling, whereas the H NMR spectra were obtained at 30.7 MHz. For details see section 2.2. 2  2  n  2  u  31  2  2  31r  40  — i  0 ppm  1  1—  0 kHz  -40  37  -2  -6  by approximately 50 ppm (a bilayer spectrum) to a spectrum with reversed  asymmetry which  is a factor of 2 narrower ( H spectrum). This transition is reflected in the corresponding H 2  n  NMR  spectra by the appearance of a narrower spectrum arising from H  DOPE  as the temperature  is raised,  compared  to the bilayer  phase [ C - H J 2  u  U  spectrum  observed at  temperatures below 5°C. The  influence  of cholesterol on the polymorphic phase preferences of various soya  PE-soya PS mixtures is illustrated in Fig. 7. The results presented there clearly illustrate an ability of cholesterol to destabilize a bilayer organization contents below 50 mol%.  of such systems, particularly at PS  Thus for the 30 mol% PS - 70 mol% PE sample a bilayer line  shape is obtained in the absence of cholesterol. Increasing cholesterol contents lead first to a structure allowing  isotropic motional averaging and subsequently to the P-NMR 31  line shape  characteristic of the H -phase. This ability of cholesterol to engender H -phase formation in n  u  PE-PS systems is also illustrated by the freeze-fracture micrographs of Fig. 8. Large fracture planes characteristic of bilayer structures are observed for the 30 mol% PS- 70 mol% PE system in the absence of cholesterol (Fig. 8A), whereas the striated pattern characteristic of H -phase n  indicated  structures  is observed in the presence of equimolar cholesterol (Fig. 8B). As  earlier (see section  1.6), the narrow  31  P-NMR  resonance may arise from  small  lamellar* structures or nonlamellar structures such as inverted micelles (de Kruijff et al., 1979) which  can play  Freeze-fracture  intermediary  roles  in bilayer-H  u  transitions  (Verkleij  et al., 1980).  studies revealed the presence of small (diameter less than 100 nm) vesicles in  these systems which appear to form spontaneously on hydration. This would be consistent with the  behaviour  of  (cholesterol/phospholipid  the  systems  containing  50  mol%  PS,  where  intermediate  ratio between 0.1 and 0.5) cholesterol contents appear to generate  formation of somewhat smaller lamellar structures, but do not induce H -phase organization n  even for cholesterol to phospholipid ratios of 1.0.  38  Fig.  7.  amounts  "P-NMR  spectra  (81.0  MHz) obtained at 30'C  of soya PS and cholesterol. C/PL refers  from aqueous dispersions of soya PE in the presence  to the molar ratio  of cholesterol  to phospholipid.  corresponds to the chemical shift of sonicated PC vesicles. For other details see section 2.2.  of varying  The 0-ppm position  Fig. 8. Freeze-fracture micrographs of soya PE - soya PS mixtures (30 mol% PS) in the presence (B) and absence (A) of equimolar cholesterol. The white bars represent 200 (A) and 100 nm (B). The direction of shadowing is indicated by the arrowhead in each micrograph. Data was collected and reproduced with permission from M. J. Hope.  40  2.3.2 Influence PE-PS  of cholesterol and Mg *  on the Ca *  2  induced  2  bilayer  to H  u  transition  in  systems.  As indicated in sections 1.6 and 2.1, bilayer to H PE-PS systems by the addition of Ca  2+  n  transitions can be triggered in  (for a reference see Tilcock and Cullis, 1981). Given  the ability of cholesterol to encourage H -phase formation, it may be expected that lower u  Ca  2+  levels are required to trigger such transitions if cholesterol is present Results showing  this to be the case are illustrated in Fig. 9 for equimolar mixtures of soya PS-soya PE in the presence of varying amounts of cholesterol. Whereas Ca /PS ratios R of 0.5 and higher 2+  are required to induce appreciable H„-phase formation in the absence of cholesterol, similar effects are observable in samples containing 25 and 50 mol% cholesterol at Ca  2+  levels as low  as R = 0.125. It has been shown previously (Tilcock and Cullis, 1981) that in contrast to Ca , Mg 2+  is unable to trigger bilayer-H Mg /PS  ratios as high  2+  Mg -induced 2+  transitions in equimolar soya PS-soya PE dispersions even at  n  as 10.0. However, the presence of equimolar cholesterol enables  bilayer-Hii  transitions to occur as illustrated  in Fig. 10 for 8 mM Mg " 2  concentrations. This leads to the possibility that lower levels of Mg  2+  with Ca * to produce Ca -induced phase transitions at lower net Ca 2  2+  2+  2+  can act synergistically concentrations. Results  supporting this possibility are illustrated in Fig. 11 for equimolar soya PS-soya PE systems, which were dispersed in the presence of 2 mM Mg buffer containing 2 mM The addition of Ca  2+  Mg  2+  2+  and subsequently dialyzed against a  and various concentrations of Ca  2+  (see section 2.2 for details).  had little effect on the structural preference of systems containing no  cholesterol. Even at 8 mM  Ca  2+  levels such systems evidenced largely lamellar  31  P-NMR  lineshapes with a small (10%) H„-phase component superimposed (see Fig. 11). some H -phase n  formation  is visible  for Ca  concentrations as low as 0.25 mM  2+  equimolar (with respect to phospholipid) cholesterol is present Thus Mg  2+  However, when  and cholesterol in  combination can act as important adjuncts for Ca -induced phase changes in PS-PE systems. 2+  41  Fig.: 9. "P-NMR spectra (81.0 MHz) obtained at 30° C from aqueous dispersions of equimolar •i mixtures of soya PS and soya PE in the presence of varying amounts of cholesterol and Ca">. The ratio C/PL refers to the molar ratios of cholesterol to phospholipid, whereas the R ; refers to the molar ratio of Ca to PS. For details of sample preparation see section 2.2. The. Ca * was added to the (0.7 ml) aqueous lipid dispersions as aliquots from a 100 mM stock solution of CaCl . 2  2+  2  2  C/Pl=  AO  0  0  0.2  -AO  >  AO  0  -AO  ppm  H  —>-  42  0.5  ' AO ' 0  '-A0 '  :Fig. 10. P-NMR spectra (81.0 MHz) obtained at 30°C from aqueous dispersions of equimolar mixtures of soya PS and soya PE after dialysis against the indicated concentrations of\.Mg . The ratio C/PL refers to the molar ratio of cholesterol to phospholipid. For details of sample preparation see section 2.2. 31  2+  C/Pl =0  C/Pl -1.0  [Mg2 ] +  8.0mM  A.OmM  2.0mM  1.0mM  OmM 1  i  AO  r — — i  0 ppm  1  1  T  r  -AO  r-  AO  43  -i  0 ppm  1  1  -AO  r  Fig.- 11. P-NMR spectra (81.0 MHz) obtained at 30° C from aqueous dispersions of equimolar mixtures of soya PS and soya PE after dispersion in the presence of 2 mM Mg and.' subsequent dialysis against a buffer containing 2 mM Mg and the indicated concentration of Ca . The ratio C/PL refers to the molar ratio of cholesterol to phospholipid. For other details see section 2.2. 31  2+  j  2+  2+  C/PL=0  ppm  C/PL = 1.0  ppm  The  ability of Mg  2+  to induce polymorphic  bilayer-H transitions when cholesterol is u  present and its inability to segregate PS in PE-PS systems in the absence of cholesterol suggests either of two possibilities. First, it may be that cholesterol facilitates PS segregation. Alternatively Mg  2+  may act to decrease the local charge density at the membrane surface,  thereby facilitating H „ phase formation (see section 2.4). If the latter possibility is correct, high salt concentrations should also be able to exert similar effects. This is indeed the case as indicated in Fig. 12, where it is shown that 1 M proportion  of the PE-PS-cholesterol  NaCl concentrations cause a large  (1:1:2) dispersions to adopt  the H  n  configuration,  behaviour which does not occur for the systems not containing cholesterol.  2.3.3 Influence  The  of cholesterol on the mixing  properties  of DOPC/DOPE  mechanism whereby cholesterol promotes H  u  systems  phase formation in mixed PE-PC  systems is not understood. One possibility would be that it associates preferentially with the bilayer-stabilizing PC species (van Dijck et al., 1976) and that the resulting PC-cholesterol complex is more readily incorporated into the H  n  phase matrix. Alternatively, it is conceivable  that cholesterol induces a lateral segregation of PC, allowing the PE to adopt the H  n  phase  it prefers in isolation. In order to investigate these possibilities, the H-labeled (C - H )DOPE 2  2  n  and  (C - H )DOPC  lipids were employed. The P  2  u  31  2  (C - H )DOPE-DOPC 2  n  2  and H  (4:1) and DOPE-(C - H )DOPC 2  n  2  2  NMR  2  results obtained from  (4:1) (at 40° C) in the presence of  varing amounts of cholesterol are shown in Fig. 13. There are several features of interest First the P NMR 31  spectra for mixtures containing H-labeled DOPC or DOPE are virtually 2  identical, both with each other and with the corresponding mixtures containing unlabeled lipids. Second, the H 2  NMR  spectra arising from (C„- H )DOPE or (C - H )DOPC 2  2  2  n  2  in mixtures  containing the same proportions of PE, PC, and cholesterol are also very nearly identical. Thus, DOPC and DOPE appear to partition with equal probability between the various phases (bilayer, H , or isotropic) present in the sample. This result demonstrating u  (liquid-crystalline) DOPE and DOPC  miscibility of  even when different phase structures are present is  rather surprising as it may have been expected that DOPC would be present at higher levels  45  Fig. 12. P-NMR spectra (81.0 MHz) obtained at 30° C from equimolar mixtures of soya PS and soya PE in the presence and absence of 1 M NaCl. The ratio C/PL refers to the molar ratio of cholesterol to phospholipid. 31  Fig.  13.  81.0  MHz  31  P  NMR  dioleoylphosphatidylethanolamine molar  ratio  of the  4:1  labeled  at  sample  preparation  where  and  the  MHz and  n  DOPE  2  H  *0  1  T  0  PPm  1  1  -40  The  see  N M R spectra  at  2  H  labeled  ratio  section  R  at  refers  40' C  arising  (DOPC)  the  C  to  the  u  from  and  position molar  ([C - Hj u  6  1  4  of  1  DOPE)  cholesterol  :  i  0  at  or  to  D'OPE C|,- H 4  2  2  dispersions (CHOL)  of  mixtures  a  DOPE:DOPC  the  DOPC.  DOPC For  is  details  of  2  H of  2.2.  1  rI  aqueous  cholesterol  ratio  DOPE = D O P C 4  -r-  H  is.  !  manipulation  ;  dioleoylphosphatidylcholine  ([C - HjDOPC).  data  C „ -  30.7  (DOPE)  either  position  Cu  and  1  -2  1  -4  1  -6  1  6  1  4  1  2  1  1  0 -2 kHz  :  i.T  -4  -6  D O P C  ?  1  1  40  i  1  0 PP"'  , r-40  in the bilayer phase component, and DOPE in the H A point of interest regarding the H NMR 2  quadrupolar 2  2  phase component  spectra of Fig. 13 concerns the values of  splitting observed. First, the quadrupolar  and (Cn- H )DOPC  n  splittings ( A Q ) of the (dr- H )DOPE 2  2  are equivalent in both the bilayer and H  n  organization. It may be noted  that for the cholesterol contents correponding to R = 0.1, the maximum A Q expected for the ( C „ - H ) D O P C (if all the cholesterol was associated with the DOPC) would be 6.3 kHz (Fig. 2  2  14). The value of 8.1 kHz observed both for the labeled DOPC and for DOPE indicates that bilayer phase DOPE  has a more ordered  hydrocarbon  region than  DOPC  and that this  increased order is also experienced by the acyl chains of the DOPC. This is again consistent with a well-mixed system. Second, even in the presence of the highest cholesterol levels used (R=0.25), the A Q of the bilayer component of the labeled DOPC sample remains at 8.3 kHz. If the cholesterol present was preferentially associated with the PC, the results of Fig. 14 suggest that the A Q should be increased by almost 3 kHz. Thus the results of Fig. 13 give no support for the notion of preferential association of cholesterol with PC over PE.  2.4 Discussion  The data presented here shows clearly that the presence of cholesterol in (unsaturated) PE-PS or PE-PC systems promotes formation of H -phase structure. This is expressed either n  as a direct result of the presence  of cholesterol, or as an increased sensitivity  of  the  structural preferences of PE-PS systems to the presence of divalent cations or increased ionic strength. The mechanism whereby cholesterol causes such effects will be discussed first and subsequently the potential biological ramifications of these effects will be considered. The ability of cholesterol to destabilize bilayer structure in PS-PE systems is consistent with an ability to exert similar effects in (unsaturated) PC-PE systems (Cullis and de Kruijff, 1978; Cullis et al., 1978, see also Fig. 13). In these systems, it was suggested that cholesterol has  a net cone shape (for disscusion of the shape  concept  see section  1.7) which in  combination with PC results in a complex more compatible with H -phase structure. Such a n  rationale may also apply to the effects of cholesterol on the PS-PE systems, where the  48  Fig?' 14. Influence of cholesterol on the quadrupolar splitting A Q observed for dipleoylphosphatidylcholine (LX)PC) H labeled at the C postion of the acyl chains ( [ G i j - ' H J D O P C ) . The H N M R spectra from which these data were obtained at 30.7 MHz and at a temperature of 40° C. For details of sample preparation and N M R data manipulation see section 2.2. 1  J  u  2  :  15-  m o l e °/  0  49  Cholesterol  cholesterol has been suggested to associate preferentially with the PS component (Demel et al., 1977). There are other factors which must be taken into account, however. First, cholesterol may  be expected  to exert  a spacing  effect in the mixed  systems, thus reducing the  electrostatic repulsion between the negatively charged serine head groups and thereby reducing the effective size of the head group. Second, although the hydration state of cholesterol in a phospholipid  matrix is not known, it may be expected to be relatively poorly hydrated in  comparison  to phospholipids.  Cholesterol  in water,  for example,  monohydrate form. Given that lipids which readily adopt the H  a  adopts  a (crystalline)  phase hydrate poorly in  comparison with "bilayer" phospholipids, the reduction in bound water per unit of membrane surface due to the presence of cholesterol could also play a role in promoting H -phase n  structure. The  ability of cholesterol to lower the amount of Ca  required to trigger bilayer-H  2+  n  transition in PS-PE dispersions poses interesting problems with regard to mechanism. Tilcock and Cullis (1981) showed that in the absence of cholesterol Ca " appears to trigger H -phase 2  n  formation in these systems by segregating PS into "cochleate" (Papahadjopoulas et al., 1975a) domains, leaving the phosphatidylethanolamine free to adopt the H isolation. Such  Ca  2+  induced  segregation  may occur to some extent  cholesterol, but the results obtained when Mg particular, high levels (50 mM) of Mg  2+  u  2+  phase it prefers in in the presence of  is present suggest an alternative possibility. In  are ineffective for inducing lateral segregation of PS  in mixed PS-PC systems (Ohnishi and Ito, 1974) and cannot trigger bilayer-H  u  transition in  mixed PS-PE systems (Tilcock and Cullis, 1981 and Fig. 10). Thus the ability of 4.0 mM and  higher  Mg  2+  concentrations  to induce H -phase structure when cholesterol is present n  either suggests that cholesterol facilitates the lateral segregation Mg -dependent incorporation of the PS into the H 2+  u  of PS or that it promotes  phase. The latter possibility appears the  most likely as there is no evidence for a Mg - induced segregation 2+  may  be estimated that more than 80% of the phospholipid  systems containing equimolar cholesterol adopt the H Mg  2+  50  in the soya PS-soya PE (1:1)  configuration in the presence of 8 mM  n  (Fig. 10). As soya PS in the presence of Mg  of PS. In particular, it  2+  maintains the bilayer organization (as  does equimolar PS-cholesterol in the presence of Mg ; M.J. Hope unpublished observation), 2+  Mg -induced lateral segregation of PS in the sample of Fig. 10 should leave 50% of the 2+  lipid in lamellar organization, contrary to experiment. It is suggested that the presence of Mg  2+  acts to decrease the charge density at the lipid-water interface. This may be considered  to reduce the electrostatic repulsion between serine head groups, thus reducing the effective size of the serine head group and producing a net molecular "shape" more compatible with the H  phase. This would allow the H -phase preferences of the PE to predominate and  n  would  n  correspond  to  the  situation  for  Ca -triggered  H  2+  phase  n  formation  in  phosphatidylglycerol-PE systems (Farren and Cullis, 1980). The  Ca -induced triggering 2+  of H -phase n  formation  in the soya PS-PE-cholesterol  (1:1:2 on a mole basis) systems may have a similar basis as the Mg -induced transition. The 2+  situation  where 2 mM  Mg " 2  acts as an adjunct to the Ca * 2  effect, lowering  the Ca  2+  concentrations required to 0.25 mM, w^uld be particularly likely to proceed via this charge neutralization mechanism. This is because such concentrations of Ca  2+  are well below those  required to induce lateral segregation of PS in mixtures with PE and even below those required to trigger formation of "cochleate" crystalline PS-Ca  2+  (2:1) complexes in pure PS  systems (Tilcock and Cullis, 1981). The ability of high salt concentrations to induce Hu-phase structure in PS-PE-cholesterol (1:1:2) systems also corresponds to charge neutralizations effects, and it therefore appears possible that the cation-dependent macroscopic structural alterations of these systems can proceed by a common mechanism which does not necessarily involve lateral segregation of individual components. This proposal would be supported by the investigations examining the mixing properties of DOPE/DOPC systems where cholesterol influences the polymorphic phase properties of the lipids (see Fig. 13). The results presented here suggest that cholesterol is not preferentially associated  with  the PC component  in these systems, arguing  against  the possibility that  cholesterol is associated with PC to form a "cone"-shaped (see Cullis and de Kruijff (1979)) complex  more  compatible  with  the H  n  phase. Similarly, the observation  DOPE-DOPC-cholesterol systems which exhibit regions of bilayer and H  51  n  that in mixed  phase organization  (as well as structures allowing isotropic motional averaging) the DOPC and DOPE appear to partition  into these  segregation  structures with  equal probability argues against  involving  of different lipid species into different phase structures. More recent experiments  et al., 1984) have demonstrated, by employing H  (Tilcock  explanations  2  NMR  (and x-ray diffraction  techniques) in a similar fashion to that illustrated in Fig. 13, that DOPS can adopt the H organization  when Ca  is added  2+  to mixed  systems composed  n  of DOPE/DOPS containing  cholesterol. This result provides more direct evidence for the conclusions derived here from the soya PE-soya PS mixed systems. The  resulting conclusion that these lipids remain ideally mixed even when such diverse  structural alternatives are available to them suggests that they will also behave in a miscible fashion in less extreme situations. In particular, it has been argued that local clusters with differing lipid compositions from the surrounding bulk lipid may exist in biological membranes and  that these microenvironmcnts may modulate local function (see section 1.3). The results  presented here would argue against such a hypothesis, suggesting that such clusters would not exist for times longer than the NMR The processes, erythrocyte  time scale (10" sec). 5  results presented here also have important implications for Ca - induced fusion 2+  as well  as the compositions  and properties  membrane. These areas will be discussed  cholesterol and cytosol levels of Mg  2+  of the inner  monolayer  of the  in turn. First, the observation  can reduce the Ca  2+  that  concentrations required to induce  nonbilayer structures in appropriate systems to 250 yyM or less supports the contention that membrane fusion processes in vivo proceed by intermediate formation of inverted micelle and (or) inverted cylinder lipid configurations (section 1.6.2 and Cullis and Hope, 1978; Verkleij, 1984;  Hope et al., 1981). Previously, the high (nonphysiological) Ca  more required to induce nonbilayer  2+  levels of 2 mM or  structures in and induce fusion between mixed PE-PS  vesicle systems posed major problems for extrapolating to in vivo situations. It should be noted that other  factors may act to reduce the Ca  include proteins such as synexin increase the effective Ca  2+  +2  levels required  even further. These  (Creutz et al., 1978; Hang et al., 1981), which appear to  concentration for fusion between model systems.  52  The monolayer  second point concerns the composition of  the  erythrocyte  membrane. First,  polymorphic preferences of this lipid composition  and  structural preferences of the inner  it is clear that  the  sensitivity  of  the  to the presence of divalent cations will be  markedly enhanced by the presence of cholesterol. This is in agreement with observations that Ca  2+  cannot trigger H -phase formation n  composition  in model systems mimicking the inner monolayer  in the absence of cholesterol (M.J. Hope, unpublished observations). Although the  transbilayer distribution of cholesterol in the erythrocyte membrane has not been established, it is  likely  that  cholesterol is present  at  levels  approaching  equimolar  (with  respect  to  phospholipid) proportions, given the ability of cholesterol to redistribute rapidly across bilayer membranes (Kirby and Green, 1977). Thus the structural preferences of the inner monolayer may  be  dictated by relatively low  cytosol levels of Ca  content  53  2+  or even by the local cholesterol  PRODUCTION OF  LARGE UNILAMELLAR VESICLES BY  PROCEDURE: CHARACTERIZATION OF TRAPPED VOLUME, AND  A RAPID EXTRUSION  SIZE DISTRIBUTION,  ABILITY TO  MAINTAIN A  MEMBRANE POTENTIAL!  3.1 Introduction As  described  in section 1.4, model membrane systems are in one  aqueous dispersions of phospholipids biological membranes. Obviously  form or another  that are designed to mimic some of the properties of  MLVs (multilamellar vesicles) have little relation to biological  membranes, yet they have been useful in determining the physical characteristics of a variety of lipid species. For example, the previous chapter utilized MLV show that different lipid species may and/or H  n  model membrane systems to  assume different polymorphic phases such as the bilayer  configurations depending on  the presence of such factors as cholesterol and/or  divalent cations. L i respect to understanding the role of lipids in regulating the permeability properties of membranes, however, the MLV  model membrane system is of limited value (see  section 1.4) and other systems must be employed. These model membrane systems must satisfy several stringent criteria, with the basic requirements that such model vesicles be closed and unilamellar and that the vesicle be reasonably large to enclose an appreciable trapped volume and to avoid problems associated with very small, highly curved systems (Schuh et al., 1982). These demands have either proved difficult to fulfill (see section 1.4 or for review see Szoka and Papahadjopoulos, 1980) or have not yet been attempted. A major problem involves the use  of lipid solubilizing agents (organic solvents or detergents). These techniques often  require lengthy dialysis procedures which can never completely remove the solvent or detergent employed. Further, a variety of protocols are required  depending on  the lipid species. For  example, the limited solubility of certain lipids (eg., cholesterol, phosphatidyl ethanolamine (PE))  tThis chapter has been based on the references Hope et al. (1984) and Bally et al. (1984).  54  in ether or ethanol requires modification of techniques employing these solvents. Alternatively, detergent dialysis procedures employing non-ionic detergents such as octylglucoside are tedious to apply as they can involve several days of dialysis. Before studies can membranes  a  general  be  and  designed to analyze the  straightforward  protocol  permeability  for production  functions of  LUVs  of lipids in is required.  Preferably, such a technique would avoid the use of solubilizing agents, would produce vesicles of a relatively homogeneous size and  would be  relatively rapid. In this work a procedure  which satisfies most of these criteria is presented. Further, these LUVs have been utilized to model a basic feature of biological membrane systems - namely the presence of a transbilayer membrane potential (  )• The  work of Olson et al. (1979) who  procedure for generating  LUVs involves an extension of the  describe a technique whereby the trapped volume of large  multilamellar vesicles can be increased by extrusion under relatively low 80 psi) through polycarbonate filters of 0.2  ym  pressures (less than  pore size. These vesicles are multilamellar  with a reasonably homogeneous size distribution centred about the pore size of the membrane. It was  reasoned that extrusion through smaller pores may  result in vesicles sufficiently small  that the presence of inner lamellae is unlikely. It is demonstrated that repeated extrusion under moderate pressures filters of 0.1 y m  (less than 500  psi) of multilamellar vesicles through polycarbonate  pore size results in a relatively homogenous population  vesicles. These LUVETs (large unilamellar vesicles by  of unilamellar  extrusion techniques) can be produced  rapidly (less than 15 min) in a manner which is relatively independent of lipid composition. Further, LUVETs can be generated at high lipid concentrations (up to 300  U mol/  ml)  and  can exhibit relatively high trapping efficiencies (up to 30%). These vesicles are employed to obtain a model system exhibiting a membrane potential. In addition, certain aspects of the influence of phospholipid composition on the maintenance of the permeability are investigated.  55  and  regulation of ion  3.2 Materials and Methods 3.2.1 Lipid  preparation  Egg PC  and soya PC  corresponding PE  and PS  were prepared as described in sections 2.2.1 and 2.2.2 and the  derivitives were prepared from the respective PC  exchange capacity of phospholipase D  using the base  (see section 2.2.1). PE and PS were purified and the  PS's were converted to the sodium salt as described previously. Cholesterol (Sigma, St Louis, MO)  was  used  without  further  purification. All lipids  were greater than  99%  pure as  determined by TLC.  3.2.2 Vesicle  preparation  Multilamellar vesicles (MLVs) were prepared by vortexing dry lipid in the presence of the appropriate aqueous buffer. The device (produced  by  resulting MLV  dispersion was  then transferred into a  Sciema Technical Services Ltd., Richmond, B.C.)  extrusion of the MLVs through standard 25 mm  polycarbonate filters with 0.1  (Nucleopore Corp., Pleasanton, CA). Briefly, MLVs (in 2-5 chamber above the polycarbonate filters, and  which  allowed the ym  pore size  ml) were injected into a central  nitrogen pressure applied via a standard gas  cylinder fitted with a high pressure (0-4000 psi) regulator. The vesicles were extruded through the filter employing pressures of 100-500 psi resulting in flow rates of 20-60 ml/min, and were collected and re-injected. Unless stated differently, the LUVET preparations were passed through  two  (stacked) filters  10 times. When the freeze-thaw procedure  was  utilized, the  vesicles were sized as above, freeze-thawed (employing liquid nitrogen) and then sized again through new filters. A total of two freeze-thaw cycles were usually employed.  3.2.3  Determination  of trapped volumes  To determine trapped volumes, the multilamellar vesicles were prepared in the presence of 1 u Ci of Na or C-inulin (NEN, 22  14  Canada) and the LUVETs made as indicated above.  56  An  aliquot (100 y 1) was  then loaded onto a Sephadex G-50  column packed in a 1 ml  disposable syringe, and vesicles eluted by centrifugation of the column at 500g for 3 min. In the case of Na this was sufficient to remove all the untrapped material. To remove all the 22  untrapped inulin, however,, this procedure was removed using a 2 ml  Ultragel (AcA  repeated twice or the untrapped inulin  34, LKB,  was  France) column. Aliquots of the eluted  material were assayed for lipid phosphorus (Bottcher et al., 1961, see following paragraph), trapped  22  Na  inulin was  was  determined employing a Beckman 8000 gamma counter and trapped  14  C  estimated using a Phillips PW-4700 liquid scintillation counter. Trapped volumes  calculated are expressed as y 1 of trapped volume per y mol of phospholipid. For  the  phosphate  assay,  samples  (containing  phospholipid) were digested in 0.6 ml of 70% HC10  4  between  0.05  and  0.2  ymole  at 190° C for 1 to 2 hr. Subsequently,  7 ml of ammonium molybdate reagent (0.22%, w/v, ammonium molybdate in 2% H S0 , v/v) 2  and  0.6 ml  of Fiske-Subbarrow  reagent (30 gms  l-amino-2-napthol-4-sulphonic acid  in 200  ml  NaHSO„,  1 gm  water at 40° C  Na S0 2  4  and  4  0.5  gms  to aid solubilization. The  reaction mixture was incubated at 100° C for 15 min, subsequently the sample was cooled to room temperature and the absorbance at 815 nm 3.2.4 Freeze-fracture  and  negative  was determined.  staining  For freeze-fracture, vesicle preparations were mixed with glycerol (25% by volume) and frozen in a freon slush. Samples were fractured and replicated employing a Balzers BAF 400D apparatus, and micrographs of replicas were obtained using a Phillips 400 electron microscope. Vesicle size distributions were determined by measuring the diameter of fractured vesicles that were 50% shadowed according to the procedure of van Venetie et al. Negative staining  was  treated  grids, using  2.5%  EM-10  electron microscope.  performed  ammonium  on  samples  placed  on  Formvar  coated, bacitracin  molybdate. Micrographs were obtained using a Zeiss  57  3.2.5  31  P  Nuclear  31  P  NMR  magnetic  resonance  was employed  to provide an indication of the extent to which the vesicle  preparations were unilamellar. Briefly, Mn umol phospholipid per ml in a 15 mm broaden beyond  detection the P  the  addition  of  Mn .  tube) at levels (5 mM)  unilamellar then 50%  The  2+  to the vesicle dispersion (3 ml, 30 sufficient to  signal from those phospholipids facing the external  medium. If the vesicles are large and following  added  diameter NMR  NMR  3l  was  2+  impermeability  of  of the signal should remain the  vesicles  to  Mn  was  2+  straightforward to demonstrate by following the timecourse of the signal intensity, which for the PC  systems investigated here was stable over a period of days. Spectra were obtained  employing section  a Bruker WP  200  NMR  spectrometer operating at 81.0  2.2.4. Signal intensities were measured  by  cutting  triphenylphosphite (in a small central capillary in the NMR  3.2.6 Membrane  potential  and  permeability  In order to produce the Na -K +  +  out and  MHz  weighing  studies  chemical gradients required to establish a membrane  which  NaCl, 20 mM was  membrane  lipophilic  HEPES, pH  pre-equilibrated  (Sigma, St. Louis, MO) The  potential  generated  was  G-50  measured  cation H-methyltriphenylphosphonium 3  dispersion which and  G-50  solution. Where employed, valinomycin  was added in ethanol to a concentration of 1 yg/y mol phospholipid.  3  withdrawn  mM  7.4) by passage through a Sephadex  with the NaCl  Canada). Routinely, 1 u Ci of H-MTPP LUVET  buffer (155  HEPES, pH 7.4). Subsequently, the untrapped buffer was exchanged for a NaCl  buffer (150 mM column  spectra with  tube) as a reference.  potential, LUVETs (20 u mol phospholipid per ml) were made in a KC1 K.C1, 20 mM  as described in  was  +  by  iodide  in 1 uul  determining the distribution of the ( H-MTPP\ obtained 3  ethanol was  added  from  to 1-2  then incubated at 20° C. At various times an  NEN,  ml of a aliquot  was  the untrapped H-MTPP* removed by passage through the 1 ml Sephadex 3  columns described above. The trapped H-MTPP was determined by liquid scintillation 3  counting and the phospholipid determined by phosphate assay. A potential difficulty with this  58  procedure  concerns the possibility that trapped H-MTPP 3  However, similar results were obtained employing below  for  42  K  in  the  presence  Knowledge of the trapped ((MTPP )/) and +  potential may Axp (mV)  =  +  and  an  equilibrium  dialysis  technique.  volume (see above) allows the concentrations of MTPP inside  be calculated employing the Nernst equation: -59 log [MTPP ]//[MTPP ]o +  36  determined  +  and CI" across the LUVET membranes subsequent to establishing  +  distributions (K  and C1" (NEN, was  valinomycin  the ultrafiltration technique detailed  +  +  +  both  leaks out while on the column.  outside ((MTPP » the vesicles to be calculated, from which the membrane  The flux of Na , K the Na /K  of  +  +  inside) were determined  employing  the radioisotopes Na , K , 22  +  42  +  Canada) in the absence and presence of valinomycin. Briefly, influx of Na by addition of Na 22  +  +  (2 y Ci/ml) to the external medium. Aliquots (100 y 1)  were then removed at various time intervals, the untrapped Na  +  removed by passage over the  1 ml Sephadex columns, and the Na influx determined by gamma counting. Efflux of K* in 22  the absence of valinomycin was  determined  by a similar procedure  where K 42  was  initially  trapped inside the vesicles. However, when valinomycin was added the column technique could not be  employed  due  to efflux of K  technique. In this case a 10 ml fitted  with  a YM  LUVETs from  10  Diaflo  while on  +  the column, necessitating an alternative  ultrafiltration unit (Amicon Corp.) operated at 5 psi and  (Amicon  Corp.) membrane  the external medium. Vesicles (40 y mol  volume) were made in the presence of K 42  buffer. Valinomycin was  added and  +  was  employed  to separate the  phospholipid per ml, 20 ml total  and the outside medium exchanged for the NaCl  for each time point 1 ml of the vesicle dispersion was  removed and placed in the ultrafiltration unit Aliquots (100 y 1) of the (untrapped) buffer were removed (within 30 sec) and the K  content determined. Knowledge of the initial  42  levels, the lipid concentration and trapped volume then allowed the distribution of K calculated.  59  +  42  K  to be  The CI" flux in and out of the LUVET systems was determined by similar procedures as the K  and Na  +  +  flux in the absence of valinomycin. In addition FT flux was estimated  using the procedure of Nicolls and Deamer (1980), where the outside pH function of time, which was susequently converted to H  +  was measured as a  (or OH") flux (see section 1.8).  3.3 Results 3.3.1 Characterizations  The  influence  of  LUVETs  of repeated passages  (MLVs) through polycarbonate filters of 0.2 EPC  of (initially) large ym  and 0.1  sequestered away from externally added Mn  vesicles passed through the 0.2  ym  2+  ym  multilamellar  EPC  vesicles  pore size on the amount of  is illustrated in Fig. 15. In the case of  filter the signal intensity drops to approximately 65%  after five passes then remains relatively constant This suggests an appreciable population of multilamellar vesicles, a conclusion supported by freeze fracture results (see Fig. 17(d) and related discussion). Extrusion of MLVs through the 0.1 y m filter, on the other hand, results in reduction in signal intensity to 50% after five or more passes, suggesting the presence of primarily unilamellar vesicles. This is illustrated in the negative stain micrographs of EPC LUVETs (Fig. 16) which indicate that after a single pass (Fig. 16(b)), a substantial number of multilamellar vesicles are still present in comparison to samples passed ten times through the filters (Fig. 16(c)). This conclusion is also supported by freeze-fracture studies for LUVETs formed from a variety of phospholipids as indicated in the micrograph of Fig. 17. Vesicles formed from SPC, SPC-SPS (1:1), and SPE-SPS (1:1) (Fig. 17 (a), (b), and (c) respectively) do not exhibit a significant number of cross-fractures (less than 0.1%) indicating the absence of inner lamellae. This contrasts with the SPC  vesicle system formed employing a 0.2  ym  filter (Figure 17(d)) where such cross-fractures are readily observable. The size distribution of SPC LUVETs was determined from freeze-fracture micrographs according to the procedure of van Venetie et al. (1980) and is illustrated in Fig. 18. The half-tone columns represent SPC 0.1 y m  LUVETs prepared by passing MLVs through two (stacked)  pore size filters ten times. Assuming an area per phospholipid molecule of 0.6  60  nm  2  Fig.-- 15. P NMR signal intensity arising from egg PC multilamellar vesicles (in the presence of -5 mM MnCl ) as a function of the number of extrusions through polycarbonate filters with 100 nm (#) and 200 nm (•) pore sizes. The error bars represent standard deviations (n = 6 for the point at 10 extrusions through the 100 nm filter; n = 3 for the point at 30 extrusions). All other experimental points represent the average obtained from two separate experiments. The lipid concentration was 50 mg/ml. For other details see section 3.2. 31  ;  2  -II-  100 r  D c  CO  40 20  0 0  2  4  6  8  10  Number of Passes  61  30  Fig.  16.  Micrographs  multilamellar mg/ml  vesicles  phospholipid.  of (A)  negatively through  100  stained nm  (2.5%  (w/v)  polycarbonate  Sonicated (small) unilamellar vesicles  ammonium filters (D)  are  twice  molybdate) (B)  included  egg  PC  times  vesicles  or  ten  (C)  for  size comparison.  on  prepared lipid  by  systems  extrusion containing  of 100  Fig. 17. Freeze-fracture micrographs of vesicles prepared by repeated extrusion of multilamellar vesicles of varying lipid composition through polycarbonate filters: (a) soya PC MLVs extruded through a 100 nm filter; (b) soya PC-soya PS (1:1) MLVs extruded through a 100 nm filter; (c) soya PE-soya PS (1:1) MLVs extruded through a 100 nm filter; (d) soya PC MLVs extruded through a filter with 200 nm pore size. The arrow in part (d) indicates a cross fracture revealing inner lamellae. All micrographs have the same magnification and the direction of shadowing is indicated by the arrowhead in the bottom right corner of each section. The extrusion procedure was repeated 10 times on lipid systems containing 50 mg/ml phospholipid. Data collected and reproduced with permission from M. J. Hope.  Fig. 18. Size distribution of soya PC vesicles obtained after 10 extrusions through a polycarbonate filter with 100 nm pore size. The vesicle diameters were measured from freeze-fracture micrographs employing the technique of van Venetie et al. (1980). The half-tone columns represent vesicles that did not undergo a freeze-thaw cycle, the black columns represent vesicles subjected to the freeze-thaw protocol outline in section 3.2.2.  i  1  i  25  50  1  i  70  Vesicle  64  90  i  115  Diameter  i  140 (nm)  i  160  (Schieren et al., 1978) and a bilayer thickness of 4 nm are unilamellar, the trapped  volume and  (Blaurock, 1982) and that the vesicles  amount of inner monolayer phospholipid can  be  calculated for such a distribution. These estimates can be compared with the experimentally observed  trapped  volumes and  amount of inner monolayer phospholipid  to determine the  proportion of unilamellar vesicles present For the vesicle size distribution presented in Fig. 18 (half tone), it can be calculated that such a vesicle population (if unilamellar) would have an "inner monolayer" signal intensity (after the addition of Mn ) of approximately 2+  original intensity and may  that the trapped volume would be approximately  be compared with the measured values of sequestered  volume (1.2 ±0.2  yl/y mole). The  agreement between  1.6  43%  y l/|j mole. This  phospholipid (48%) and  measured  and  of the  trapped  calculated results is  perhaps reasonable given the number of assumptions, in particular the area per phospholipid molecule which can greatly affect the trapped volume expressed as y l trapped per y mole of phospholipid. However, as shown in Table 2, LUVETs composed of SPC  and EPC consistently  exhibit trapped volumes smaller than those expected from a unilamellar vesicle of the same diameter assuming an charged  phospholipid  area/molecule of 0.6  2  and  a bilayer thickness of 4 nm.  species such as phosphatidylserine  volume is achieved. Two SPC  nm  is present  the  theoretical  If a trapped  possible reasons for the low trapped volumes observed for EPC  and  LUVETs are that there are a significant number of multilamellar vesicles present in the  population, or that there are a greater proportion of small vesicles present than estimated from the freeze-fracture micrographs. It has been noted (Pick, 1981) that sonicated vesicles increase in size, but remained unilamellar, following a freeze-thaw cycle. For  this reason  SPC  and  > subjected to a freeze-thaw cycle followed by re-sizing through 0.1 y m  EPC  pore size filters in  an attempt to reduce the proportion of smaller vesicles in the preparation. The distribution for freeze-thawed SPC  distribution  measured values for SPC  resulting size  LUVETs is given in Fig. 18 (black columns). The mean  diameter of the population increased by approximately for this vesicle  LUVETs were  is 2.3 y 1/y mole which  20 nm.  The  calculated trapped volume  is in excellent agreement with  the  LUVETs following a freeze-thaw cycle which fall within the range  65  Table 2 Physical characteristics of vesicles produced by extrusion of a variety of lipid mixtures through filters with a pore size of 100 nm. Lipid  % Intensity!  Mean Diameter ±S.D. (nm)  Mean Trap Volumett +S.D. ( u 1/ UTtiole)  1  EPC SPC EPC/EPS (2:1) SPC/SPS (2:1) SPE/SPS (2:1) SPS EPS  48 48 46 ND ND ND ND  71 70 73 73 79 ND ND  EPC (Freeze-thaw) SPC (Freeze-thaw)  51 48  77 + 16 94 ± 26  2.2 ± 0.5 (17) 2.2 ± 0.1 (12)  EPC (Octylglucoside) EPC (REV)  49 50  ND ND  1.2 ± 0.1 (3) 1.2 (2)  + ± ± ± ±  24 23 25 20 36  1.1 1.2 1.5 2.4 2.0 2.3 2.2  t Intensity of P-NMR signal remaining in the presence of 5 mM Mn \ f t u 1/umole phospholipid (number of experiments in parenthesis). 31  2  66  ± 0.1 (64) ± 0.2 (13) (2) (2) (2) (2) (2)  of 2.0-2.5 y l/y mole (Table 2). These observations lead to the conclusion that the large majority of vesicles produced by repeated extrusion of PC of  a freeze-thaw  MLVs through a filter with 0.1 y m  pore size in the absence  step are unilamellar, even though the trapped  expected. In order to reinforce this conclusion, EPC which are widely accepted  volume is smaller than  LUVs were prepared  to produce unilamellar vesicles and  by two  procedures  subjected them to the same  sizing procedure employed here. As indicated in Table 2, the trapped volumes measured for EPC and  LUVs produced by the octylglucoside detergent dialysis procedure (Mimms et al., the reverse phase evaporation procedure (Szoka and  Papahadjopoulos, 1980), which were  subsequently extruded (10 times) through the filter with 0.1 the trapped volumes obtained for the EPC octylglucoside procedure was  1981)  m  pore size, are comparable to  LUVETs. It is pertinent to note that when the  employed to make vesicles consisting of EPC/cholesterol (1:0.25),  multilamellar vesicles were formed. An  important parameter of LUV  volume of trapped hydrate  buffer expressed  preparations is their trapping efficiency, ie. the total  as a percentage of the initial buffer volume used to  the dried lipid film, is particularly important  either expensive,  as is the case  when the agents to be trapped are  for many drugs, or have low  preparations described above have a trapped volume of 1-3 preparations  described  in  Papahadjopoulos, 1980)  and  the  literature  have  therefore might be  higher  expected  solubilities. The  LUVET  y 1/ ymole phospholipid. Other trapped  volumes  (Szoka  and  to allow better trapping efficiency.  However, an advantage of the LUVET protocol is that very high lipid concentrations can be employed. This is demonstrated in Fig. 19 where the percentage of aqueous volume trapped inside the LUVETs  is plotted against lipid  concentrations of up  to 300  concentration. Preparation of LUVETs at lipid  moles/ml is quite possible, giving rise to a 30%  u  trapping  efficiency even though the trapped volume of the vesicles are only 1 u 1/ umole lipid. It is interesting to note that the freeze-thaw cycle only gives rise to significant increases in trapped volume per observations  ymol have  of lipid been  at lipid  reported  concentrations  elsewhere  67  below  150  mg/ml. Similar types of  (Pick, 1981). It should  be  noted  that  the  Fig. 19. The trapping efficiency of LUVETs prepared without (#) and with (O) freeze-thawing. C- inulin was used as a trap marker and LUVETs were prepared at various concentrations of EPC as described in section 3.2. 14  unilamellar nature (as determined by NMR at lipid concentrations greater than 50 3.3.2  Characterization  The  of LUVETs  and freeze-fracture techniques) of vesicles prepared  umoles/ml has not been well characterized.  with a  membrane  potential  LUVET system as characterized above would appear to have a wide range of  application due  to the  preparation  the generality and  and  absence of contaminants (detergents,  organic  solvents), the  reproducibility of the technique. As an illustration of this  utility, a simple model membrane system exhibiting a transmembrane potential Axp developed and  ease of  characterized. Reasons for such a choice  include the  has been  demonstrated biological  importance of AI/J in a variety of processes (see section 1.8) and the present lack of a well characterized unilamellar model system exhibiting a membrane potential, which would serve as an  initial step  in modelling  the  more complicated  biological situation. For  example, the  potentials exhibited by biological membranes correspond to very large electric field gradients (a typical value of 100 may  be  mV  corresponds to a transbilayer electric field of 250  kV/cm),  which  expected to influence the transmembrane distributions of charged lipid and protein  components. A appropriate  simple model system incorporating  system  for studying  a membrane potential would provide an  these interesting possibilities as is illustrated  in the  two  subsequent chapters. The  particular model system characterized is a SPC  transmembrane chemical gradients (K KC1  buffer and  LUVET preparation where NaVK  interior) are established by preparing  +  then exchanging the untrapped KC1  +  the vesicles in a  for a NaCl buffer by  gel filtration.  Questions addressed concern whether such systems exhibit a membrane potential in the absence of agents (valinomycin) to enhance K valinomycin is, how and  how  quantify  +  the magnitude of  permeability  (see section 1.8), what the influence of  AiJ> is related to transmembrane NaVK* distributions  the stability of the potential may  be influenced by lipid composition. In order to  Aip the radiolabeled lipophilic cation [ H]-MTPP was 3  been used extensively to obtain measures of and Kaback, 1975;  Lichtshtein et al., 1979;  +  ATJ> in energized  Young et al., 1983).  69  employed. This agent has  biological systems (Schuldiner  As Na /K +  +  illustrated  chemical  determination (interior  in Fig. 20, the LUVET  gradient (K  of outside  negative)  preparations  experiencing  a transmembrane  interior) do exhibit a membrane potential as detected by  +  and inside concentrations  of -20 mV  of [ H]-MTPP . A 3  is apparent at the first time  +  interval  (30 min). This  AI/J  gradually  decreases to -30 mV over eight hours. It may be noted that the equilibrium distribution of MTPP  +  across the vesicle membranes was achieved within 10 min. As also indicated in Fig.  20, the Na /K +  +  chemical  gradients remain  constant  within  the limits  of  experimental  detection over the incubation period. This reflects the low permeability of these ions across the  vesicle membrane (P  value  less than  observations (see section 1.8). The Na  +  limits even after a 24 hr incubation  10"  cm/sec) and is consistent with  10  previous  influx into the SPC LUVETs remained below detection at 20° C.  These results indicate that a membrane  potential is established in the SPC LUVET system in the presence of a transmembrane concentration gradient of NaVK . This potential presumably arises from a difference in the +  permeability  {P  potential if K  +  values) of SPC LUVETs  to Na  leaks out faster than Na  +  and K , which can create a diffusion  +  +  leaks in (see section 1.8 for details). This is  consistent with the larger permeability coefficients observed for K  +  than for Na  +  in a variety  of membrane systems (Jain and Wagner, 1980; Papahajopoulos and Bangham, 1966). When NaVK  +  the K  +  ionophore  valinomycin  is added  to the SPC LUVET system  chemical gradients, much different behaviour is observed as illustrated in Fig. 21. The  (absolute) values of |A^|as reported by t H]-MTPP 3  +  are much larger (120 mV after 30 min  incubation in the presence of valinomycin at 20° C), and AifJ  decays fairly rapidly for longer  incubation periods. In addition, this decay is accompanied by significant Na efflux. These results indicate that, as expected, valinomycin enhances the K  +  influx and K  +  permeability which results in dissipation of the K  +  +  permeability and  +  thus gives rise to larger (absolute) values of | AI(J,| but also that valinomycin Na  with  enhances the  electrochemical gradient as Na  +  leaks  in. In order to demonstrate that the values of  reported by MTPP  +  reflect the K  +  diffusion potential generated, the potentials calculated from the measured interior and exterior  70  Fig. 20. Time course at 20° C of the membrane potential (A) and inside K (#) and Na (•) concentrations in soya PC LUVETs (20 umole lipid/ml) on application of a Na /K chemical gradient (KC1 interior; NaCl exterior). The gradient was established by preparing LUVETs in 150 mM KC1 (pH = 7.0) and subsequent pasage through a Sephadex G-50 desalting column equilibrated with 150 mM NaCl (pH = 7.0). The membrane potential was determined by measuring the transmembrane distribution of [ H]-MTPP (see section 3.2.6) whereas the interior Na and K concentrations were determined employing Na and K. radioisotopes. Non-specific binding of the probe to LUVETs was measured in control experiments where K was present inside and outside the vesicles, the experimental points were corrected accordingly. +  +  +  3  +  +  22  +  Hours  71  42  +  Fig. 21. Time course at 20° C of the membrane potential (A), interior K (#) and Na (•) concentrations in soya PC LUVETs (20 y mole lipid/ml) on application of a Na /K chemical gradient (KC1 interior, NaCl exterior) and in the presence of valinomycin (1 yg/ ymol phospholipid). For details see Fig. 20 legend. +  +  +  ;  i  i  1  1  1  i  i  i  i  i  0  1  2  3  4 Hours  72  1  _i  5  1  1  i  i  i  i  6  7  8  +  concentrations of K  ([K]z and  +  [K]o) were determined  section 3.2.6. As illustrated in Fig. 22, the values of in  good  agreement and  emphasized  exhibit  similar  time  ATJJ  dependent  employing  42  K  as indicated in the  obtained from both procedures are behaviour. This point  is further  by data illustrated in Fig. 23. Using [ H]-MTPP to determine the valinomycin 3  induced membrane potential exhibited by EPC  +  LUVET systems (regular and freeze-thawed),  one observes a reasonably close agreement between the value of ATJ> calculated on the basis of known transmembrane K  +  distributions and the transmembrane distribution of [ H]-MTPP . 3  +  There is some error observed at the high absolute values of | AIJJ [ (greater than 100 mostlikely due to loss of MTPP The employing  inward C1  36  and  +  during separation on the Sephadex columns.  outward flux of CI" in these SPC  systems was  also characterized  (see section 3.2.6). The permeability coefficients calculated from the observed  influx and efflux of CI" are similar, P values of approximately 7.0 x 10"  11  that the transmembrane diffusion of CI" is electrically silent permeability sonicated  mV),  coefficients  EPC  vesicles  for CI" are in close (Toyoshima  and  It may  agreement with  Thompson,  1975)  and  cm/sec, indicating  be  noted  results obtained EPC  LUVs  that the employing  prepared  by  detergent dialysis employing octylglucoside (Mimms et al., 1981), yet inconsistent with studies which have demonstrated rapid (P value of greater than 10") transmembrane equilibration of s  CI"  ions when  the ion is present on  Preliminary studies examining  both  sides of the membrane (see section 1.8).  the permeability of H  +  in EPC  LUVET systems indicate that  this ion is not readily permeable across the vesicle bilayer, giving P values in the range of 10"  11  to 10"  14  cm/sec. Interpretation of these P values are complicated by the fact that these  systems exhibit a decrease proton  H  +  diffusion  potential  flux. This is indicated  (negative interior) which in Chapter  5  which  membrane potential can be established by a transmembrane H  73  +  would  demonstrates  ion gradient  be  expected to that a stable  Fig. 22. Time course of the membrane potential as determined by [ H]-MTPP (•) and K distribution (•) in soya PC LUVETs on application of a NaVK transmembrane chemical gradient and subsequent introduction of valinomycin (see Fig. 20 legend and section 3.2.6 for details). 3  +  0 r  > E -  -60 •  c O  C D _Q  £  -120 -  -180  74  +  +  , ; ••<• Fig. 23. Comparison between the membrane potentials obtained for various transmembrane K chemical gradients as detected by [ H]-MTPP for regular (O) and freeze-thawed (•) . LUVETs and the theoretical potentials (#) predicted by the Nernst equation. EPC LUVETs were prepared in a K glutamate buffer (169 mM KGlu, 10 mM HEPES (pH = 7.5)), and .. the untrapped (exterior) buffer replaced by an isoosmotic NaCl buffer (150 mM NaCl, 10 mM HEPES (pH = 7.5)) containing various amounts of KGlu. The membrane potential AIJJ was determined employing [ Hj-MTPP (see section 3.2.6) in the presence of valinomycin after a 6 hr incubation at 20° C to ensure an equilibrium transmembrane distribution of MTPP . The solid line indicates the theoretical potential given by the Nernst equation: Aip (mF) = -59 log ([K]//[K]o). 3  3  +  +  +  —i  1  —  i  i  -240  -200 -  o •  >  .3 - 1 6 0  _  o  <  Q -120  So  M  °  -80  -40  a  •  1  <  1  3  2  75  4  +  3.4 Discussion  This work presents have  been  employed  potential. The  to  a new  procedure for generating  develop  a  simple  system  exhibiting a  K  +  diffusion  LUVET procedure per se will be discussed first and subsequently the utility of  a model membrane exhibiting a well defined The  unilamellar  large unilamellar vesicles which  LUVET  system  obtained  polycarbonate filters of 100 nm systems produced by  by  AI[J will be discussed.  extrusion  of large multilamellar  vesicles through  pore size appear to possess significant advantages over  other means. One  LUV  of the most important is the absence of residual  organic solvent or detergent While it is arguable whether the low levels of such contaminants present  after extensive  dialysis  significantly  perturbs  such  properties  as  permeability  and  dynamic behaviour of component lipids, the situation is clearly less ambiguous when such agents are not present  at all. In addition, in applications such as the generation  of drug  carrier systems, the total absence of these potentially toxic agents is obviously beneficial. A second advantage of the LUVET procedure concerns the generality of the technique. All  lipid  systems thus far investigated, which give rise to large multilamellar vesicles on  mechanical agitation (vortexing) in the presence of buffer, can subsequently be converted to LUVET form employing the extrusion procedure. Further, these systems exhibit a relatively constant  size distribution and  associated trapped volume, allowing  direct comparisons of the  properties of unilamellar systems with differing lipid compositions. These features, coupled with the ease and  speed of preparation, the range of lipid concentrations that can be employed,  the high trapping efficiencies that can be achieved  and  the relatively gentle nature of the  procedure establish it as a most attractive protocol. One low  drawback of the LUVET systems as detailed here, however, concerns the relatively  trapped volumes (1-3  ul/umol  lipid) that can  be  achieved. Although high  efficiencies can be attained by increasing the lipid concentration, situations can be  trapping envisioned  where it is desirable to emp'loy unilamellar systems with increased trap to lipid ratios. In this regard  preliminary  studies on  soya PC  and  76  egg  PC  systems show that a freeze-thaw of  LUVETs prepared by extrusion through the 0.1 y m  pore size filters, followed  by extrution  through filters with larger (0.2 ym) pore size results in larger systems with trapped volumes on the order of 10 y 1/ ymol lipid. The achieving  LUVET systems exhibiting a large membrane potential are an important step in  more sophisticated and accurate models of biological membranes and understanding  the functional roles of individual components. Aside from obvious applications in examining possible roles of various lipid species (such as cholesterol) in the maintenance of as a simple test and calibration system  ATJJ  as well  for development of fast and accurate probes of  membrane potential, this system exhibiting a AX/J should allow several aspects of membrane structure and function influence of of  to be examined  in a direct manner. Three  examples include the  on transbilayer distribution of charged lipid and protein components, the role  membrane  potential in protein  relationships between  insertion (Wickner, 1983; Zwizinski, 1983) as well as  Axp and certain membrane transport processes. With regard to the latter  area, the massive uptake of safranine and other biologically relevant lipophilic cations (drugs and biogenic amines) into LUVET systems in the presence of a membrane potential has been demonstrated  and is described  membrane potential or pH  in the following  gradient  to LUV  chapters.  Finally, the application  systems reconstituted  of a  with specific membrane  transport proteins could also provide simple assay procedures for transport mechanisms relying on K  +  or H  +  symport and antiport processes.  77  U P T A K E O F SAFRANINE A N D O T H E R LIPOPHILIC CATIONS INTO M E M B R A N E S Y S T E M S IN RESPONSE T O A M E M B R A N E  MODEL  POTENTIALt  4.1 Introduction  As  shown in the previous chapter, membrane permeable lipophillic cations, such as  [ H]-MTPP\ 3  can be accumulated  by cells or organelles exhibiting a membrane potential.  Subsequent determination of the interior and exterior concentrations of these cations can allow estimates of  to be made. In addition to MTPP, there are a variety of other probe  molecules including those which exhibit optical and fluorescent responses (such as safranine 0) when accumulated into the interior of the energized system. These agents have been utilized to determine Aip  in mitochondria  and chloroplasts (Skulachev, 1971; Waggoner, 1979), vesicles  derived from prokaryotic and eukaryotic membranes (Schuldiner and Kaback, 1975; Ramos et al., 1979) as well as a variety of intact cells (Lichtshtein et al., 1979; Young et al., 1983). An  interest in this uptake of lipophilic cations was stimulated by two observations.  First, the accumulation of these components by energized  systems can result in substantial  differences between the concentrations of the probes in the external and internal compartments. A  membrane potential of -100 mV as detected by MTPP\ for example, reflects an interior  probe concentration  which  is 50 times higher  than in the external environment  As the  redistributions of the probes across the membranes can occur within minutes, it is clear that accumulation of lipophilic cations in response to Axp  reflects a rather effective membrane  transport process. Second, a large variety of biologically active agents such as certain biological amines and many drugs are essentially lipophilic cations. Thus processes involved in the uptake of lipophilic cations employed as membrane potential probes may well be of more general significance to metabolite and drug distributions in vivo.  t  This chapter has been based on the references Bally et al. (1984), Mayer et al. (1984a),  and Mayer et al. (1984b).  78  In this work the ability of LUVET systems to accumulate selected lipophilic cations in response to a membrane potential, is characterized with three objectives in mind. First, to show that the large changes in absorbance observed on accumulation of optical probes such as safranine in biological preparations can also be observed in model systems exhibiting a Aip , and  to  characterize  membrane  the  potential are  mechanisms present  involved.  at as  electrochemical gradient giving rise to  low  Second, as  levels  as  usually  possible  to  employed, avoid  probes  perturbing  of the  AI|J. From the transport point of view, it is of interest  to determine whether higher exterior concentrations can lead to high absolute concentrations of probe accumulated in the vesicle interior. Finally, to determine whether representative drugs can  also be  accumulated by  vesicles exhibiting a membrane potential, which may  provide  information regarding the non-specific uptake and distribution of these agents in vivo. It  is demonstrated  phosphatidylcholine  (EPC)  that the  safranine  optical  response  vesicle systems exhibiting a K  +  can  be  observed  diffusion potential, and  marked absorbance changes observed appear to correspond to precipitation of the vesicle interior. Further, both safranine and MTPP than 75 mM)  for  egg  that the dye in  can be concentrated to high levels (greater  +  in the vesicle interior for initial exterior concentrations in the range 1-2  mM.  Finally, it is shown that a variety of different classes of drugs, including the local anaesthetics chlorpromazine and  dibucaine, the adrenergic  drugs vinblastine and  adriamycin,  systems in response to  can  blocking agent propranolol, and  also be  accumulated  the anticancer  into model membrane vesicle  •  4.2 Materials and Methods 4.2.1  Materials  Egg  phosphatidylcholine (EPC)  and  soya PC  (SPC)  were isolated from hen  egg yolk  and crude soya lecithin (Sigma) respectively employing procedures described in chapter 2. Egg phosphatidylserine (EPS) was  obtained from EPC  79  utilizing the headgroup exchange capacity of  phospholipase D (see section 2.2.1). Cholesterol, MTPPBr, chlorpromazine, dibucaine, propranolol, vinblastine, valinomycin  and HEPES  buffer were obtained  from  Sigma. Safranine O was  obtained from MCB whereas methoxy [ C]-inulin and [ H]-methyltriphenylphosphonium iodide 14  3  ( H-MTPP) were obtained from NEN (Canada) and K C L from Amersham. Adriamycin was 3  42  the generous gift of Dr. A.C. Eaves. For ease of reference, the structures of safranine, MTPP , chlorpromazine, dibucaine, propranolol, adriamycin and vinblastine are given in Fig. 24. +  All lipids employed were more than 99% pure as determined by TLC, and all other reagents were employed without further purification. 4.2.2  Vesicle  preparation  Vesicles were prepared  according to the LUVET prodecure  detailed in the preceding  chapter which involved hydration of a dry lipid film to produce large multilamellar vesicles (50 100  ymol phospholipid/ml)  which  were subsequently  extruded  10 times  through two  (stacked) polycarbonate filters with 100 nm pore size (Nucleopore). Lipids were hydrated in either the potassium glutamate (KGlu) and NaCl buffers contained 20 mM HEPES adjusted to pH  7.5 employing NaOH (final Na  +  concentration 10 mM) and were adjusted to a common  osmolarity of 310 mOsm/kg (NaCl concentration 150 mM, KGlu concentration 169 mM).  4.2.3  Generation  of a membrane  Transmembrane LUVETs in the KGlu  Na -K +  potential  chemical  +  gradients (K* inside) were generated  buffer and subsequently  employing a Sephadex G-50 column. Defined K  +  exchanging the untrapped  by forming  KGlu  for NaCl  gradients were generated by pre-equilibrating  the G-50 columns with isoosmotic NaCl buffers containing the appropriate concentration of KGlu. Where employed, the K  +  ionophore valinomycin was added to achieve a concentration  of 0.5 u g/u mol lipid.  80  Fig. 24. Structures of safranine, methyltriphenylphosphonium (MTPP*), chlorpromazine, dibucaine, propranolol, vinblastine, and adriamycin.  METHYLTRI PHENYL PHOSPHONIUM  5AFRANINE  ? ?  H  CH CHCH H HN—CH ° PROPRANOLOL CH 2  2  / C  3  N  3  81  4.2.4 Uptake  The  of  safranine  uptake of safranine was  interval 560-460 nm related text). The  (qualitatively) monitored spectrophotometrically over the  employing a Pye Unicam SP8-200 spectrophotometer (see Fig. 25 and actual amount of safranine accumulated  adding safranine from a saturated (96.8 mM (2-10  was  determined quantitatively by  at 20° C) stock solution to a LUVET dispersion  ymol lipid/ml) to achieve a 2 mM  safranine concentration. Subsequently, at appropriate  time intervals, the unsequestered dye was removed by passing aliquots of the vesicles through small (1 ml) Sephadex G-50 (w/v)  Triton  X-100  columns, and aliquots of the effluent were mixed with 0.5%  to disrupt  the  vesicles  and  release  sequestered safranine. Safranine  concentrations were then determined from the absorbance at 516 nm,  and the phospholipid  phosphorus assayed by the technique described 3.2.3.  4.2.5 Determination  of membrane  potential  and  Membrane potentials were determined the  efflux of K  +  measured employing  42  K  K  +  efflux  employing  [ H]-MTPP 3  (see section 3.2.6) and  +  in combination with the ultrafiltration technique  described in the preceding chapter (see section 3.2.6). 4.2.6 Uptake  of assorted drugs  Uptake determined  of chlorpromazine, dibucaine, propranolol,  quantitatively  chlorpromazine was  employing  similar  procedures  as  monitored in a system containing 200  u Ci/ml [ H]-chlorpromazine) which was 3  vinblastine,  added to EPC  and  adriamycin  for safranine. Accumulation yM  was of  chlorpromazine (containing 2  LUVETs (1  ymol phospholipid/ml).  After various incubation times, the vesicles were separated from unsequestered chlorpromazine (employing  1  ml  Sephadex  G-50  columns).  The  effluent  was  counted  to  assay for  chlorpromazine and phospholipid was assayed as described previously (Ames and Dubin, 1960). Dibucaine uptake was measured in a system which contained 100  yM  dibucaine and 1  mM  phospholipid. The vesicle associated dibucaine was measured fluorometrically (excitation, 330nm;  82  emission, 416  nm)  employing  a  Perkin-Elmer  disruption of the vesicles in 0.5% fluorometrically incubated  (excitation, 292  adriamycin, vesicles (1 mM)  Triton X-100. Accumulated  nm;  for various times with  650- 10S fluorescent spectrophotometer  emission, 337 M  200  propranolol was  also assayed  after the vesicles (1 mM)  nm)  after  were  propranolol. In the case of vinblastine and  were incubated with a 200  \iM  solution of the respective drug,  and the LUVET associated adriamycin and vinblastine were assayed (after disruption with 0.5% Triton X-100  or 93% (v/v) ethanol) spectropotometrically at 480 nm  and 265 nm, respectively.  4.3 Results  4.3.1  Characterization  of safranine  accumulation  by  LUVETs  Large absorbance changes can occur when safranine is incubated in the presence of energized biological systems such as mitochondria (Colonna et al., 1973) and vesicles derived from E. coli membranes (Schuldiner and Kaback, 1975). Such a "safranine response" has been correlated  with the presence of a membrane potential  observed  for EPC  LUVET  systems  experiencing a  . This response K  +  diffusion  potential  can  also be  induced  by  valinomycin as illustrated in Fig. 25. The addition of valinomycin to LUVETs prepared with asymmetric  Na -K +  transmembrane distributions (K  +  +  inside) and incubated in the presence of  safranine results in a marked time dependent decrease in absorption in the region of 516  nm,  which is combined with a shift in the absorbance maximum ( A max)  nm.  The change in the absorbance at 516 nm the situation is then relatively stable as  (/\ A ) J16  AA  m s  from 516 to 472  is essentially complete after 20 min and  decreased by less than 20% over a 24 hr  time course (results not shown). As  may  be  expected, the  safranine response were found  extent of the absorbance  changes associated  to be sensitive to the vesicle and  with the  safranine concentrations  employed. In order to determine optimum conditions, the (normalized) absorbance changes were monitored  as  corresponding  a  function  to 0.5  mM  of  safranine  concentration for  phospholipid. As  83  indicated  a  fixed  vesicle concentration  in Fig. 26, the normalized optical  Fig. 25. Spectrophotometxic response observed for safranine incubated at 20 C in the presence of EPC LUVETs exhibiting an electrochemical NaVK gradient (K interior) in the presence of •valinomycin. The LUVETs were prepared in the KGlu buffer, and the untrapped buffer exchanged for the NaCl buffer as described in section 4.2.2. Subsequently the vesicles were diluted to achieve a concentration of 0.5 mM phospholipid in 3 ml of NaCl buffer which contained 60 u M safranine. Spectra were taken before (upper trace) and at various times (in seconds(s) and minutes(m)) after the addition of valinomycin (0.5 ug/umole phospholipid). +  150-  460  4&0  500  520  540  W a v e l e n g t h (nm)  84  560  response then exhibits a maximum in the region of 60 y M  safranine. Other investigators (for  review see Zanotti and Azzone, 1980) have suggested that the safranine response arised from active uptake of safranine followed by a membrane associated "stacking" phenomenon, which gives rise to the observed absorbance changes. This process (which, as indicated below, appears to correspond to precipitation of safranine in the vesicle interior) could explain the behaviour noted in Fig. 26. At vesicle interior may  low  safranine concentrations  the  dye  concentrations  achieved  in the  not be sufficient to result in precipitation or "stacking", whereas at higher  exterior safranine levels, the spectral response for accumulated material will be swamped by the absorption from excess exterior safranine. The the EPC  results of Figs. 25 and  26 are consistent with an accumulation of safranine into  LUVET system which is driven by a K  +  diffusion potential. It is of interest to  quantify the extent of safranine uptake in response to a given investigated;  first, where the  safranine  was  in  accumulated safranine could be characterized, and therefore allowing a determination were  prepared  corresponding  with  varying  safranine  per  ymol  transbilayer K  over  a  for 30 min  phospholipid  Sephadex  leads  to  vesicle-associated  two  G-50  conclusions.  safranine  reaches  with  +  concentration  gradients  which  levels of limiting,  0.2  column  (see  phospholipid First,  levels  when  section  ymol  give  rise  and  0.05  ymol  safranine  per  ymol  then removed by passing the 4.2.4).  Determination  of  the  resulted in the data shown in Fig. 27, the  in excess of  amount  of  0.04  ymol  safranine safranine  is  limiting,  per  ymol  (interior negative) indicating trapping efficiencies in  a resulting transmembrane safranine gradient in excess of 500  fold.  Second, when safranine is in excess, extremely high levels of accumulated safranine can achieved  (greater than 0.12  to  (see 3.3.2, Fig. 23).  in the presence of valinomycin  untrapped safranine was  phospholipid for A\JJ greater than 80 mV excess of 80%,  maximum  second, where the safranine was  (safranine limiting) or  vesicle-associated safranine per y mol which  the  obtained on addition of valinomycin  phospholipid (safranine in excess). The vesicles  that  situations were  of the efficiency of the trapping process. Briefly, LUVETs  variations in the Atp  These vesicles were incubated  excess so  Axp • Two  ymol safranine per  85  y mol  be  phospholipid). Given the measured  Fig. 26. Influence of increasing safranine concentrations on the (normalized) safranine response obtained in the presence of EPC LUVET systems (0.5 mM phospholipid). The LUVET systems were prepared with NaVK electrochemical gradients as indicated in the legend to Fig. 25 (and section 4.2.4) and the normalized safranine response (A A max/A§ ) is measured as the difference between the initial absorbance (A? ) at 516 nm (before the addition of valinomycin) and the absorbance observed 20 min after the addition of valinomycin divided by +  J16  u  16  • A"•sit0  25  50  75  [Safranine] p M  86  100  Fig. 27. Levels of LUVET associated safranine obtained as a function of (initial) transmembrane potential AI|J . The vesicles were prepared from EPC dispersed in the KGlu buffer, and the external medium was replaced by a NaCl buffer containing various concentrations of KGlu (see legend of Fig. 23) to establish a range of K gradients. The membrane potential Aifj developed in the presence of valinomycin was assayed employing [ H]-MTPP (see section 3.2.6). Subsequently, the amount of safranine accumulated by the LUVETs 30 min after incubation in 0.2 y mol safranine/ umole lipid (#) and 0.05 y mol safranine/umol lipid (•) in the presence of valinomycin was monitered as described in section 4.2.4. In the case of the system containing 0.2 u mol safranine per y mol lipid the safranine was in excess (not all of it could be accumulated by the LUVET systems) whereas at 0.05 y mol safranine per ymol phospholipid the amount of safranine available limited the uptake. +  3  +  -i  87  r  trapped volumes (1.5 y l/ymol phospholipid) of these LUVET systems, this indicates interior safranine concentrations of 100 mM 96.2  mM,  this  suggests  that  or more. As a saturated solution of safranine at 20° C is  the marked  changes  in safranine  absorption  result  from  precipitation of the dye in the vesicle interior. In order to determine whether this may be the  case,  the safranine  transmembrane K  +  response  (A A max) J16  was measured  for LUVETs  with  various  gradients (as for Fig. 27, 0.2 ymol safranine/ ymol phospholipid) allowing  a correlation to be obtained between A A max and the amount of safranine accumulated. As 516  indicated  in Fig. 28(a),  a  dramatic  vesicle-associated safranine corresponding  increase  AA max  in  J16  occurs  for amounts of  to interior concentrations of 100 mM  or more. This  is clearly consistent with the proposal that the safranine response reflects a precipitation of safranine inside the vesicles, and results in a non-linear relation between  A A  5  U  and  (Fig. 28(b)). It is useful to characterize the stability of the vesicle systems containing high levels of safranine. A LUVET preparation (K inside) was incubated in the presence of safranine (0.2 +  y mol  safranine/ ymol phospholipid) and valinomycin,  and the amount of vesicle associated  safranine determined at various time intervals. As shown in Fig. 29, the safranine accumulated in the presence of valinomycin  reaches a plateau after 2 hr and remains constant at 0.14  y mol safranine per y mol phospholipid  for 8 hr or more. It is interesting to note that  significant safranine uptake is achieved, albeit at a much slower rate, in the absence of valinomycin. Accumulation of lipophilic cations such as safranine likely proceeds in exchange for an efflux of K  +  (Harris and Baum, 1980), and thus the slow valinomycin independent  uptake may arise in response to passive efflux of K . +  To further characterize the relation between safranine uptake and K of K  +  +  effux, the release  from vesicles on uptake of safranine was monitored employing the radioisotope K. As 42  illustrated in Fig. 30, limited release of K  +  is observed on addition of valinomycin  arises in response to valinomycin-facilitated influx of Na  +  which  (see Fig. 21 previous chapter). The  subsequent additon of safranine results in a rapid release of the remaining entrapped K , and +  the time course of this release is similar to that observed for safranine uptake (Fig.  88  29).  Fig. 28. Magnitude of the maximum safranine response (A A max) as: (A) a function of the internal concentration of safranine inside EPC LUVETs and (B) as a function of the membrane potential measured from a known K diffusion potential (see Fig. 23 previous chapter). The system of Fig. 27 containing 0.2 u mol safranine per umol phospholipid was employed in both cases and the A A i max determined 30 min after addition of valinomycin. The safranine concentration inside the vesicles was calculated from the results of Fig. 27 employing a measured trapped volume of 1.15 u 1/umol phospholipid. 516  +  5  20  AO  6  60  80  100  [ S A F R A N I N E l i n (mM)  AT(mV) 89  120  Fig. - 29. Time course for accumulation of safranine by EPC LUVET systems experiencing a Na>VK transmembrane electrochemical gradient (prepared as described in the legend of Fig. 25) in the presence (•) and absence (•) of valinomycin (0.5 ug/umol phospholipid). The safranine taken up into the vesicles was determined by removing untrapped safranine on a gel filtration column (see section 4.2.4). The open symbols indicate background uptake in the absence of an electrochemical gradient (KGlu buffer on both sides of the membrane) and in the presence (O) and absence (p.) of valinomycin. +  90  Fig: 30. Demonstration of K release from EPC LUVETs on addition of safranine. The -LUVETs were prepared in the presence of K.C1 where the untrapped buffer was replaced by the NaCl buffer, and were subsequently incubated in an Amicon ultrafiltration cell in the presence of valinomycin as indicated in section 3.2.6. The K released from the vesicles was determined in the filtered eluate from the ultrafiltration cell by scintillation counting. This eluate did not contain measurable levels of phospholipid. +  42  42  91  +  These  observations  are  consistent with  electroneutral safranine-K  +  The  a  safranine  uptake  mechanism  which  involves  an  exhibiting a  K  exchange process.  results presented  to this stage establish that EPC  LUVETs  +  diffusion potential accumulate the lipophilic cation safranine in a manner which is consistent with  a safranine- K  safranine and  transmembrane exchange mechanism. In order  +  for this to occur  the  the K -valinomycin complex must traverse the hydrocarbon region, suggesting +  that such transport should be sensitive to the acyl chain composition  and  "order" in the acyl  chain region (see section 1.5). That this is the case is illustrated in Fig. 31 for LUVETs composed of SPC, EPC  in the presence of varying amounts of cholesterol, and EPC/EPS (1:1)  mixtures. Uptake into the relatively unsaturated unsaturated  than egg PC, due  SPC  system (soya PC  to the high content of linoleic acid in soya PC  Cullis, 1981)) is faster than for the more saturated EPC presence  of  is considerably more  cholesterol results in a  progressive  LUVET system and  inhibition  of  safranine  (Tilcock and the additional  accumulation. At  equimolar cholesterol levels little or no uptake occurs within 1 hr at 20° C. This inhibition could result from a decreased permeability of safranine or a reduced effectiveness of the  K  +  ionophore in less fluid membranes. Further, the presence of the (negatively charged) egg phosphatidylserine  (PS)  enhances the  redistribution  of safranine, possibly arising  from  an  increased partitioning of the safranine into the lipid bilayer.  4.3.2  Uptake  The K  +  of methyltriphenylphosphonium  results obtained  (MTPP*)  for safranine uptake into model membrane systems exhibiting a  diffusion potential show that safranine can be efficiently accumulated to high levels into  the vesicle interior. It is important to determine how  general these findings are, and therefore  the AiJ; -dependent uptake of a variety of other lipophilic cations has Initially  MTPP  was  investigated  because  transmembrane  been characterized.  redistributions of  this  agent in  radiolabeled form ([ H]-MTPP ) can be detected for very low concentrations (2 x 10" 3  +  8  This leads to minimal perturbations of the electrochemical gradients giving rise to has therefore been utilized to obtain quantitative measures of  92  ATJJ  M).  , and  in energized membrane  Fig.^ 31. Influence of lipid composition on the safranine response (L\ A max measured as the difference in absorbance at '516 nm between the spectrum obtained at zero time and the spectrum obtained at various times after addition of valinomycin: All vesicle systems were prepared and spectra recorded under conditions similar to those indicated for Fig. 25. The various LUVET systems employed were composed of EPC (A); SPC(B); EPC/EPS (1:1; # ); EPC containing 10 mol% cholesterol (•), 25 mol% cholesterol (O), and 50 mol% cholesterol 516  (A).  10  15 T I M E (min.)  93  20  60  systems (Schuldiner and Kaback, 1975; Lambardi et al., 1974; also, see previous chapter). The  uptake  valinomycin-induced 10"  8  M  of K  [ H]-MTPP 3  into  +  EPC  LUVET  systems  in  response  to  a  diffusion potential, employing an (initial) exterior concentration of 2 x  +  [ H]-MTPP is illustrated in Fig. 32. Equilibrium is achieved after 2 hr indicating a 3  membrane potential of greater than 100 mV slower valinomycin  (negative interior). It is interesting to note that a  independent uptake of MTPP* also occurs, presumably by  involving passive K*  a mechanism  efflux similar to the valinomycin-independent accumulation of safranine  (Fig. 29). Again, the uptake process is markedly sensitive to lipid composition the SPC  (for example  systems illustrated in the previous chapter achieved probe equilibration in less than  15 min) and is inhibited by equimolar levels of cholesterol (results not shown). These MTPP  uptake studies were extended to determine the levels of accumulated  MTPP* obtained for higher (initial) external MTPP  +  concentrations to ascertain whether massive  uptake (similar to that observed for safranine) could be achieved. As shown in Fig. 33, the presence of 2 mM  external MTPP* results in valinomycin-induced  accumulation of MTPP* to  levels which correspond to concentrations in the vesicle interior which approach 75 4.3.3  Uptake  of charged  lipophilic  mM.  drugs  It is of interest to extend these uptake studies to include lipophilic cations with acknowledged  biological  activities. A  large  proportion  of  commonly  employed  drugs  are  lipophilic cations. This is presumably because most drugs must traverse the plasma membrane of cells in order to exert their biological effects, and protein  this will  be  facilitated  if the  drug  has  in the absence of a specific transport  lipophilic and  cationic characteristics. In  particular, it is possible that the membrane potential could then encourage drug accumulation in a manner similar to that observed here for safranine and MTPP*. Five representative drugs were studied, including chlorpromazine (a local anaesthetic with application in treatment of schizophrenia), dibucaine (a local anaesthetic), propranolol (a beta adrenergic blocking agent), and vinblastine and  adriamycin  (both of which are anticancer  drugs). Structures of these agents are given in Fig. 24. All of these compounds could be  94  Fig:>. 32.. Time course of the accumulation of H labelled methyltriphenylphosphonium ([ H]-MTPP) by EPC LUVETs prepared as indicated in section 4.2.2 with KGlu in-and NaCl out The [ H]-MTPP (50 Ci/mmol) was added to achieve a concentration of'20 X 10" 1 M (1 p Ci/ml) of MTPP in a dispersion of EPC LUVETs (10 p mol phospholipid/ml). The accumulation of labelled MTPP was monitered in the presence (#) and absence •(•) of valinomycin as described in section 4.2.4. The open symbols indicate the uptake in the absence of an electrochemical NaVK gradient 3  3  3  8  +  +  :  95  Fig.- 33. Time course of the accumulation of methyltriphenylphosphonium (MTPP) into EPC LUVETs under the- conditions ^of;.'Fig.'; 32' but where the initial exterior concentration of MTPP was 2 mM. The labelled H]-MTPP was diluted into cold MTPP to achieve: radioisotope levels corresponding to 1 y Ci/ml. The uptake was determined in the presence (9) and absence (•) of valinomycin. The open symbols indicate the uptake obtained in the absence of a NaVK electrochemical gradient. 3  +  T i m e (hrs.)  96  accumulated into LUVET systems displaying a K 35, and  36. In the  systems experiencing  case of the Na /K +  +  +  diffusion potential as illustrated in Figs. 34,  local anaesthetics  chemical gradients  dibucaine  accumulated  and  chlorpromazine, LUVET  these agents to levels of  nmole/ ymol lipid and 75 nmole/ ymol lipid, respectively. Vesicles with the K and  out exhibited much lower levels (less than 5 nmoles/y mol  uptake  were unaffected  valinomycin  was  by  the  presence of valinomycin.  that a  these levels of  It is interesting to  can be  AIJJ  (see previous chapters) as well as altered K  +  buffer inside  note that  not essential for the uptake of the local anaesthetics. This behaviour would  be consistent with the previous observations efflux of K  lipid) and  +  55  +  established by the passive  permeabilities due to the presence  of the anaesthetic itself (McLaughlin, 1975). Similarly, the rate of propranolol and vinblastine uptake (to levels of 90  nmoles and  40  nmoles / y mole phospholipid,  respectively)  was  enhanced by the presence of valinomycin, however these agents were also accumulated in the absence of this ionophore. Adriamycin was than 180  accumulated most efficiently (to levels of greater  nmoles/y mole lipid, representing  a trapping  efficiency of better than 90%),  an  uptake process that was observed to be markedly sensitive to the presence of valinomycin.  4.4 Discussion The  results presented here provide new  information on the mechanisms of action of  indicators of membrane - potential such as safranine and  have important implications for the  transmembrane distributions of lipophilic cationic molecules in model and  biological systems.  These two areas will be discussed in turn. The  optical response of safranine has been employed to estimate Aip  in mitochondrial  preparations (Colonna et al., 1973; Zanotti and Azzone; 1980; Harris and Brown, 1980). These studies indicate that safranine is accumulated by an energy dependent mechanism and that the spectral changes occur as a result of a "stacking" of the dye on the inner surface of the membrane. This stacking was  proposed to involve an association with negatively charged lipids  in the inner monolayer. The  results presented here are consistent with the optical response of  safranine to  arising from stacking or precipitation of the dye, which may  97  occur at the  Fig. 34. Time course of the accumulation of (A) chlorpromazine and (B) dibucaine into EPC LUVETs experiencing a NaVK electrochemical gradient The chlorpromazine uptake was determined for LUVETs (1 u}mol phospholipid/ml) incubated in the presence of 200 u M chlorpromazine (2 yCi/ml [ H]-chlorpromazine) and the vesicle-associated drug determined subsequently as indicated in section 4.2.6. Similarly, dibucaine uptake was determined for LUVETs (1 u mol phospholipid/ml) incubated with 100 u M dibucaine. Dibucaine accumulation was quantitated as described in section 4.2.6. Both uptake experiments were conducted in the presence (#) and absence (•) of valinomycin (0.5 u g/u mol phospholipid. The open symbols indicate uptake observed in the absence of a NaVK chemical gradient Data for the dibucaine figure was collected and "reproduced with permission from L. D. Mayer. +  3  +  60  90  TIME (min)  O  E c "o u _Q  o £ c  0  20  40  60  Time(m:n.) 98  120  Fig. 35. Time course of propranolol uptake by LUVETs with a transmembrane Na /K chemical gradient Egg PC LUVETs (1 ymol phospholipid/ml) were incubated in the presence of 200 y M propranolol. Vesicle preparation and quantitation of vesicle-associated propranolol was accomplished as described in section 4.2.6. Uptake was measured in the presence (#) and absence (•) of valinomycin. The open symbols represent accumulation in the absence of a electrochemical gradient. Data collected and reproduced with permission fromT. Redelmeier. +  99  +  Fig. 36. Time course for the accumulation of (A) vinblastine and (B) adriamycin into EPC LUVETs experiencing a Na /K electrochemical gradient The vinblastine uptake was determined for LUVETs (1 ymol phospholipid/ml) incubated with 200 yM vinblastine sulphate and the vesicle-associated vinblastine was determined by removal of untrapped material and subsequently assayed as described in section 4.2.6. The adriamycin uptake was measured in a similar fashion, as described in section 4.2.6. Both uptake experiments were conducted in the presence (#) and absence (B) of valinomycin (0.5 y g/y mol phospholipid). The open symbols indicate uptake observed in the absence of a' Na-/K* chemical gradient +  +  50  0  20  40  60  80  100  120  T i m e (minutes) 200  0  '  2 0  4  0  6 0  8 0  Time (minutes) 100  100  12C  inner monolayer-water interface. However, it is clear that negatively charged lipids are not required. The limitations of the optical response of safranine as a quantitative indicator of the membrane potential are equally clear due to the inherent non-linearity of the stacking or precipitation events giving rise to this response, which give rise to non-linear absorbance changes as Atp is increased. The  mechanism whereby safranine is accumulated by the LUV systems is of particular  interest The results presented here are consistent with an electroneutral "antiport" K -safranine +  transmembrane exchange mechanism (modeled in Fig. 37), as indicated by release of entrapped K* on safranine accumulation as well as the fact that the maximum accumulated are comparable to the initial levels of trapped K  +  levels of safranine  (greater than 100 mM  as  compared to 150 mM). Such a proposal is not new (Harris and Baum, 1980) - indeed, it is difficult to imagine any other  process that could  drive safranine uptake in the relatively  simple model systems investigated here. The important points are that the lipophilic cation appears to "flip" across the bilayer in a charged form in response to the  ATJJ  dependent  electric field gradient, and that extremely high levels of internalized safranine can be achieved. This clearly reflects a rather efficient and effective transport process which, as indicated below, may be of general significance for the distributions of lipophilic cations in viva In the case of MTPP*, the ability to employ low concentrations of the radiolabeled form allows reasonable correlations to be obtained experimentally  between Aip  determined interior and exterior concentrations  K* diffusion potential (see Figs. 22 and 23, preceding  calculated on the basis of  of [ H]-MTPP* and the actual 3  chapter). Alternatively, higher (2 mM)  initial exterior levels of MTPP* lead to accumulation of high (greater than 75mM) internal concentrations of MTPP* (Fig. 33), behaviour which corresponds to that observed for safranine. Before discussing the similar uptake characteristics of the biologically active lipophilic cations investigated here, it is of interest to note problems that may be involved in obtaining accurate measures of Axp  derived from equilibrium transmembrane redistributions of lipophilic  cationic probe molecules such as MTPP* or safranine. A  particular difficulty concerns the  length of time required for equilibrium redistributions to occur, which may be on the order  101  rFig: > 37. Model of safranine uptake. It is suggested that the uptake process would involve (1) •partitioning of safranine into the lipid bilayer, subsequently the molecule would "flip" across the:, bilayer in the charged form in response to the AIJJ dependent electric field gradient (2). The.: transport step would be associated with an electroneutral "anti-port" K -safranine transmembrane exchange that would be facilitated by the presence of the K -ionophore 'valinomycin (3), but could also occur in response to the passive efflux of K through the bilayer (4). +  +  +  102  of  hours. Further, in less  redistributions may reasonable  fluid  be sufficiently  membranes, such slow  as those  as to preclude  time frame (see Fig. 31). This may  containing  cholesterol, these  achievement of equilibrium in a  reflect reduced partitioning of the probe  molecule into the membrane or inhibition of the transmembrane "flip" process in response to the electrochemical gradient  In any event a requirement for an extended time course to  determine whether probe uptake has achieved equilibrium is apparent particularly for systems such as plasma membranes containing high levels of cholesterol. Gross underestimates of may otherwise result The  observation  that  lipophilic  cationic  drugs  such  as chlorpromazine,  dibucaine,  propranolol, adriamycin, and vinblastine can be accumulated to high levels within LUV systems exhibiting  a  membrane  chlorpromazine the  mechanism  potential  has  far reaching  implications  in four  areas.  First  and dibucaine are local anaesthetics. A fundamental problem in understanding whereby  local  anaesthetics  induce  their  effects  has been  that  clinical  concentrations of anaesthetics have little influence on the physical properties of lipid systems, even though available evidence  suggests that these agents exert their effects via the lipid  component of membranes (Seeman, 1972). The results presented here suggest that the presence of a membrane potential could lead to local anaesthetic concentrations on the interior of a nerve membrane, for example, which are more than two orders of magnitude higher than plasma concentrations. Additional studies (Mayer et al., 1984) characterizing the uptake of dibucaine in response to a b\> suggest that this is the case. This investigation indicated that the protonated  form of the anaesthetic is transported in response to the NaVK  +  chemical  gradient leading to inner monolayer anaesthetic concentration an order of magnitude larger than predicted on the basis of anaesthetic lipid-water partition coefficient It is of interest to determine whether similar considerations apply to the accumulation of naturally ocurring lipophilic cations, such as biological amines. The chromaffin granule of the adrenal medulla, for example, concentrates dopamine to high levels. Since dopamine has lipophilic and cationic characteristics, the possibility exists that dopamine uptake could proceed in  association with  cation efflux, without  103  a requirement  for a specific  transport protein.  Previous studies (Nichols and Deamer, 1976) have demonstrated the accumulation of biogenic amines in LUVs with a transmembrane pH gradient, a process that is characterized in greater detail in the following chapter. The  third and fourth areas concern drug design and efficent loading of liposomal drug  carrier systems. Model systems such as those employed here (and described in detail in the previous chapter) clearly provide convenient systems for evaluating the possible influence of a given  structural alteration on non-specific  potential. concentrated  Alternatively, LUV  systems  uptake into cells in response  containing  high  to a membrane  levels of lipophilic  cationic drugs  in response to Atp may well have application as vehicles for drug delivery (see  Poste, 1983). Reasons for this include the high drug trapping efficiencies that are possible for many anticancer  agents, such as the cosdy antitumor alkaloids. Further  vinblastine and adriamycin show that trapping  studies employing  efficiencies in excess of 95% can easily be  achieved (Mayer et al., 1984). In summary, the studies presented in this chapter reveal a remarkable ability of LUV systems to accumulate safranine and other lipophilic cations in response to a K potential. This  ability  may  have  important  and general  implications  transmembrane distributions of biologically active cations in vivo.  104  +  diffusion  for the equilibrium  UPTAKE OF DOPAMINE AND  OTHER BIOGENIC AMINES INTO LARGE  UNILAMELLAR VESICLES IN RESPONSE TO A MEMBRANE POTENTIAL: ACTIVE TRANSPORT IN THE  ABSENCE OF A CARRIER PROTEINt  5.1 Introduction In a variety of biological systems biogenic amines are stored in high concentrations within  specialized  organelles. Examples  include  catecholamines  norepinephrine) which are stored at high concentrations (0.6 M) the  adrenal medulla (Winkler, 1976;  (ie. dopamine,  epinephrine,  in the chromaffin granules of  Smith, 1968), serotonin which is stored in serotonin  granules of blood platelets (Wilkins et al., 1978;  Fukami et <z/.,1978), and  which are stored in neural synaptic vesicles (Martin, 1973). The  neurotransmitters  mechanism by which these  amines are accumulated in these organelles has been the subject of intense investigation (for reviews see Njus el al., 1981; Kanner, 1983). Previous investigations have indiaued that catecholamine accumulation  in the chromaffin  granule occurs by a reserpine sensitive carrier-mediated process which is directly coupled to hydrolysis (Kanner et al., 1979;  ATP  that the uptake of amines was was  Schuldiner et al., 1978). A  variety of data, indicating  dependent on the formation of ion gradients (Holz, 1979) and  sensitive to the presence of proton  ionophores (Johnson  et  al., 1981), supported  chemiosmotic mechanism of biogenic amine transport whereby accumulation gradient inwardly  Briefly, it was directed  proposed that the carrier mediated  proton-translocating  ATPase  which  is  is driven by an ion  transport process  responsible  a  for  involves an  maintaining  a  transmembrane electrochemical proton gradient (acid interior) that provides the driving force for the uptake of catecholamines (Schuldiner et al., 1978; Maron et al., 1983; Knoth et al., 1980; Johnson et al., 1978). Further, it was to H  +  suggested that the inward flux of amines was  coupled  efflux providing a H /amine antiport type of mechanism that is coupled through the +  tThis chapter has been based on the reference Bally et al. (1984).  105  et al., 1981;  carrier protein (Njus  Johnson et al., 1981). A  similar mechanism has been  proposed for the transport of serotonin, where it has been shown that vesicles accumulate this amine  in exchange  for K  +  (Kanner, 1983)  and  for the  uptake  of neurotransmitter  by  synaptosomes, which appear to employ a Na -coupled transport system (Fukami et al., 1981). +  The  role of the various components (pH  gradient, ATPase, carrier protein) which are  involved in catecholamine transport are still not understood at the molecular level. It has been suggested that the characteristics of biological amines as weak bases, allow the molecules to gradient (Schuldiner et  accumulate across the membrane according to the transmembrane pH  al., 1978), where the neutral form of the amine permeates across the lipid bilayer. This theory  was  supported  by  data  that  demonstrated  a  variety of catecholamines could  accumulated in egg  PC  vesicles in response to a pH  Deamer, 1976). The  difficulty with this proposal lies in the fact that the pKa  gradient (interior acid; Nichols and values of the  biological amines are significantly higher than physiological pH. For example the pKa amino group of dopamine is approximately of about 8.8. Therefore at the pH  be  of the  9.9, and the catechol hydroxyls demonstrate a pKa  values examined in many of the reports which demonstrate  the transport of biogenic amines, the amine exists predominatly addition, if amine transport occurs  by  the  proposed  in the protonated  form. In  H /amine antiport mechanism, +  ATP  independent uptake of the amine by chromaffin granule ghosts would result in alkalinization of the vesicle. This does not appear to be the case (Knoth et al., 1983), suggesting that the amine may In quaternary LUVs  be transported in a charged (protonated) form. the  previous  chapter  it was  suggested  that the  lipophilic  cation safranine, a  amine which exists exclusively in a positively charged form, is accumulated by  exhibiting a  membrane potential (interior  negative). Since  at physiological pH  biological amines have both lipophilic and cationic characteristics it may  the  be possible that these  molecules could be accumulated by a similar, membrane potential driven, uptake process. In this  investigation the  composed  of  egg  uptake of dopamine in model  phosphatidylcholine  (EPC)  which  large unilamellar vesicles (LUVETs)  have  a  transmembrane  potential (interior negative) in the presence and absence of a pH  106  electrochemical  gradient (interior acid) has  been characterized. The  results clearly demonstrate that a membrane potential can provide the  driving force for biological amine transport by a process that is sensitive to the presence of CCCP (a H  ionophore) and  +  valinomycin  (a K  +  ionophore). The  results are consistent with  the proposal  that dopamine crosses the lipid bilayer in a charged form and  presence  an  of  aqueous trap  for  dopamine, such  as  ATP,  accumulation of dopamine to levels in excess of 120 mM 20  this  uptake  that in the  results in the  against an exterior concentration of  yM.  5.2 Materials and Methods 5.2.1  Materials  Egg phosphatidylcholine  (EPC)  was  2.2.2. Dopamine, 5-hydroxytryptamine Sigma (St Louis, MO).  isolated from hen  (serotonin), and  egg yolk as described in section  epinephrine  were all obtained  from  [ H]-inulin, [ C]-cholesterol oleate, [ H]-methyl triphenylphosphonium 3  14  iodide (MTPP), [ H]-dopamine HCL  3  and [ H]-epinephrine were obtained from NEN  3  3  (Canada).  For ease of reference the structures of the various biogenic amines are shown in Fig. 38. 5.2.2  Preparation  of  vesicles  Vesicles were prepared utilizing the  LUVET  (large unilamellar vesicles by  techniques) procedure described in section 3.2.2. Briefly a dry lipid film was appropriate  buffer to produce multilamellar vesicles (25-100  extrusion  hydrated with an  u moles lipid/ml) which were  extruded 10 times through two (stacked) polycarbonate filters with a defined pore size of 100 nm  (Nucleopore).  Subsequently the  vesicle preparation  through the 100 nm filters 5 times. The with an  average diameter of 90  microscopy  (see  nm  section 3.3.1). The  was  freeze-thawed twice and resized  resulting vesicle suspension was  as determined by vesicles exhibited  ul/umole phospholipid as determined by [ H]-inulin. 3  107  an  largely unilamellar  freeze fracture and average trapped  negative stain volume of  1.5  Fig. 38. Structures of Dopamine, Epinephrine, and Serotonin.  0n H0\  /VCH CH NH 2  2  DOPAMINE  OH HO'  CHCH N< ?  z  i  OH  C H  u  3  H  EPINEPHRINE  CH CH NH 2  SEROTONIN  108  2  2  Transmembrane Na -K +  buffer (169 mM  K  utilizing  a  +  glutamate, 20 mM  mOsm/kg) and subsequently buffer (150 mM  chemical gradients (interior K ) were formed by trapping a K  +  exchanging the untrapped  NaCl, 20 mM  Sephadex  HEPES adjusted to pH 7.5 with 1M  G-50  K  +  +  NaOH; 310  buffer for an iso-osmotic Na  +  HEPES adjusted to pH 7.5 with 1M NaOH: 310 mOsm/kg) desalting column  (as described  in section 4.2.2). Similarly,  transmembrane pH gradients (interior acid) were formed using a trap buffer with low pH (150mM  KOH, 175 mM  glutamic acid pH 4.65; 290 mOsm/kg) which was then exchanged  with a high pH buffer (150 mM  KOH, 125 mM  glutamic acid, 30 mM  NaCl pH 7.5; 290  mOsm/kg) on a Sephadex G-50 column. Defined pH gradients were generated the low pH  buffer and subsequently  passing  the vesicles down  pre-equilibrated with buffers containing 150 mM EPPS (pKa= (pKa=  8.0), 10 mM  CAPS (pKa=  KOH, 10 mM  by trapping  Sephadex G-50 columns  MES  (pKa= 6.15), 10  mM  10.4) and varying concentrations of glutamic acid  4.2) and NaCl to give iso-osmotic buffers of the desired pH. A similar buffering  mixture (glutamic acid, MES, EPPS) was utilized when the pH was varied under conditions of a constant NaVK  ion gradient (interior K ). Where ATP was utilized as a trap, the internal  +  +  buffer consisted of 150 mM  KOH, 175 mM  glutamic acid and 125 mM  Na ATP giving a 2  pH of 4.3 (470 mOsm/kg). This was exchanged against either a buffer containing 150 KC1, 20 mM  glutamic acid, and 80 mM  NaCl (adjusted to pH 4.3 with 1 M  mOsm/kg) for control samples or 220 mM with 1 M  NaCl and 20 mM  NaOH; 470  Hepes (adjusted to pH 7.5  NaOH; 450 mOsm/kg) for experimental samples. All biogenic amine uptake studies  were preformed with buffers containing 5 mM employed  mM  the K  ionophore valinomycin  +  y g/ ymole lipid and the H  +  sodium ascorbate as an antioxidant Where  was added to give a final concentration of 0.5  ionophore CCCP was added to give a final concentration of 20  y M. The same concentrations of each were used in samples that contained both ionophores.  5.2.3 Determination  of membrane  potential  The membrane potential was measured by determining the distribution of the lipophilic cation  [ H]-MTPP 3  +  as described  in section 3.2.6. Briefly, 1  109  yCi of [ H] -MTPP 3  +  (50  Ci/mmole) was added to a 1 ml LUVET dispersion which was subseqently incubated at room temperature for 1 hr before an aliquot was  withdrawn and the untrapped [ H]-MTPP was 3  removed by passing over a small (1 ml) Sepadex G-50  column. The  trapped [ H]-MTPP 3  +  was determined by liquid scintillation counting employing a Phillips PW-4700 liquid scintillation counter and  the phospholipid was  determined  by  a phosphate  assay (see section 3.2.2).  Utilizing the trapped volume, as determined with [ H]-inulin (see section 3.2.2), the internal 3  and external concentration of MTPP* was calculated and the membrane potential was estimated employing the Nernst equation (see section 3.2.6). 5.2.4 Uptake  assays  The amount of dopamine accumulated was determined by adding dopamine from a 2 mM  stock solution which contained [ H]-dopamine (45.4 Ci/ mmole) to a LUVET dispersion 3  to achieve a 200 y M  dopamine concentration with 1 yCi/ ml labeled amine and a 1  mM  lipid concentration. Subsequently, at appropriate time intervals, the unsequestered amine  was  removed  by  passing aliquots of the vesicles through  1 ml  Sephadex G-50  columns as  described in the preceding chapter (section 4.2.6). Aliquots of the effluent were counted to assay for dopamine and phospholipid phosphorous ATP  was  was assayed by standard techniques. Where  used as a trap, [ C]-cholesterol oleate (56.6 mCi/mmole) was 14  used (1 y Ci/20  y moles lipid) as a probe to assay for lipid . The uptake of epinephrine and serotonin was determined employing similar procedures as for dopamine. Accumulation of epinephrine was monitored using [ H]-epinephrine in systems 3  containing 200  y M  amine and  concentrations of amine and  lipid  1 mM  EPC  LUVETs. In the case of serotonin, similar  were utilized  assayed fluorometrically (excitation, 309  nm;  and  the LUVET  emission, 340  nm)  associated serotonin  employing  was  a Perkin- Elmer  650-10S fluorescent spectrophotometer after disruption of the vesicles with 0.5% Triton X-100.  110  5.3 Results  5.3.1  Accumulation  The  of dopamine  in response to a transmembrane  electrochemical  previous chapter demonstrated that a variety of molecules  potential  with  lipophilic  and  cationic characteristics can be accumulated in a vesicle (LUVET) preparation in response to a transmembrane membrane potential (interior negative) generated by a Na /K +  inside) across  the  lipid  bilayer. The  mechanism  of  uptake  electroneutral K /lipophilic cation exchange process that was +  appeared  +  ion gradient (K to occur  facilitated by the K  +  by  +  an  ionophore  valinomycin. It is of interest to determine whether the uptake of biolgical amines, which have lipophilic and cationic characteristics at neutral pH, could also be accumulated by a similar mechanism. This is shown to be the case in Fig. 39 where it is shown that the accumulation of dopamine by EPC The  LUVETs can be driven by a transmembrane K  uptake is dependent on the presence of the K  +  ionophore  +  ion diffusion potential.  valinomycin, and  levels of  greater than 50 nmoles dopamine/ umole lipid are acheived within a 1 hr incubation. This level remains constant for periods greater than 8 hr (data not shown). In the absence of valinomycin the uptake is reduced 5 fold to 12 nmoles dopamine/ pmole lipid. Uptake levels of  only 3 nmoles dopamine/ P mole lipid  are obtained for samples which lack a Na /K +  electrochemical gradient (equal concentration of K  +  inside and outside). Assays employing the  membrane potential probe [ H]-MTPP revealed a potential of greater than 100 mV 3  negative) for these vesicles in the presence ionophore a potential of -40  to -60  mV  These data indicate that the accumulation electrochemical potential and  +  (interior  of valinomycin, while in the absence of the  was  detected (for example, see preceding chapter).  process is dependent on both the transmembrane  the efflux of K  +  which is facilitated by valinomycin, and are  qualitatively similar to previous results demonstrating  that K*  efflux is coupled to uptake of  the lipophilic cation safranine. Dopamine uptake in the presence  of valinomycin resulted in a 200  fold or larger  concentration gradient of the amine across the membrane (based on an trapped volume of 1.5  111  :Eig.a 39. Time course for accumulation of dopamine by EPC LUVET systems experiencing a . Na*/K electrochemical gradient The LUVETs were prepared in a K glutamate buffer, and the.,',, untrapped buffer was exchanged for a NaCl buffer as described in section 5.2. Subsequently the vesicles were diluted to achieve a concentration of 1 mM phospholipid in a NaCl buffer containing 200 y M dopamine (1 yCi/ml [ H]-dopamine). The dopamine taken up into the vesicles was determined using liquid scintillation counting after the untrapped dopamine was removed by gel filtration. The open circles represent uptake in control samples which have equal concentrations of K on both sides, of the membrane. The other symbols represent uptake in the absence of added ionophores (•), and in the presence of valinomycin (0.5 yg/ymole lipid; • ) , or CCCP (20 y M; #). or both ionophores (•). +  3  +  Time(hours)  112  y 1/ ymole  lipid).  The  gradient  formed  is sensitive  to the initial  external  dopamine  concentration, as similar levels of uptake were obtained when external dopamine concentrations were as high as 1 mM. the  Fig. 39 also indicates that the uptake process can be uncoupled by  presence of both valinomycin and CCCP. The presence of these ionophores initially results  in a more rapid accumulation of dopamine to levels of greater than 50 nmole/ ymole lipid. Subsequently there is a rapid efflux of the entrapped amine to control levels within 1 hr. A similar time course is observed for the transmembrane membrane potential assayed by MTPP , +  where a maximum of -60 mV is obtained at 15 min decreasing to less than 5 mV (negative interior) at 4 hours. Addition of CCCP alone to vesicles with a NaVK  +  ion gradient also  facilitated transport of dopamine, albeit at a much reduced rate. Since chromaffin granules maintain a transmembrane pH gradient (Johnson et al., 1978) and it has been demonstrated that this pH gradient can drive the uptake of dopamine and other  biogenic amines  in these  membrane  preparations and  in model  vesicle  systems  (Schuldiner et al., 1978; Johnson et al., 1981; Nichols and Deamer, 1976), it was of interest to determine whether dopamine uptake in LUVETs could also be driven by a pH gradient (interior acid) in the absence of a K  +  ion gradient This is demonstrated to be the case in  Fig. 40, where levels of 40 nmoles dopamine/ ymole lipid are obtained (representing a 150 fold gradient across the membrane) within 30 min after addition of the amine to vesicles with a transmebrane  pH gradient of 3 units. This level of uptake is stable for periods  exceding 8 hours and appears to be unaffected by the exterior concentration of dopamine. The H  +  diffusion potential generated by vesicles with this transmembrane pH gradient was  determined to be greater than 100 mV (interior negative) and was stable for periods in excess of 8 hr. The presence of the H by  +  ionophore CCCP results in a rapid accumulation followed  efflux of the amine to control levels by 2 hr. It is interesting that the membrane  potential exhibited by these CCCP containing vesicles is stable, maintaining a potential of greater than 100 mV (interior negative) for periods in excess of 8 hr. As was shown for vesicles with a transmembrane K  +  diffusion potential, the presence of both a K  ionophore uncouples the uptake process indicating that K V H  113  +  +  and a H  +  exchange eliminates the driving  Fig.  40.  Time course for  LUVETs (pH  7.5)  were as  prepared described  represent  control  presence  of  concentrations |  no  in  samples added  accumulation in  a  low  section with  a  5.2. low  ionophore  specified in Fig.  of  pH  dopamine  buffer The  pH  (O).  (pH  by EPC L U V E T 4.65),  conditions buffer  CCCP  for  and  the  uptake  systems untrapped  were  inside  and  outside  the  (#),  and  CCCP  and  as  experiencing buffer  described  vesicle.  a  was in  p H gradient  exchanged Fig.  The  other  valinomycin  (A).  39.  for  The  symbols  (interior  acid).  a  p H buffer  high  open  represent  Ionophores  were  square  The  symbols  uptake  in  used  at  the the  39. 1  1  1  1  1  force for dopamine uptake in these model systems. Similar results have been obtained for chromaffin granule preparations which discharge entrapped catecholamines in systems where the pH gradient is uncoupled by the presence of both these ionophores (Bangham et al., 1980). 5.3.2 Influence  of external  pH  on the uptake of dopamine  It has been suggested that weak bases, such as dopamine, cross the lipid bilayer in a neutral rather than charged form (Johnson et al., 1978; Johnson et al., 1981) which implies that  these molecules  distribute  across a membrane  according to the transmembrane  pH  gradient However, if dopamine is accumulated in LUVETs by a similar mechanism described in the previous chapter for the positively charged safranine, then it may be expected that it is the charged form of dopamine which is translocated across the membrane. If this is the case the accumulation  should  be sensitive  to the pH  of the surrounding medium. To  investigate this aspect of transport further, dopamine, uptake in LUVETs was examined under two situations: a) where the transmembrane electrochemical potential was held constant utilizing a defined NaVK  ion gradient in the presence of valinomycin while the external and internal  +  pH was varied (no pH gradient was present; Fig. 41A); b) where the electrochemical potential was varied by creating different transmembrane pH gradients (in the absence of a K ion +  gradient), maintaining an internal pH of 4.5 and varying the external pH (Fig. 41B). The first situation  clearly  demonstrates  distribution of [ H]-MTPP 3  +  a pH  optimum  in these systems  (-120mF) was established by the K  +  for uptake  of dopamine by LUVETs. The  indicated that a constant membrane potential  ion gradient in the presence of valinomycin at all pH  values examined. The maximum level of dopamine uptake occurs in the range of pH 7.5-8.5, which is similar to the pH optimum (pH 8.3) demonstrated for biological amine transport by granule membranes (Maron  et al., 1979). Above  pH  8.5 the levels of uptake decrease  markedly, presumably due a decrease in the availability of the protonated form of the amine. This is supported by data which indicate that at pH 9.5 the rate of dopamine uptake is reduced,  therefore  longer  incubations would  be  required  to obtain  levels  of uptake  demonstrated at pH 8.0. Lowering the pH below 6.5 results in a decrease in the level of  115  Fig. 41. Influence of external pH on dopamine uptake. (A) Dopamine uptake by egg PC LUVETs with a constant membrane potential established by a valinomycin induced K ion diffusion potential and no pH gradient (initially external pH = internal pH). The buffer system utilized is described in section 5.2. The symbols represent uptake in the presence of valinomycin. (B) Dopamine uptake by vesicles with varying membrane potential established by trapping a low pH (pH 4.65), and exchanging the untrapped buffer with buffers of varying pH (see section 5.2). The symbols represent uptake in the absence of added ionopohores. Uptake was evaluated in both experiments after a 2 hr incubation at room temperature. +  T  45  r  t5 63  Z5 &5 external pH  116  93  amine uptake where at pH 4.5 control (systems which lack a NaVK uptake are obtained. This is also reflected  +  ion gradient) levels of  in Fig. 41B, however  in this  situation the  magnitude of the membrane potential is varying according to the pH gradient present. This is indicated in the data presented  in Fig. 42, which demonstrate the relationship between the  membrane potential measured by the distribution of [ H]-MTPP 3  +  and the value calculated  based on the initial transmembrane H* gradient Deviations from the ideal potential probably result from dissipation of the pH gradient due to H gradient is not an accurate assessed  with  measure of the pH  MTPP . This pH +  +  permeability such that the initial pH  gradient at the time the potential was  dependency of dopamine uptake may  reflect the reduced  permeability of dopamine at the lower pH values, arguing that the protonated  form of the  amine can not permeate across the lipid bilayer and thus cannot be transported. Evaluation of the passive permeability of entrapped  dopamine (at a concentration  of 40 mM)  indicate,  however, that at pH 4.5 and 7.5 (where greater than 99% of the dopamine is present in a protonated form), 80% of the entrapped amine is released within 1 hr.  5.3.3 Influence  of trapped ATP  on dopamine  accumulation  It has been proposed by several investigators that the large (125 mM) concentration of ATP  within the chromaffin granule may effectively trap internalized catecholamines in the form  of mixed amine-nucleotide across the membrane 1969;  Berneis  aggregates allowing for accumulation  of amines as they diffuse  against an effective "downhill" concentration gradient (Berneis et al.,  et al.; 1974). The influence of trapped  transport of dopamine by LUVETs  ATP on the electrochemical driven  is shown in Fig. 43. The presence of trapped  ATP  increased the level of dopamine accumulation five fold compared to the similar situation in the absence of ATP, obtaining levels of 180 nmoles dopamine/y mole lipid in vesicles with a transmembrane pH gradient (interior acid) and a trap of 125 mM  ATP. This  accumulation  appears to be dependent on the transmembrane electrochemical gradient since control samples with trapped  ATP and no pH gradient, either 4.5 or 7.5 inside and outside, showed no  accumulation  of dopamine indicating that ATP is not capable of acting as a passive trapping  117  Fig. 42. Comparison between the membrane potentials obtained for various pH gradients as detected by [ H]-MTPP and the theoretical potentials based on the PP ion gradient as predicted by the Nernst equation. LUVETs were prepared as described in Fig. 41B and section 5.2. The membrane potential was determined employing [ H]-MTPP (see section 3.2.6) in the presence (•) and absence (•) of CCCP after a 2 hr incubation at room temperature to ensure equilibration of the probe. The dashed line indicates the theoretical potential given by the Nernst equation: A i p (mF) - -59 log ([H ]//[H ]o) 3  3  +  A pH ini fri  118  +  Fig.  43.  described as the  of  trapped  in section  described  gradient by  Effect in  (pH  presence vesicles  5.2,  Fig.  39.  such  =  of  CCCP  both  that  The  interior  with a  ATP  on 125  closed  p H exterior). and  transmembrane  dopamine  accumulation  m M A T P was circles The  represent  other  valinomycin (A) p H gradient  trapped uptake  symbols at  and no  by  LUVETs  within  the  composed vesicles.  by  control  samples,  represent  uptake  in  concentrations trapped  described  A T P (see  Fig.  the  of  EPC.  LUVETs  The conditions which absence  in Fig.  39.  had  for  The  dashed  prepared  dopamine uptake  trapped  of added  were  A T P with  ionophores line  (O).  represents  were  no o r  3  pH i n  uptake  40).  2 Time(hours)  as  4  agent Under the conditions of the uptake assay greater than 80% was  associated with the vesicles, resulting in the formation  of the available dopamine  of a concentration gradient in  excess of 500 fold. As before the concentration gradient formed appears to be dependent on the external concentration of amine utilized since similar levels of uptake were obtained when the outside dopamine concentration was both  valinomycin  and  CCCP  can  500  u M.  Fig. 43 also shows that the presence of  uncouple this ATP  enhanced accumulation  of dopamine,  suggesting that if an ATP-dopamine aggregate is formed, the resulting complex can dissociated under conditions where the pH 5.3.4  Electrochemical  driven  gradient is dissipated.  uptake of other biogenic  amines  It is of interest to determine whether the accumulation dopamine is general  and  could  result in the  accumulation  process shown to occur for  of other  biogenic amines. In  paricular, it has been demonstrated that a K* ion gradient (Kanner, 1983) and transmembrane pH  gradient (interior acid; Bangham et al., 1980;  Carty et al., 1981)  play a role in the  transport of serotonin by granule membranes. In addition, both uptake processes appear to be sensitive to a variety of similar inhibitors, such as reserpine. Fig. 44 shows the uptake of epinephrine  (Fig. 44A)  transmembrane NaV acid; Fig. 44  A2  K  +  and  and  serotonin  ion gradient (K B2). The  presence of ionophores as was  by  EPC  LUVETs  interior; Fig.44 A l and Bl) or pH  uptake of these demonstrated  transport of both epinephrine and may  +  (Fig. 44B)  with  may  be  a  a  gradient (interior  amines show similar sensitivity to the  for dopamine uptake. Valinomycin  facilitated  serotonin, indicating that a similar mechanism of transport  be responsible for the uptake of these amines. Differences in the level and  accumulation  either  reflection  of  the  hydrophilicity  of  the  amine. For  rate of example,  epinephrine, which contains a hydroxyl on the methylene group adjacent to the catechol ring (see Fig. 38)  is accumulated  at a  slower rate and  dopamine, which lacks this group.  120  saturates at lower levels than does  Fig.  44.  NaVK* Open uptake  Time ion  symbols in  course  gradient  the  for (1)  represent presence  the or  accumulation a  uptake  of  transmembrane in  control  of valinomycin (•)  epinephrine pH  samples and  gradient  (A) (2).  which lacked  in the  absence  and  serotonin  Gradients a  (B)  were  transmembrane  of added  by ion  ionophores  20  1  Q_  10  _CD 0  E  \ CCD  20  E  < d) 10 0  E  Time  EPC  formed  hours  as  LUVET described  gradient. (#).  systems in  T h e ' other  with  Figs.  39  symbols  either and  a 40.  represent  Discussion This investigation clearly demonstrates that biogenic amines can be accumulated into large unilamellar vesicles systems in the absence of a specific carrier protein. This transport appears  to be driven  by an electrochemical  potential gradient  established  by  either a  transmembrane NaVK* ion gradient (K interior) or a pH gradient (acid interior). There are +  two areas of interest generated by the data presented here. The first concerns the mechanism whereby biological amines are accumulated in LUVETs and the second has to do with the physiological relevance of this accumulation process with regard to the transport, and inhibition of transport, of biological amines in vivo. Each of these areas will be discussed in turn. Previous 1981; on  investigators (Schuldiner  et al., 1978; Johnson et al., 1978; Johnson et al.,  Maron et al., 1983) suggested that the mechanism of dopamine uptake was dependent  the amine distributing across the vesicle membrane according  to the transmembrane pH  gradient, resulting in accumulation of the amine inside an acidic vesicle. This mechanism implied that the neutral form of the weakly basic amine permeates the membrane and was supported by investigations demonstrating that liposomes composed  of soyabean lipids were  impermeable to the protonated form of biological amines. Others (Knoth et al., 1980; Maron et al:, 1979) have argued that amines are transported in the protonated form rather than the neutral form, a hypothesis which is supported by data that showed chromaffin granule ghosts are not permeable to the neutral species of the amine and that ATP dependent uptake of biological amines by chromaffin  granule ghosts did not result in alkalinization of the vesicle  (Knoth et al., 1983). In addition, since the pKa of the amine group of these molecules is relatively high (for dopamine the pKa is approximately 9.9), the predominant species present at physiological pH is the protonated form and hence the neutral form would not be as available for transport. For numerous reasons the results presented here are consistent with the concept that dopamine is transported in a charged form. First, dopamine uptake can be driven by a K  +  ion diffusion potential (negative interior), which is initially established in the absence  of a pH gradient (see Fig. 39). This accumulation is facilitated under conditions where K  122  +  efflux occurs, ie. in the presence of valinomycin,  suggesting  an electroneutral K /dopamine +  exchange process similar to that postulated in the previous chapter for safranine. It should be noted, however, that it is possible that valinomycin  may engender a H  +  the charge gradient  gradient  by a H V K  +  established  by addition of  exchange mechanism. Second,  if  dopamine uptake relies on the movement of the neutral species and the resulting distribution is dependent on the pH gradient, then one would expect that larger exterior concentrations of dopamine would result in increased accumulation. This does not appear to be the case as saturation levels of 40 nmoles dopamine/ ymole lipid were obtained utilizing exterior dopamine concentrations  from 200 y M  to 1 mM.  Third, since other biological amines are also weak  bases, the uptake of these amines should  be similar under conditions where the amine  accumulates in accordance to the pH gradient The data in Fig. 44 suggest that this does not occur, epinephrine and serotonin are accumulated at different rates and to a lesser extent than dopamine in response to LUVETs with similar transmembrane pH gradients. Finally, it was demonstrated that dopamine is permeable at a pH where the amine exists primarily in a protonated form (pH 4.5 and 7.5), indicating that this species is capable of crossing the egg PC  bilayer. This data would also suggest that an additional factor, other than a low internal  pH, is required for maintaining the level of dopamine which has been accumulated. In order to investigate the role of charge on the transport of dopamine, uptake was investigated under conditions where there was a constant membrane potential (established by a K  ion gradient in the presence of valinomycin) and no pH gradient (Fig. 41A) or a variable  +  electrochemical  potential (Fig. 41B) as established  by changes in the transmembrane pH  gradient These data indicate that at pH 4.5, where the amine is present in the protonated form, there is no transport of the molecule, even under conditions where a substantial (greater than 100 mV) negative potential is present. This result is clearly inconsistent with the model of uptake based on the protonated form of the amine being transported across the vesicle membrane. However, it can be suggested (Fig. 45) that the charged permeant species is a HA  + 2  complex formed between a protonated amine and a neutral amine. Therefore at pH 4.5  uptake of the amine would be limited due to the reduced presence of the neutral species.  123  Fig. 45. Postulated mechanism for the uptake of dopamine by large unilamellar vesicles.  124-  Further, optimal uptake would occur at a pH close to the pKa of the amine examined, as indicated in Fig. 41 A. Formation of a HA  complex has been postulated previously as a  + 2  mechanism for the permeation of local anesthetics through bilayer membranes (McLaughlin, 1975). This model of uptake still suggests that biological amine transport in LUVETs occurs by an electroneutral cation (H or K )/ cation (HA ) antiport mechanism that is driven by +  +  +  2  the  membrane potential (interior negative). In addition, the presence of the membrane potential  (negative  interior) would  dissociation of the HA the  + 2  inhibit  the efflux  of the positively  charged  amine, therefore  complex in the vesicle interior would "trap" the protonated form of  amine resulting in net accumulation. The neutral amine would be free to shuttle across  the membrane. It is difficult to differentiate between the mechanism suggested here, and the previous mechanisms based on the equilibration of the amine according to the pH gradient. In all the uptake experiments presented in this chapter the presence of a negative membrane potential was maintained, whether the result of a H  +  or a K  +  ion diffusion potential. Similarly, in  membrane systems which have demonstrated ATP independent uptake of biogenic amines in response to a pH gradient (Schuldiner et al., 1978; Johnson et al., 1978; Knoth et al., 1980; Johnson  et al., 1981; Maron et al., 1983; Nichols and Deamer, 1976; Carty et al.,  1981;  Maron et al., 1979), the pH gradient present would also result in formation of a negative potential. It has been demonstrated  that in the absence of ATP the membrane potential  present across the chromaffin granule membrane approximates the Fr equilibrium potential of -70 mV (Holz., 1979). Further, the results present here do not preclude the possibility that dopamine may be translocated in a protonated form in association with a counterion such as Or.  Although the accumulation of dopamine by LUVET systems shows similarities to the uptake of biological amines in vivo, it is difficult to relate the two situations. It has been demonstrated, for example, that the chromaffin granule membrane supports a positive (interior) membrane potential in the presence of ATP and Mg amines. The model suggested here clearly  125  2+  (Holz, 1979) yet can still transport  depends on the presence of a transmembrane  potential  that  is negative  interior. In addition, the uptake  of dopamine  could  not be  demonstrated in vesicles which had a transmembrane pH gradient (interior acid) and a positive (interior) membrane potential (40 mV; established by a CI" ion diffusion potential, ie. a glutamateVCl" gradient (CI interior); unpublished  observation). It is not clear from these  -  experiments whether the pH gradient is maintained under conditions where a positive (interior) membrane potential exists. Further, the rate of uptake by vesicles composed  of EPC is  substantially slower than that demonstrated for amine uptake in chromaffin granules. This rate, however, would be markedly sensitive to the lipid composition  and temperature as indicated in  the previous chapter (see section 4.3.1), and may be enhanced if a lipid composition similar to that of the chromaffin granule membrane is utilized at 37° C. The chromaffin  role of ATP as a trapping agent responsible for the storage of catecholamines in granules is supported by data presented  suggests that formation  in this report. This data (see Fig. 43)  of the ATP-dopamine complex increases the vesicle's capacity for  accumulating biogenic amines. The mechanism of uptake, however, would still be dependent on an  electrochemical  potential  (interior  negative)  driven  cation/cation  exchange  mechanism  postulated previously. This is indicated by data which demonstrated that dissipation of the electrochemical gradient by addition of both a H  and a K  +  +  ionophore inhibited uptake of  dopamine in these ATP containing vesicles. This would also suggest that the ATP-amine complex allows for rapid exchange with the free amine pool and could not act as a passive trapping agent as suggested by Berneis et al. (1974). The previous  general  chapter,  mechanism  based  of lipophilic  cation transport  on an electrochemically  driven  presented  uptake  process  here  and in the  suggests that the  presence of a transmembrane potential could have an important role in regulating the uptake of biological amines in vivo. It is demonstrated that both epinephrine  and serotonin can be  accumulated in vesicles in response to a membrane potential (Fig. 44), suggesting  that the  uptake process is relatively non-specific. In addition, it is interesting to note that a variety of inhibitors of biogenic amine transport in biological systems, such as reserpine and imipramine, are lipophillic cations. Due to the greater hydrophobicity of these cations, they may be able  126  to compete with or block amine uptake and/or release. Generally, a single inhibitor shows a broad specificity for a number of different amine transport systems. Reserpine, for example, inhibits catecholamine uptake in chromaffin granules (Kanner et al., 1979) as well as serotonin uptake in serotonin granules of blood platelets (Maron et al., 1979). Further, it has been shown that the putative transporter molecule for biogenic amines also has a broad specificity for a variety of substrates, such as hydroxyindolamines, hydroxyquinolines, reserpine  like  alkaliods (Maron  et  al.,  1983), which  all have  tetrabeozine  lipopillic  and  and  cationic  characteristics. Finally, the influence of local anesthetics, which are accumulated in a similar manner as described for dopamine (Mayer et al., 1984), on the uptake and release of these biologically active neurotransmitters and hormones is of obvious interest  127  SUMMARIZING DISCUSSION  The  studies presented in this thesis illustrate the utility of model membrane systems to  investigate the simplest  structural and  functional properties of lipids in biological  model system which can  be  generated involves hydrating  membranes.  a dry  The  lipid film. The  resulting multilamellar vesicles (MLVs) are useful for characterizing the structural properties of lipids in membranes. This is demonstrated by the investigations of Chapter 2 which examine the  influence of cholesterol on  the polymorphic phase properties of lipids. These studies  support the surprising conclusion that cholesterol engenders formation of non-bilayer H  phase  u  structures and hence serves to destabilize the bilayer structure of membranes. This property is illustrated by  model systems containing  PS  and  PE  conjunction with physiological concentrations of Mg  2+  where the presence of cholesterol, in (2 mM),  can reduce the level of Ca  required to trigger H -phase structure in these systems from 4 to 0.25 n  mM,  2+  a concentration  which more closely approximates that observed in vivo. These results suggest that a role of cholesterol  in membranes  may  non-lamellar structures, as may With structures  regard  in  to  the  membranes  be  to  facilitate  processes that  the  formation  of  be the case in Ca * induced membrane fusion events. 2  ability  the  of phospholipids  results of  Chapter  multicomponent systems individual phospholipids  to assume formation 2  have no  particular phase in samples where bilayer, hexagonal suggests that in complex  require  lipid  systems, such as  the  also  demonstrate  of  that  non-bilayer in  complex  tendency to associate with any and  "isotropic"  phases coexist  one This  biological membrane, that different,  functionally significant, structural phases could be assumed without the segregation of individual lipid species into distinct domains. The  next area of investigation concerns the barrier properties of lipid membranes and  the influence of a membrane potential on  certain transport processes. For  studies such as  these MLVs are of limited value, thus a more sophisticated large unilamellar vesicle model system was  (LUV)  developed and utilized. The procedure, described in Chapter 3, required the  128  repeated  extrusion  of multilamellar  vesicles under moderate pressures  through polycarbonate filters with a defined  pore size of 100  nm.  (less than The  500  psi)  resulting LUVETs  (large unilamellar vesicles by extrusion techniques) are shown to be unilamellar with diameters in the range of 60-100 nm  and trapped volumes in the region of 1-3  yl/ ymole. Particular  advantages of this procedure are that lipid solubilizing agents (such as organic solvents and detergents) are not different lipid  required  species and  and  vesicles can  be  prepared reproducibly  from a variety of  mixtures. This model large unilamellar vesicle system, therefore,  provides a very attractive model system. This vesicle preparation was  used to model a basic feature of biological membranes,  namely the presence of a transmembrane potential. Vesicles exhibiting a membrane potential were obtained  by imposing a NaVK* ion gradient (K  This ion gradient resulted in the formation  of a  +  A^  inside) across the vesicle membrane. (as detected by  the lipophilic cation  MTPP ), the magnitude of which correlated well with the transmembrane concentration gradient +  and showed a time dependent decay which reflected the flux of Na  +  This model system exhibiting a transmembrane potential was the role of accumulate  into the vesicles. employed to investigate  A\p in certain transport functions of membranes. In particular an a  variety  of  molecules  which  have  lipophilic  demonstrated. In Chapter 4 this accumulation process was  and  ability to  cationic characteristics is  characterized using the dye safranine  O. It is concluded that accumulation of this molecule proceeds by a antiport K.H—safranine exchange process that is driven by the transmembrane potential. The rapid formation  of a large (greater than 100  transport resulted in the  fold) transmembrane concentration  gradient of  safranine and clearly reflected a very efficient transport mechanism. The  generality of this transport process is shown in chapters 4 and  demonstrated that a variety of biologically active lipophilic cations can  be  5, where it is accumulated  by  vesicles to acheive interior cation concentrations which are more than two orders of magnitude higher than exterior concentrations. These cations include the drugs chlorpromazine, dibucaine, propranolol, vinblastine and epinephrine.  Biogenic  adriamycin; and  the  amine accumulation occurred  129  biogenic  amines dopamine, serotonin  and  in response to vesicles exhibiting either  transmembrane H  +  or K  +  diffusion potentials, a result which indicates that the uptake of these  molecules in vivo could proceed without a requirement for a specific transport protein. The  observations  made in this thesis define several areas of research that warrant  further investigation. In particular, the model membrane system described here exhibiting a Aip  can be utilized to examine other  involved in metabolite distribution of charged  Aifi  dependent transport processes, such as those  and protein transport, as well as protein insertion and transbilayer lipid  components. Research  within  the immediate  future, however,  should concentrate on evaluating the relationship between the physical and structural properties of lipids and the permeability of membranes to small ions. 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