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The effects of anaesthetics on lipid polymorphism Hornby, Antony Paul 1981

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THE EFFECTS OF ANAESTHETICS ON LIPID POLYMORPHISM by ANTONY PAUL HORNBY B.Sc, The University of Wales, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF March (c) Antony Paul BRITISH COLUMBIA 1981 Hornby, 19 81 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of V?CL-V-ev^ \^r-\^ j The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i ABSTRACT The e f f e c t s of r e p r e s e n t a t i v e a n a e s t h e t i c s on the polymorphic phase be h a v i o r of p h o s p h o l i p i d s , p a r t i c u l a r l y p hosphatidylethanolamine, i s s t u d i e d . The s e r i e s o f normal a l c o h o l s and alkanes d e s t a b i l i z e the b i l a y e r s t r u c t u r e of hydrated phosphatidylethanolamine causing the l i p i d t o adopt hexagonal ( H J J ) s t r u c t u r e s a t lower temperatures than c o n t r o l By e x c e p t i o n , e t h a n o l and b u t a n o l s t a b i l i z e the b i l a y e r c o n f i g u r a t i o n . Chloroform a l s o i n i t i a t e s b i l a y e r d e s t a b i l i -z a t i o n , producing t h i s e f f e c t a t c l i n i c a l l y r e l e v a n t concen-t r a t i o n s of a n a e s t h e t i c . The charged a n a e s t h e t i c s t e s t e d show the a b i l i t y t o s t a b i l i z e the b i l a y e r c o n f i g u r a t i o n of phosphatidylethanolamine to h i g h e r temperatures than c o n t r o l s t h i s occurs i n a s i n g l e or mixed l i p i d system ( i e . phospha-t i d y l e t h a n o l a m i n e / p h o s p h a t i d y l s e r i n e ) . In a l l cases the a b i l i t y of an a n a e s t h e t i c to a l t e r the polymorphic phase behavior of phosphatidylethanolamine i s a f u n c t i o n of i t s i n d i v i d u a l potency. The observed e f f e c t s of a n a e s t h e t i c agent on l i p i d polymorphism can be i n t e r p r e t e d i n r e l a t i o n t o the molecular shapes of the l i p i d s and a n a e s t h e t i c s i n v o l v e d . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF FIGURES v ACKNOWLEDGEMENTS . v i CHAPTER 1. I n t r o d u c t i o n 1 2. M a t e r i a l s and Methods 9 2a G e n e r a l 10 2b N e u t r a l A n a e s t h e t i c s 11 2c Charged A n a e s t h e t i c s 12 3. R e s u l t s 17 3a G e n e r a l 18 3b N e u t r a l A n a e s t h e t i c s 18 3c Charged A n a e s t h e t i c s 34 4. D i s c u s s i o n 36 BIBLIOGRAPHY 49 V LIST OF FIGURES Figure Page 31 1. Phase properties, shape, and P NMR spectra for representative phospholipids 14 2. Comparison of control, ethanol, and decanol spectra 20 3. Determination of the b i l a y e r to hexagonal (H.^) phase t r a n s i t i o n temperature of Egg phosphatidylethanolamine 22 4. E f f e c t s of alcohols of the polymorphic phase behavior of phosphatidylethanolamine 25 5. E f f e c t s of alkanes on the polymorphic phase behavior of phosphatidylethanolamine 2 7 6. E f f e c t s of chloroform on the polymorphic phase behavior of Soya phosphatidylethanolamine 29 7. E f f e c t s of charged (amine) anaesthetics on Egg phosphatidylethanolamine polymorphic phase behavior 31 8. Dibucaine e f f e c t s on PE/P9 mixtures 33 -1-CHAPTER 1 INTRODUCTION -2-Anaesthetic molecules provide a p a r t i c u l a r l y useful, yet seldomly applied, t o o l for the study of membrane structure. I t i s s u r p r i s i n g to many that very l i t t l e i s known about how anaesthetics work. The term anaesthetic applies to a diverse range of molecular structures which have a single common property - they are invariably l i p i d s o l u ble r Since these agents i n t e r a c t with c e l l membranes and have a large e f f e c t on c e l l function (most noticeably i n nerve c e l l s ) they provide a means f o r gaining much information on how membranes are structured and how t h i s structure relates to function. I t is. commonly accepted that both general and l o c a l anaesthetics act i n a general manner with the l i p i d or l i p i d - p r o t e i n component of the c e l l membrane rather than a s p e c i f i c i n t e r s action with a membrane receptor (1). This hypothesis i s based on a large v a r i e t y of documented studies from which a number of generalizations can be drawn, for example: 1.) greater l i p i d s o l u b i l i t y constitutes a more potent anaesthetic, 2.) a vast v a r i e t y of chemically disparate drugs are able to produce nerve block, and 3.) action potentials i n a r t i f i c i a l l i p i d b i l a y e r s are blocked by anaesthetics such a cocaine or chlorpromazine (see R e f . l ) . The general rule of Meyer and Overton applies for most anaesthetic studies, i t states that, "narcosis commences when any chemically i n d i f f e r e n t substance has attained a ce r t a i n molar concentration i n the l i p o i d s of the c e l l " (2 and 3). In other words, anaesthesia i s produced when a c r i t i c a l volume i s occupied by an anaesthetic i n the membrane: -3-under conditions of l o c a l anaesthesia t h i s average volume occupation i s 0.3% of the membrane volume. S i m i l a r l y , high membrane concentrations of anaesthetics, which correspond to around one anaesthetic molecule per f i v e phospholipid molecules, are inva r i a b l y associated with the threshold of membrane l y s i s (1). By d e f i n i t i o n , anaesthesia constitutes action p o t e n t i a l blockade without a measurable a f f e c t on the resting membrane pote n t i a l ; anaesthetics are not, however, se l e c t i v e between nerve and other c e l l types. Action potential' i n i t i a t i o n and propagation requires i n f l u x of sodium ions across the c e l l membrane through a proposed sodium channel; subsequently, an e f f l u x of potassium ions occurs, the r e s u l t being a depolarization of the c e l l membrane. Anaestheics presumably block t h i s process by i n t e r f e r i n g with, or i n some way disrupting, the sodium channel. Seeman (1) has stated that "for any p a r t i c u l a r hypothesis to be termed a 'theory of anaesthesia', i t i s necessary for that hypothesis to explain the manner i n which the sodium conductance channel i s in h i b i t e d or the manner i n which synaptic transmission i s modified. Otherwise, i f the hypothesis merely shows a c o r r e l -ation of anaesthetic potency with some physiochemical parameter, the hypothesis i s e s s e n t i a l l y only a rule of anaesthesia". Since disruption of the sodium channel by anaesthetic i n t e r a c t i o n occurs, wholly or i n part, within the l i p i d component of the membrane, i t i s not surprising that most investigations i n t h i s area have focused t h e i r - 4 -attention on the l i p i d element i n e f f o r t s to determine the anaesthetic mechanism. Many of these studies have demonstrated the a b i l i t y of anaesthetics to increase the f l u i d i t y (decrease the g e l - l i q u i d c r y s t a l l i n e hydrocarbon t r a n s i t i o n temperature) of the hydrophobic enviroment of the b i a l y e r , leading to suggestions that t h i s e f f e c t s either a protein or l i p o p r o t e i n conformation with subsequent i n a c t i v a t i o n of the sodium channel. Models, such as that due to Lee ( 4 ) , have suggested that the sodium channel i s surrounded by an annulus of gel state l i p i d , and penetration of anaesthetic molecules into t h i s annulus causes an increase i n f l u i d i t y leading to disruption and i n a c t i v a t i o n of the channel. This sort of model seems u n l i k e l y considering the unsaturated nature of nerve membrane l i p i d (5) (causing greater f l u i d i t y ) as well as the fac t that there i s no evidence for the existence of gel-state l i p i d s i n eukaryotic c e l l membranes i n general. Secondly, the anaesthetic concentrat-ions required to induce s i g n i f i c a n t " f l u i d i z a t i o n " are usually an order of magnitude larger than those required to i n h i b i t the action p o t e n t i a l i n vivo (6). In f a c t , such concentrations often have l y t i c e f f e c t s . A l l anaesthetics whether general or l o c a l i n h i b i t sodium conduction at concentrations low enough not to a f f e c t the resti n g p o t e n t i a l of the nerve c e l l ( 1 ) . By t h i s general mechanism anaesthetic agents, i n some way, disrupt the sodium channel and d r a s t i c a l l y a l t e r the function of the neuronal membrane. Anaesthetics are of p a r t i c u l a r i n t e r e s t to membrane b i o l o g i s t s because of t h e i r apparent a b i l i t y to r a d i c a l l y -5-p e r t u r b membrane s t r u c t u r e and f u n c t i o n and c o n s e q u e n t l y t h e y p r o v i d e a u s e f u l t o o l t o e x p l o r e t h e p o s s i b l e f u n c t i o n a l r o l e s o f l i p i d s i n b i o l o g i c a l membranes. I n r e c e n t y e a r s i t has become i n c r e a s i n g l y e v i d e n t t h a t models f o r membrane s t r u c t u r e such as t h e f l u i d m o s a ic model o f S i n g e r and N i c h o l s o n (13) o r t h e e a r l i e r u n i t membrane model (14) do n o t e x p l a i n t h e many o b s e r v e d membrane f u n c t i o n s . I t i s i m p l i e d i n t h e f l u i d m osaic model t h a t t h e l i p i d component assumes a c l o s e d b i l a y e r s t r u c t u r e , t h u s r e a l i z i n g b o t h a s t r u c t u r a l m a t r i x w i t h w h i c h f u n c t i o n a l p r o t e i n s may be a s s o c i a t e d as w e l l as an i n t e r n a l environment w h i c h may be r e g u l a t e d and c o n t r o l l e d . T h i s model a l s o p r o v i d e s f o r l a t e r a l d i f f u s i o n o f membrane components i n t h e p l a n e o f t h e membrane, as w e l l as p e n e t r a t i o n o f p r o t e i n s i n t o o r t h r o u g h t h i s m a t r i x . T h i s c o n c e p t o f membrane s t r u c t u r e does n o t , however, a l l o w e x p l a n a t i o n o f c e r t a i n membrane-mediated p r o c e s s e s such as c e l l f u s i o n , exo- and e n d o c y t o s i s , t r a n s b i l a y e r movement o f l i p i d s ( ' f l i p - f l o p ' ) , f a c i l i t a t e d t r a n s p o r t as w e l l as p r o t e i n i n s e r t i o n and o r i e n t a t i o n . More r e c e n t l y , a l t e r n a t i v e e x p l a n a t i o n s t o some f u n c t i o n a l a s p e c t s o f membrane b i o l o g y have become p o s s i b l e t h r o u g h advances i n t e c h n i q u e s such as X-ray and n u c l e a r m a g n e t i c resonance w h i c h have shown membrane dynamics p r e v i o u s l y unknown. Fo r t h e p a s t twenty y e a r s i t has been known t h a t h y d r a t e d l i p i d s have t h e a b i l i t y t o adopt a v a r i e t y o f phases i n a d d i t i o n t o t h e commonly a c c e p t e d b i l a y e r phase. P a r t i c u l a r l y s i g n i f i c a n t c o n t r i b u t i o n s t o t h i s r e s e a r c h -6-a r e a have been made by L u z z a t t i and coworkers (7-9) empl o y i n g X-ray t e c h n i q u e s t o s o l v e i n d e t a i l t h e s t r u c t u r a l c h a r a c t e r i s t i c s o f t h e s e a l t e r n a t i v e s . I n a d d i t i o n , t h e s e (10) and o t h e r (11 and 12) i n v e s t i g a t o r s i m m e d i a t e l y r e c o g n i z e d t h e p o s s i b i l i t y t h a t t h e s e n o n - b i l a y e r s t r u c t u r e s may be r e l a t e d t o membrane s t r u c t u r e and f u n c t i o n . I n t r o d u c t i o n o f 31 P NMR added a new d i m e n s i o n t o membrane r e s e a r c h xn terms o f l i p i d p olymorphism and a l l o w e d f u r t h e r r e e v a l u a t i o n o f e x i s t i n g membrane t h e o r i e s . I n a d d i t i o n , more s o p h i s t i c a t e d f r e e z e - f r a c t u r e t e c h n i q u e s , improved i s o l a t i o n and p u r i f i c a t i o n o f l i p i d s , p l u s b e t t e r d e f i n e d model systems has a l l o w e d c o n f i r m a t i o n o f e a r l i e r o b s e r v a t i o n s made w i t h X-ray and NMR, thus h e l p i n g t o p r o v i d e more d i r e c t e v i d e n c e f o r t h e e x i s t e n c e on n o n - b i l a y e r phases i n b i o l o g i c a l membranes. B i o l o g i c a l membranes a r e composed o f more th a n a hundred d i f f e r e n t s p e c i e s o f l i p i d . T hat a l l t h e s e m o l e c u l a r t y p e s a r e r e q u i r e d t o m a i n t a i n t h e s t r u c t u r a l i n t e g r i t y o f t h e membrane b i l a y e r seems u n l i k e l y when t h i s same s t r u c t u r e can be formed by a s i n g l e l i p i d s p e c i e s such as p h o s p h a t i d y l -c h o l i n e . T h i s s t r u c t u r a l v i e w seems even more u n l i k e l y c o n s i d e r i n g t h e f i n d i n g s t h a t a number o f l i p i d s p e c i e s , i n c l u d i n g u n s a t u r a t e d p h o s p h a t i d y l e t h a n o l a m i n e s ( 1 5 ) , c a r d i o -l i p i n and p h o s p h a t i d i c a c i d i n t h e pr e s e n c e o f c a l c i u m i o n (16, 1 7 ) , p r e f e r e n t i a l l y adopt n o n - b i l a y e r phases when 31 h y d r a t e d (as i l l u s t r a t e d w i t h c o r r e s p o n d i n g P NMR s p e c t r a i n F i g u r e 1) and i t i s l o g i c a l t o assume t h a t t h e s e phases may be v i t a l t o such membrane f u n c t i o n s as ' f l i p - f l o p ' ( 1 8 ) , -7-membrane f u s i o n ( 1 9 ) , exo- and e n d o c y t o s i s , as w e l l as f a c i l i t a t e d t r a n s p o r t ( 2 0 ) . A d d i t i o n a l l y , t h e e x i s t e n c e o f n o n - b i l a y e r l i p i d s i n b i o l o g i c a l membranes has been s t r o n g l y 31 s u g g e s t e d w i t h P NMR and f r e e z e - f r a c t u r e t e c h n i q u e s i n t h e ( r a t l i v e r ) e n d o p l a s m i c r e t i c u l u m (21,22) and f u s i o n o f e r y t h r o c y t e membranes (23) . I n v i e w o f t h e proposed g e n e r a l mechanism o f a n a e s t h e t i c a c t i o n , t h a t i s , t h e a b i l i t y o f a n a e s t h e t i c m o l e c u l e s ( s o - c a l l e d membrane p e r t u r b a n t s ) t o r a d i c a l l y a l t e r membrane f u n c t i o n , i t became c l e a r t h a t a s t u d y o f a n a e s t h e t i c e f f e c t s on l i p i d p o lymorphism would l e n d v a l u a b l e i n s i g h t t o t h e p o s s i b l e f u n c t i o n a l r o l e o f n o n - b i l a y e r l i p i d s as w e l l as a b e t t e r u n d e r s t a n d i n g o f a n a e s t h e t i c a c t i o n . L i p i d s used f o r models i n t h i s s t u d y were: egg p h o s p h a t i d y l e t h a n o l a m i n e , p h o s p h a t i d y l c h o l i n e , . p h o s p h a t i d y l -s e r i n e , and p h o s p h a t i d y l g l y c e r o l . These l i p i d s , e x c e p t p h o s p h a t i d y l e t h a n o l a m i n e , s p o n t a n e o u s l y adopt and m a i n t a i n a b i l a y e r c o n f i g u r a t i o n when h y d r a t e d . The l a t t e r , however, undergoes a b i l a y e r t o h e x a g o n a l (H.^) phase t r a n s i t i o n a t 30°C. A number o f r e p r e s e n t a t i v e a n a e s t h e t i c s were chosen o r t h e s t u d y i n c l u d i n g t h e s e r i e s o f n - a l c o h o l s and n - a l k a n e s ; t h e l o c a l a n a e s t h e t i c s , d i b u c a i n e , t e t r a c a i n e , and p r o c a i n e ; t h e t r a n q u i l i z e r , c h l o r p r o m a z i n e ; p l u s t h e g e n e r a l a n a e s t h e t i c , c h l o r o f o r m . The s e r i e s o f n - a l c o h o l s and n - a l k a n e s were found t o c o n v e r t b i l a y e r p h o s p h a t i d y l e t h a n o l a m i n e t o t h e h e x a g o n a l phase a t h i g h c o n c e n t r a t i o n s , w i t h t h e e x c e p t i o n o f e t h a n o l -8-and b u t a n o l , w h i c h produced s t a b i l i z a t i o n o f t h e b i l a y e r . The c h a r g e d l o c a l a n a e s t h e t i c d i b u c a i n e s t a b i l i z e s t h e b i l a y e r s t r u c t u r e o f egg p h o s p h a t i d y l e t h a n o l a m i n e above t h e c o n t r o l b i l a y e r t o h e x a g o n a l phase t r a n s i t i o n t e m p e r a t u r e , b u t has no e f f e c t on egg p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l -s e r i n e , o r p h o s p h a t i d y l g l y c e r o l . The o t h e r c h a r g e d a n a e s t h e t i c s t e s t e d : p r o c a i n e , t e t r a c a i n e , and c h l o r p r o m a z i n e a l s o produced b i l a y e r s t a b i l i z a t i o n s i m i l a r t o t h e e f f e c t s o f d i b u c a i n e and t h i s a b i l i t y was f o u n d t o c o r r e l a t e w i t h t h e a n a e s t h e t i c p o t e n c y o f t h e i n d i v i d u a l d r u g s . F u r t h e r m o r e , i n a mixed l i p i d system (80% PE/ 20% P S ) , a d d i t i o n o f s m a l l amounts o f C a + + i n d u c e s t h e h e x a g o n a l phase, whereas subsequent a d d i t i o n o f d i b u c a i n e a t p h a r m a c o l o g i c a l l y r e l e v e n t c o n c e n t r a t i o n s was found t o r e e s t a b l i s h a b i l a y e r s t r u c t u r e . The above r e s u l t s a r e d i s c u s s e d i n t h e c o n t e x t o f r e c e n t e v i d e n c e f o r t h e e x i s t e n c e o f n o n - b i l a y e r phases i n b i o l o g i c a l membranes p l u s t h e i r p o s s i b l e r e l e v a n c e t o anaes-t h e t i c mechanisms. A model f o r l i p i d / a n a e s t h e t i c a c t i o n i s p r o p osed i n terms o f t h e m o l e c u l a r shapes o f t h e s e m o l e c u l e s , and a d d i t i o n a l l y , s p e c u l a t i o n s made on t h e p o s s i b l e r o l e s o f n o n - b i l a y e r phases i n i o n t r a n s p o r t and how t h i s f u n c t i o n may be d i s r u p t e d by a n a e s t h e t i c m o l e c u l e s . A l s o , t h e p o s s i b i l i t y o f l i p i d - p r o t e i n i n t e r a c t i o n s b e i n g d i s r u p t e d by a n a e s t h e t i c m o l e c u l e s i s d i s c u s s e d . CHAPTER 2 MATERIALS and METHODS 2a. G e n e r a l Egg p h o s p h a t i d y l e t h a n o l a m i n e and egg p h o s p h a t i d y l c h o l i n e were p u r i f i e d from a t o t a l egg l i p i d e x t r a c t i o n u s i n g h i g h p r e s s u r e l i q u i d chromatography (Waters P r e p LC 500) w i t h s i l i c a c i d p r o v i d i n g t h e s t a t i o n a r y phase, t h e l i p i d b e i n g s e p a r a t e d w i t h a m i x t u r e o f c h l o r o f o r m and met h a n o l . Egg p h o s p h a t i d y l s e r i n e and p h o s p h a t i d y l g l y c e r o l were p r e p a r e d by a c o n v e r s i o n t e c h n i q u e a c c o r d i n g t o C o m f u r i u s and Zwaal (24) u s i n g p h o s p h a l i p a s e D t o change t h e headgroup from egg PC t o PG o r PS. The i n d i v i d u a l l i p i d s were f u r t h e r p u r i f i e d by column chromatography u s i n g s i l i c a c i d . I n each case t h e p u r i t y o f t h e l i p i d was a s s e s s e d by t h i n -l a y e r chromatography and, f o r egg PE, by c o n s i s t e n c y i n t h e b i l a y e r t o h e x a g o n a l phase t r a n s i t i o n t e m p e r a t u r e o f t h e l i p i d w h i c h i s v e r y s e n s i t i v e t o PE d e g r a d a t i o n . S t o r a g e o f pure p h o s p h a t i d y l e t h a n o l a m i n e i n c h l o r o f o r m a t -20°C i s n o t s u f f i c i e n t t o m a i n t a i n p u r i t y ( i e . s t a b l e Tbh) and w i t h i n t h r e e days t h e phase t r a n s i t i o n t e m p e r a t u r e can i n c r e a s e as much as 10°C. The r e a s o n f o r d e g r a d a t i o n o f p h o s p h a t i d y l e t h a n o l amine, i n p a r t i c u l a r , i s not known, however, i t i s r e a s o n a b l e t o s u s p e c t t h a t i t may be a r e s u l t o f o x i d a t i o n o f a r e l a t i v e l y l a r g e p o r t i o n o f t h e h i g h l y u n s a t u r a t e d 22:6 f a t t y a c i d c h a i n s found i n egg PE ( a p p r o x i m a t e l y 15% by w e i g h t ) . P h o s p h a t i d y l -e t h a n o l a m i n e s from o t h e r s o u r c e s w h i c h do n o t c o n t a i n such h i g h l y u n s a t u r a t e d f a t t y a c i d s a r e l e s s s u b j e c t t o such e f f e c t s To a v o i d t h i s p r o b l e m o f d e g r a d a t i o n t h e l i p i d was k e p t under n i t r o g e n as much as p o s s i b l e d u r i n g p r e p a r a t i o n , and -11-i m m e d i a t e l y a f t e r p u r i f i c a t i o n was s e a l e d i n p l a s t i c c y r o -t u b e s and immersed i n l i q u i d n i t r o g e n . 2b. E x p e r i m e n t s w i t h N e u t r a l A n a e s t h e t i c s Samples were p r e p a r e d from d r y 100 mg. p o r t i o n s o f p u r i f i e d egg p h o s p h a t i d y l e t h a n o l a m i n e . The l i p i d was d i s s o l v e d i n 1 ml. c h l o r o f o r m and t r a n s f e r r e d t o a 10 mm. . NMR tube and s u b s e q u e n t l y d r i e d under a stream o f n i t r o g e n . A l l t r a c e s o f c h l o r o f o r m were removed by f u r t h e r d r y i n g t h e l i p i d under h i g h vacuum f o r a minimum o f 2 h o u r s . To t e s t an i n d i v i d u a l c o n c e n t r a t i o n o f c h l o r o f o r m , a l c o h o l , o o r a l k a n e , 0.6 ml. ii2° c o n t a i n i n g 100 mM N a C l , 10 mM T r i s / 2 a c e t i c a c i d , 2 mM EDTA, p H 7.0 was added t o t h e d r y l i p i d . A s m a l l amount o f a n a e s t h e t i c was i n j e c t e d d i r e c t l y i n t o t h i s s o l u t i o n u s i n g a H a m i l t o n m i c r o p i p e t t e t o make up t h e r e q u i r e d c o n c e n t r a t i o n , and t h e e n t i r e m i x t u r e was V o r t e x mixed t o form l i p o s o m e s and ensure maximum p a r t i t i o n o f a n a e s t h e t i c i n t o t h e s e s t r u c t u r e s . E q u i v a l e n t r e s u l t s were o b t a i n e d when l o n g e r c h a i n l e n g t h a n a e s t h e t i c s were added i n c h l o r o f o r m , w h i c h was s u b s e q u e n t l y e v a p o r a t e d . 2c. E x p e r i m e n t s w i t h Charged (Amine) A n a e s t h e t i c s P r e p a r a t i o n o f samples f o r t h e s e e x p e r i m e n t s was s l i g h t l y d i f f e r e n t t h a n w i t h t h e n e u t r a l a n a e s t h e t i c s . L a r g e volumes o f b u f f e r was used t o ens u r e maximum p a r t i t i o n o f d i b u c a i n e i n t o t h e l i p i d d u r i n g l i p o s o m e f o r m a t i o n . Samples c o n t a i n i n g PC, PS, and PG were p r e p a r e d w i t h d i b u c a i n e s o l u t i o n s -12-o n l y , whereas PE was made up f o r e x p e r i m e n t s w i t h d i b u c a i n e , p l u s p r o c a i n e , t e t r a c a i n e , and c h l o r p r o m a z i n e . D i b u c a i n e HC1 was p u r c h a s e d from ICN P h a r m a c e u t i c a l s ; P r o c a i n e and T e t r a c a i n e HC1 from Winthrop L a b o r a t o r i e s ; and C h l o r p r o m a z i n e HC1 from P o u l e n c L t d . L i p i d s were d i s s o l v e d i n 1 c h l o r o f o r m and t r a n s f e r r e d t o 50 ml. SS-34 c e n t r i f u g e t u b e s , d r i e d under n i t r o g e n , t h e n h i g h vacuum f o r a minimum o f 2 h o u r s . The d r y l i p i d s were d i s p e r s e d by V o r t e x m i x i n g i n 4 m l . aqueous s o l u t i o n s o f a p p r o p r i a t e a n a e s t h e t i c c o n t a i n i n g 2 mM EDTA and 100 mM N a C l . The h y d r a t e d l i p i d was c e n t r i f u g e d a t 3000 rpm f o r 10 minutes and t h e p e l l e t : resuspended i n 0.6 m l . o f f r e s h i n c u b a t i o n medium p l u s 0.1 m l . D2O. The pH was n e u t r a l i z e d and the sample s e a l e d i n a 10 mm. NMR t u b e . Samples c o n t a i n i n g PC, PS, and PG were made i n d i b u c a i n e p r e p a r a t i o n s o n l y . By t h e same p r o c e d u r e , t h e PE/PS m i x t u r e s (125 mg.) c o n t a i n i n g 20 mol% 1 PS and 80 mol% PE were d i s p e r s e d i n 200 ml. aqueous s o l u t i o n s o f 100 mM N a C l , lOmM T r i s / a c e t i c a c i d (p 2H 7.0), 5 mM C a C l 2 p l u s t h e r e q u i r e d c o n c e n t r a t i o n o f d i b u c a i n e . The samples were c e n t r i f u g e d and p e l l e t s resuspended i n 0.6 m l . o f t h e o r i g i n a l s o l u t i o n p l u s 0.1 ml. ^^O. 31 For a l l e x p e r i m e n t s P NMR s p e c t r a were made u s i n g a B r u k e r WP 200 F o u r i e r t r a n s f o r m s p e c t r o m e t e r o p e r a t i n g a t 36.4 MHz. Temperatures were r a i s e d i n c r e m e n t a l l y t h r o u g h t h e b i l a y e r t o h e x a g o n a l t r a n s i t i o n a l l o w i n g 10 mi n u t e s 31 e q u i l i b r i a t i o n t i m e f o r each e x p e r i m e n t . P NMR s p e c t r a o b t a i n e d form p h o s p h o l i p i d membranes a r e i n t e r p r e t e d on t h e -13-FIG. 1. R e p r e s e n t a t i v e l i p i d s and c o r r e s p o n d i n g phases p l u s 31 s c h e m a t i c m o l e c u l a r shapes and P NMR s p e c t r a . The b i l a y e r s p ectrum was o b t a i n e d from aqueous d i s p e r s i o n s o f egg y o l k p h o s p h a t i d y l c h o l i n e , whereas t h e h e x a g o n a l ( H J J ) phase spectrum was o b t a i n e d from soya p h o s p h a t i d y l e t h a n o l a m i n e . The " i s o t r o p i c m o t i o n " spectrum was o b t a i n e d from a m i x t u r e o f 85 mole % soya p h o s p h a t i d y l e t h a n o l a m i n e and 15 mole % egg y o l k p h o s p h a t i d y l c h o l i n e . A l l s p e c t r a were t a k e n a t 30°C. L i p i d Phospholipid Phases Phosphatidylcholine Bilayer Sphingomyelin Phosphatidylserine x*$>>£ Phosphatidylglycerol Phosphatidylethanol- Hexagonal ;..( H Ij) amine (unsaturated) .-•/ •M* .'*::''*'' C a r d i o l i p i n - Ca Phosphatidlc acid -Ca Lysophospholipids Detergents Phases where i s o t r o p i c motion occurs a, M i c e l l a r , Inverted m i c e l l a r b, Vesicles c, Cubic Rhombic -15-b a s i s o f a number o f e s t a b l i s h e d p r i n c i p l e s . A t y p i c a l b i l a y e r spectrum ( F i g . 1) r e s u l t s from t h e l a r g e c h e m i c a l s h i f t a n i s o t r o p y o f l i p i d phosphorus i n l a r g e ( r a d i u s 2000 A) l i q u i d - c r y s t a l l i n e b i l a y e r systems. The p r i m a r y mode o f m o t i o n a v a i l a b l e t o t h e s e systems i s r a p i d r o t a t i o n o f t h e m o l e c u l e s about t h e i r l o n g a x i s so t h a t o n l y p a r t i a l a v e r a g i n g o c c u r s (20, 25-27). I n t h e p r e s e n c e of p r o t o n d e c o u p l i n g , t h i s r e s u l t s i n a c h a r a c t e r i s t i c b r o a d spectrum w i t h a low f i e l d s h o u l d e r and h i g h f i e l d peak, which a r e s e p a r a t e d by EFF ^ C S A " P P m " S i m i l a r v a l u e s a r e found f o r a l l g l y c e r o l based p h o s p h o l i p i d s ( 2 5 ) , ( w i t h t h e p o s s i b l e e x c e p t i o n o f p h o s p h a t i d i c a c i d ) i n c l u d i n g t h e mammalian phosphosphingo-l i p i d , s p h i n g o m y e l i n ; t h i s r e s u l t s i n a l m o s t e q u i v a l e n t l i n e shapes f o r t h e s e d i f f e r e n t s p e c i e s when i n t h e l i q u i d - c r y s t a l l i n e b i l a y e r c o n f i g u r a t i o n . T h e r e f o r e i n mixed l i p i d systems and b i o l o g i c a l membranes e f f e c t i v e l y a l l t h e endogenous p h o s p h o l i p i d s c o n t r i b u t e t o a c o m p o s i t e b i l a y e r l i n e s h a p e i f t h e y a r e i n t h e b i l a y e r phase. I n t h e c a s e o f s m a l l s o n i c a t e d l i p i d v e s i c l e s l a t e r a l d i f f u s i o n o f l i p i d around th e v e s i c l e and v e s i c l e t u m b l i n g o c c u r r a p i d l y -5 on t h e NMR t i m e s c a l e (10 sec.) such t h a t m o t i o n a l a v e r a g i n g o c c u r s r e s u l t i n g i n l i n e - n a r r o w i n g e f f e c t s (28). L i p i d s i n t h e h e x a g o n a l (H.^) phase e x p e r i e n c e a d d i t i o n a l m o t i o n a l a v e r a g i n g because m o t i o n a l a v e r a g i n g due t o l a t e r a l d i f f u s i o n around t h e s m a l l (20 A d i a m e t e r ) aqueous c h a n n e l o c c u r s . T h i s e f f e c t r e s u l t s i n c h a r a c t e r i s t i c NMR l i n e s h a p e s w h i c h have r e v e r s e d asymmetry compared t o b i l a y e r s p e c t r a -16-and a r e narrower by a f a c t o r o f two (2 0, 25, 2 9 ) . F u r t h e r m o r e , l i p i d s i n m i c e l l a r , i n v e r t e d m i c e l l a r , o r o t h e r phases (such as t h e c u b i c o r rhombic) c o n f i g u r a t i o n s a l l o w e f f e c t i v e l y i s o t r o p i c m o t i o n t o o c c u r , as l a t e r a l d i f f u s i o n r e s u l t s i n a v e r a g i n g o v e r a l l o r i e n t a t i o n s , l e a d i n g t o a narrow, symmetric 31 P NMR spectrum. A summary o f l i n e s h a p e s o b s e r v e d i s p r e s e n t e d i n F i g u r e 1. -17-CHAPTER 3 RESULTS -18-3a. G e n e r a l H y d r a t e d egg p h o s p h a t i d y l e t h a n o l a m i n e w i t h no a n a e s t h e t i c p r e s e n t d i s p l a y s a b i l a y e r t o h e x a g o n a l phase t r a n s i t i o n n e a r 30°C ( F i g . 2 a ) . T h i s v a l u e , termed Tbh, i s d e f i n e d as t h e t e m p e r a t u r e a t w h i c h t h e l i p i d i s b o t h 50% i n t h e b i l a y e r phase and 50% i n t h e he x a g o n a l ( h J J ) phase ( F i g . 3 ) . As t h e t e m p e r a t u r e i s i n c r e a s e d from 10°C ( F i g . 2a 31 bottom) t h e c o r r e s p o n d i n g P NMR spectrum changes from a bro a d asymmetric peak w i t h a d e f i n e d low f i e l d s h o u l d e r , r e p r e s e n t i n g l i p i d i n t h e b i l a y e r phase, t o a narrow spectrum w i t h a h i g h f i e l d s h o u l d e r and r e v e r s e d asymmetry c o r r e s p o n d i n g t o l i p i d i n t h e h e x a g o n a l ( h J J ) phase ( 2 5 ) . P h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l s e r i n e , and p h o s p h a t i d y l g l y c e r o l w h i c h were used i n e x p e r i m e n t s w i t h d i b u c a i n e , s p o n t a n e o u s l y adopt a b i l a y e r . c o n f i g u r a t i o n and m a i n t a i n t h i s phase t h r o u g h o u t i n c r e a s i n g t e m p e r a t u r e s (data n o t shown). 3b. N e u t r a l A n a e s t h e t i c s I n c o m p a r i s o n t o t h e p o l y m o r p h i c phase b e h a v i o r o f egg p h o s p h a t i d y l e t h a n o l a m i n e ( F i g . 2 a ) t h e s p e c t r a o b t a i n e d f o r t h e same l i p i d i n t h e p r e s e n c e o f 1M e t h a n o l and lOOmM d e c a n o l a r e m a r k e d l y d i f f e r e n t ( F i g . 2 b and 2 c ) . The e s t i m a t e d Tbh ( F i g . 3 ) f o r t h e s e e x p e r i m e n t s i s r a i s e d t o 41°C f o r 1M e t h a n o l and l o w e r e d t o 5°C f o r lOOmM d e c a n o l . Thus a s u b s t a n t i a l s h i f t i n t h e Tbh o f p h o s p h a t i d y l e t h a n o l a m i n e was pr o d u c e d by t h e s e a n a e s t h e t i c s and t h e degree and d i r e c t i o n o f phase s h i f t was dependent on t h e a l c o h o l employed. -19-FIG. 2. 81.0 MHz J J"P NMR s p e c t r a o f egg p h o s p h a t i d y l -e t h a n o l a m i n e a t i n d i c a t e d t e m p e r a t u r e s (a) i n t h e absence o f a l c o h o l s ; (b) i n t h e p r e s e n c e o f e t h a n o l (1 M ) (c) i n t h e p r e s e n c e o f d e c a n o l (100 mM). -20--21-F i g . 3. Method f o r d e t e r m i n i n g t h e b i l a y e r t o h e x a g o n a l (H.^) phase t r a n s i t i o n t e m p e r a t u r e (Tbh) f o r egg p h o s p h a t i d y l -e t h a n o l a m i n e . Tbh i s e s t i m a t e d as t h a t t e m p e r a t u r e where 50% o f t h e l i p i d i s i n t h e b i l a y e r o r g a n i z a t i o n and 50% i s i n t h e h e x a g o n a l ( H I T ) phase: d e c a n o l (100 mM); • - c o n t r o l ; T - e t h a n o l (1M). -22--23-The s e r i e s o f even numbered n - a l c o h o l s from e t h a n o l (&2^ t o d o d e c a n o l (C-^) were added t o i n d i v i d u a l egg PE p r e p a r a t i o n s and each a l c o h o l was t e s t e d o v e r a range o f c o n c e n t r a t i o n s ( F i g . 4 ) . The a b i l i t y o f a p a r t i c u l a r a l c o h o l t o a l t e r t h e phase p r o p e r t i e s o f t h e l i p i d i n c r e a s e d w i t h i n c r e a s i n g h y d r o c a r b o n c h a i n l e n g t h , such t h a t d o d e c a n o l ( C - ^ caused a 2 0° drop i n t h e Tbh a t a c o n c e n t r a t i o n o f 50 mM whereas the same 20° d e c r e a s e i n Tbh r e q u i r e d 12 0 mM concen-t r a t i o n o f h e x a n o l . ( C , ) . b The same p r o t o c o l as above was used f o r t h e s e r i e s o f n - a l k a n e s ( F i g . 5 ) . However, because o f t h e d i f f i c u l t y i n h a n d l i n g t h e gaseous a l k a n e s , o n l y t h o s e w i t h c h a i n l e n g t h s g r e a t e r t h a n pentane (C^) were t e s t e d . The r e s u l t s f o r t h i s s e r i e s a r e n o t as w e l l d e f i n e d as t h o s e f o r t h e a l c o h o l s , a l t h o u g h t h e i r a b i l i t y t o d e c r e a s e t h e Tbh o f t h e PE r e s e m b l e s t h a t f o r t h e a l c o h o l s ; s i m i l a r l y , an i n c r e a s e i n p o t e n c y w i t h i n c r e a s i n g c h a i n l e n g t h i s o b s e r v e d . By c o mparison t o t h e a l c o h o l s t h e a l k a n e s appear t o be a p p r o x i m a t e l y two t i m e s more p o t e n t i n t h e i r a b i l i t y t o i n i t i a t e t h e h e x a g o n a l phase, where 120 mM h e x a n o l produces a 20° drop i n Tbh, o n l y 70 mM hexane i s r e q u i r e d t o produce th e same d e c r e a s e i n Tbh. S i m i l a r t o t h e e f f e c t s o f t h e l o n g e r c h a i n l e n g t h a l c o h o l s and a l k a n e s , t h e gaseous a n a e s t h e t i c c h l o r o f o r m causes r e d u c t i o n i n t h e Tbh o f Soya PE and i n i t i a t i o n o f t h e h e x a g o n a l ( H J J ) phase a t lower t e m p e r a t u r e s as compared t o c o n t r o l s ( F i g . 6 ) . The s p e c t r a shown a r e t a k e n a t a p p r o x i m a t e l y 15°C below the c o n t r o l Tbh o f t h e l i p i d and demonstrate t h a t 5 and 10 mM - 2 4 -FIG. 4. I n f l u e n c e o f v a r y i n g amounts o f t h e normal a l c o h o l s on t h e b i l a y e r t o h e x a g o n a l ( H J J ) phase t r a n s i t i o n t e m p e r a t u r e o f egg p h o s p h a t i d y l e t h a n o l a m i n e . • - e t h a n o l ; • - b u t a n o l ; • - hexanoX A - o c t a n o l ; • - d e c a n o l ; . O - l a u r y l a l c o h o l -25-I -26-F i g . 5. The i n f l u e n c e o f i n c r e a s i n g amounts o f normal a l k a n e s o f v a r i o u s c h a i n l e n g t h s on t h e b i l a y e r t o h e x a g o n a l ( H J J ) t r a n s i t i o n t e m p e r a t u r e o f egg p h o s p h a t i d y l e t h a n o l a m i n e : • - hexane; •- heptane; O - o c t a n e ; • - decane. -28-FIG. 6. 81.0 MHz P NMR s p e c t r a o f Soya p h o s p h a t i d y l e t h a n o l -amine a t -5°C i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f c h l o r o f o r m . -29-Chloroform » cone. T i 1 1 1 1 r AO 0 -40 -30-FIG. 7. 81.0 MHz J J"P NMR s p e c t r a o f egg p h o s p h a t i d y l -e t h a n o l a m i n e a t 40°C i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f (a) c h l o r p r o m a z i n e ; (b) d i b u c a i n e ; (c) t e t r a c a i n e and (d) p r o c a i n e . F o r d e t a i l s o f sample p r e p a r a t i o n s see Methods. Chlorpromazine 1 r i r n r • "r AO 0 -AO ppm H^ > Dibucaine i i i i I I I 40 0 -AO ppm .H-* Tetracaine 1 1 1 1 r r — r AO 0 "AO ppm H -> Procaine AO 0 -AO ppm H-» -32-FIG. 8. I n f l u e n c e o f d i b u c a i n e on t h e 81.0 MHz """"P NMR s p e c t r a a t 40°C o f an aqueous d i s p e r s i o n o f 20 mol % p h o s p h a t i d y l s e r i n e and 80 mol % egg p h o s p h a t i d y l -e t h a n o l a m i n e i n the p r e s e n c e o f 5 mM C a C l ? . T— 1 1 1 1 1 r A O 0 - A O -34-c o n c e n t r a t i o n s o f t h i s a n a e s t h e t i c can produce n o n - b i a l y e r s t r u c t u r e s as i n d i c a t e d by an i n c r e a s e i n t h e h e x a g o n a l and 31 i s o t r o p i c component o f t h e P NMR s p e c t r a . F u r t h e r m o r e t h e s e e f f e c t s a r e o b t a i n e d a t near c l i n i c a l c o n c e n t r a t i o n s o f t h e a n a e s t h e t i c . T h i s l a t t e r p o i n t i s an i m p o r t a n t c o n s i d e r a t i o n s i n c e l i t t l e can be s a i d about a n a e s t h e s i a u n l e s s pharmacolo-g i c a l l y r e l e v e n t c o n c e n t r a t i o n s o f a n a e s t h e t i c a r e b e i n g employed; t h i s i s t r u e r e g a r d l e s s o f whether the system i s a model o r b i o l o g i c a l . S i n c e t h e i n i t i a l r e s u l t s w i t h t h e n e u t r a l a n a e s t h e t i c s i n d i c a t e d t h a t t h e r e was some c o r r e l a t i o n between t h e p o t e ncy o f an a n a e s t h e t i c and i t s a b i l i t y t o a l t e r membrane s t a b i l i t y , whether t h r o u g h b i l a y e r s t a b i l i z a t i o n o r d e s t a b i l i -z a t i o n , i t was o f i n t e r e s t t o o b s e r v e the e f f e c t s o f some l o c a l a n a e s t h e t i c s on l i p i d p o l y morphism and a d d i t i o n a l l y t o produce t h e s e e f f e c t s a t c l i n i c a l c o n c e n t r a t i o n s . 3c. Charged (Amine) A n a e s t h e t i c s I t was found t h a t a l l t h e charg e d a n a e s t h e t i c s t e s t e d show an a b i l i t y t o s t a b i l i z e t h e b i l a y e r c o n f i g u r a t i o n o f egg PE above t h e c o n t r o l Tbh o f 30°C. T h i s e f f e c t c o r r e l a t e s w i t h t h e r e l a t i v e p o t e n c y o f t h e s e a n a e s t h e t i c s : c h l o r p r o m a z i n e > d i b u c a i n e > t e t r a c a i n e > p r o c a i n e . The s p e c t r a f o r e x p e r i m e n t s c a r r i e d o u t a t 40° a r e shown i n F i g . 7 . Egg p h o s p h a t i d y l e t h a n o l -amine w i t h no a n a e s t h e t i c p r e s e n t n o r m a l l y b e g i n s i t s b i l a y e r t o h e x a g o n a l phase t r a n s i t i o n between 2 0 and 25°C, such t h a t the b i l a y e r s p e c t r a shown i n F i g . 7 a r e a p p r o x i m a t e l y 2 0° above t h o s e f o r c o n t r o l c o n d i t i o n s . I t i s a p p a r e n t t h a t a -35-h i g h e r c o n c e n t r a t i o n o f a n a e s t h e t i c i s r e q u i r e d t o p r e s e r v e t h e b i l a y e r s t r u c t u r e a t 40° w i t h a l e s s p o t e n t a n a e s t h e t i c such as p r o c a i n e . That i s , where a p a r t i a l b i l a y e r s t r u c t u r e i s o b s e r v e d w i t h 200 mM p r o c a i n e , l e s s t h a n 2 mM c h l o r p r o m a z i n e was found t o s t a b i l i z e the b i l a y e r c o n f i g u r a t i o n o f egg PE above 6 0°C ( d a t a n o t shown). The e f f e c t s o f d i b u c a i n e on a mixed l i p i d system (80 mol% PE/ 20 mol% PS) i n t h e p r e s e n c e o f 5 mM C a + + were o b s e r v e d ( F i g . 8 a ) . The s p e c t r a shown a r e t h o s e o b t a i n e d a t 40°C, w h i c h i s 5° above where t h e c o n t r o l PE/PS m i x t u r e i n t h e p r e s e n c e o f Ca i s n o r m a l l y h e x a g o n a l ( 8 b ) . An i n c r e a s e d s t a b i l i t y o f t h e b i l a y e r s t r u c t u r e was o b s e r v e d f o r 0.5 and 1.0 mM d i b u c a i n e , however, 0.1 and 0.2 mM c o n c e n t r a t i o n s had no e f f e c t on s t a b i l i z i n g t h e b i l a y e r above c o n t r o l v a l u e s . -36-CHAPTER 4 -DISCUSSION -37-I t i s c l e a r from t h i s and o t h e r s t u d i e s (30) t h a t a number o f l i p i d s p e c i e s have t h e a b i l i t y t o adopt n o n - b i l a y e r phases when pure and h y d r a t e d . T h i s p r o p e r t y , p l u s t h e o b s e r v a t i o n t h a t a l a r g e v a r i e t y o f l i p i d t y p e s w i t h d i v e r s e s t r u c t u r e s and shapes a r e p r e s e n t i n b i o l o g i c a l membranes s u g g e s t s t h a t l i p i d s may s e r v e f u n c t i o n a l r o l e s i n membranes r a t h e r t h a n s i m p l e m a i n t a i n e n c e o f t h e i r s t r u c t u r a l i n t e g r i t y ( 3 0 ) . L i p i d s w h i c h p r e f e r t o be n o n - b i l a y e r a t p h y s i o l o g i c a l t e m p e r a t u r e s may t h e r e f o r e be p e r f o r m i n g a f u n c t i o n a l r a t h e r t h a n a s t r u c t u r a l r o l e . T h i s was t h e b a s i c p r e m i s e o f t h i s s t u d y a l o n g w i t h t h e n o t i o n t h a t a n a e s t h e t i c s , ( s o - c a l l e d 'membrane p e r t u r b a n t s ' ) w h i c h presumably i n t e r a c t w i t h the l i p i d element o f b i o l o g i c a l membranes may, i n some way, i n f l u e n c e t h e p o l y m o r p h i c phase b e h a v i o r o f n o n - b i l a y e r l i p i d s . T h i s p o s s i b i l i t y had been i n d i c a t e d p r e v i o u s l y by work c a r r i e d o u t i n t h i s l a b o r a t o r y : model membrane systems c o m p r i s e d o f c a r d i o l i p i n , a major component o f t h e i n n e r m i t o c h o n d r i a l membrane, can be c o n v e r t e d from the b i l a y e r t o h e x a g o n a l phase by t h e a d d i t i o n o f e q u i m o l a r C a + + a t 30°C. I n the same s t u d y i t was n o t e d t h a t b o t h d i b u c a i n e and c h l o r p r o m a z i n e t r i g g e r f o r m a t i o n o f t h e he x a g o n a l phase a t a n a e s t h e t i c / c a r d i o l i p i n r a t i o s o f 2:1. Some p r e l i m i n a r y e x p e r i m e n t s had a l s o been made u s i n g e t h a n o l and c h l o r o f o r m (31). I n t h i s s t u d y t h e e f f e c t s o f a n a e s t h e t i c s on l i p i d p o lymorphism has been expanded t o a number o f o t h e r l i p i d s p e c i e s , p a r t i c u l a r l y egg p h o s p h a t i d y l e t h a n o l a m i n e . I n a d d i t i o n , a number o f r e p r e s e n t a t i v e a n a e s t h e t i c s have been t e s t e d -38-i n c l u d i n g c h a r g e d , n e u t r a l and gaseous s p e c i e s , t h e purpose b e i n g t o o b t a i n a comprehensive o v e r v i e w o f a n a e s t h e t i c e f f e c t s on l i p i d polymorphism. From t h e r e s u l t s o b t a i n e d i t i s c l e a r t h a t a number o f a n a e s t h e t i c a gents have p r o f o u n d e f f e c t s on t h e p o l y m o r p h i c phase b e h a v i o r d f egg p h o s p h a t i d y l -e t h a n o l a m i n e . I n a l l t h e e x p e r i m e n t s p r e s e n t e d above t h e egg PE sample used had a b i l a y e r t o h e x a g o n a l (H.^) phase t r a n s i t i o n t e m p e r a t u r e o f a p p r o x i m a t e l y 30°C. T h i s p r o p e r t y o f egg PE remains c o n s i s t e n t by t h e t e c h n i q u e d e s c r i b e d , however i n i t i a l d i f f i c u l t i e s were e n o u n t e r e d i n v a r y i n g Tbh's w i t h s l i g h t l y d i f f e r e n t p u r i f i c a t i o n t e c h n i q u e s and p r e p a r a t i o n s . The u s e f u l n e s s o f t h e s e r i e s o f n - a l k a n e s and n - a l c o h o l s i n s t u d i e s o f a n a e s t h e t i c mechanism (or i n e f f e c t i n g t h e p o l y m o r p h i c phase behavior; o f egg PE) i s a r e s u l t o f t h e i n c r e a s i n g p o t e n c y o f t h e s e m o l e c u l e s w i t h i n c r e a s i n g h y d r o c a r b o n c h a i n l e n g t h . E x p e r i m e n t s w i t h t h e s e r i e s , t h e r e f o r e , p r o v i d e s a c o m p a r i s o n o f i n c r e a s i n g p o t e n c y e f f e c t s u s i n g analogous m o l e c u l e s . G e n e r a l l y t h e a l c o h o l s and a l k a n e s have a n a e s t h e t i c e f f e c t s up t o c a r b o n number 10 i n a p h y s i o l o g i c a l system, a f t e r w h i c h th e l o n g e r c h a i n m o l e c u l e s a r e no l o n g e r a n a e s t h e t i c . T h i s s o - c a l l e d ' c u t - o f f e f f e c t o c c u r s because th e l o n g e r c h a i n l e n g t h a n a e s t h e t i c s a r e t o o i n s o l u b l e t o have any e f f e c t i n v i v o ; t h e c u t - o f f e f f e c t , t h e r e f o r e , i s not r e l e v e n t i n a model system where <; p h y s i o l o g i c a l p a r t i t i o n p a r a m e t e r s do not e x i s t . The r e s u l t s i n F i g u r e 4, however, do show a t y p e o f ' c u t - o f f e f f e c t between the a b i l i t y o f t h e n - a l c o h o l s up t o CA t o s t a b i l i z e -39-t h e b i l a y e r c o n f i g u r a t i o n o f egg PE, whereas Cg a l c o h o l , and g r e a t e r , cause d e s t a b i l i z a t i o n and i n i t i a t i o n o f t h e h e x a g o n a l phase. T h i s c a p a c i t y t o t r i g g e r h e x a g o n a l phase f o r m a t i o n i n c r e a s e s w i t h i n c r e a s i n g h y d r o c a r b o n c h a i n l e n g t h f o r b o t h a l c o h o l s and a l k a n e s and i n t h i s way c o r r e l a t e s w i t h t h e a n a e s t h e t i c p o t e n c y o f t h e i n d i v i d u a l d r u g s . I t i s f u r t h e r o b s e r v e d t h a t t h e s e r i e s o f a l k a n e s shows a g r e a t e r p o t e n c y t h a n t h e a l c o h o l s w h i c h i s a l s o t h e c a s e i n p h y s i o l o g i c a l s t u d i e s . The s h i f t away from t h e t e m p e r a t u r e a t w h i c h th e b i l a y e r t o h e x a g o n a l phase t r a n s i t i o n o c c u r s i n c r e a s e s w i t h g r e a t e r c o n c e n t r a t i o n s o f t h e p a r t i c u l a r a n a e s t h e t i c . A l t h o u g h t h e s e c o n c e n t r a t i o n s a r e n o t p h a r m a c o l o g i c a l l y r e l e v e n t w i t h r e s p e c t t o a n a e s t h e s i a , w i t h e x c e p t i o n o f e t h a n o l and b u t a n o l , t h e aim o f t h e s e e x p e r i m e n t s was m e r e l y t o d e m o n s t r a t e t h e a b i l i t y o f t h e s e a n a e s t h e t i c a gents t o a l t e r egg PE p o l y m o r p h i c phase b e h a v i o r . S i n c e the c a p a c i t y o f an a l k a n e o r a l c o h o l t o e f f e c t h e x a g o n a l phase f o r m a t i o n appears t o be a f u n c t i o n o f t h e h y d r o c a r b o n c h a i n l e n g t h o f t h e a n a e s t h e t i c s p e c i e s , t h i s p r o p e r t y w i l l be d i s c u s s e d l a t e r i n terms o f t h e m o l e c u l a r shape o f t h e s e m o l e c u l e s . The h e x a g o n a l phase i s a l s o e f f e c t e d by c h l o r o f o r m a t n e a r c l i n i c a l c o n c e n t r a t i o n s (lOmM). The r e s u l t s shown a r e f o r Soya p h o s p h a t i d y l e t h a n o l a m i n e b u t i d e n t i c a l e f f e c t s have been o b s e r v e d w i t h egg PE. Converse t o t h e e f f e c t s o f t h o s e a g e n t s d i s c u s s e d above, t h e l o c a l a n a e s t h e t i c s t e s t e d s t a b i l i z e t h e b i l a y e r s t r u c t u r e o f t h e l i p o s o m e s and t h i s e f f e c t t o o , i s a f u n c t i o n o f t h e a n a e s t h e t i c p o t e n c y o f t h e -40-i n d i v i d u a l s p e c i e s . I n comparing th e e f f e c t s o f t h e l o c a l a n a e s t h e t i c s on egg PE c o n c e n t r a t i o n s h i g h e r t h a n t h o s e r e q u i r e d t o produce nerve b l o c k i n v i v o were u s e d , however e x p e r i m e n t s w i t h d i b u c a i n e on a mixed l i p i d system o f PE/PS p l u s C a + + show b i l a y e r s t a b i l i z a t i o n a t p h a r m a c o l o g i c a l l y r e l e v e n t c o n c e n t r a t i o n s (0.5 - 1.0 mM) ( F i g . 8 and 9 ) . To r e c a p on..the above o b s e r v a t i o n s , i t i s c l e a r t h a t a number o f r e p r e s e n t a t i v e a n a e s t h e t i c agents can m a r k e d l y a f f e c t t h e p o l y m o r p h i c phase b e h a v i o r o f p h o s p h a t i d y l e t h a n o l -amine s i n g u l a r l y and i n a mixed l i p i d system and t h a t t h e degree o f e f f e c t d i r e c t l y r e l a t e s t o t h e p h y s i o l o g i c a l a n a e s t h e t i c p o t e n c y o f t h e agent b e i n g used. A d d i t i o n a l l y , s i m i l a r e f f e c t s can be a c h i e v e d a t p h a r m a c o l o g i c a l l y r e l e v e n t c o n c e n t r a t i o n s o f t h e a n a e s t h e t i c . A r a t h e r n a i v e b u t i l l u s t r a t i v e i n t e r p r e t a t i o n can be made f o r t h e mechanism whereby a n a e s t h e t i c m o l e c u l e s a l t e r t h e p o l y m o r p h i c phase b e h a v i o r o f c e r t a i n l i p i d s based on t h e dynamic m o l e c u l a r shape o f t h e m o l e c u l e s i n v o l v e d . Other a u t h o r s (32) have i n v o k e d s i m i l a r c o n s i d e r a t i o n s t o r a t i o n a l i z e t h e b e h a v i o r o f p a r t i c u l a r l i p i d systems. B r i e f l y , as i n d i c a t e d i n F i g u r e 1, l i p i d s assuming th e h e x a g o n a l ( H j j ) phase may be c o n s i d e r e d t o e x h i b i t a 'cone' shape, where t h e p o l a r headgroup r e g i o n i s a t the s m a l l e r end o f t h e cone. A l t e r n a t i v e l y , l y s o p h o s p h o l i p i d s may be s u g g e s t e d t o d i s p l a y an ' i n v e r t e d cone' shape where the c r o s s - s e c t i o n a l a r e a oft t h e p o l a r r e g i o n i s l a r g e r t h a n t h a t subtended towards th e end o f t h e a c y l c h a i n . T h i s shape would be -41-c o m p a t i b l e w i t h t h e m i c e l l a r phase adopted by t h e s e l i p i d s . F i n a l l y , l i p i d s w h i c h assume a more c y l i n d r i c a l shape would be most e a s i l y accomodated i n the f a m i l a r b i l a y e r phase. I n terms o f t h i s p r o p o s a l , the s m a l l e r headgroup o f PE (as compared t o PC) as w e l l as the p o s s i b i l i t y o f i n t e r m o l e c u l a r hydrogen b o n d i n g (33) would be e x p e c t e d t o r e s u l t i n a r e d u c e d a r e a p er m o l e c u l e a t the l i p i d - w a t e r i n t e r f a c e . T h i s would be c o m p a t i b l e w i t h a 'cone' shape c o n f i g u r a t i o n and p r e f e r e n c e f o r h e x a g o n a l phase f o r m a t i o n commonly found f o r PE. By the same p r e m i s e , i n c r e a s e d u n s a t u r a t i o n o f t h e a c y l c h a i n r e g i o n o f t h e m o l e c u l e would e f f e c t i v e l y i n c r e a s e t h e c r o s s - s e c t i o n a l a r e a o f t h i s p o r t i o n o f t h e m o l e c u l e as would i n c r e a s e d t h e r m a l m o t i o n a t h i g h e r t e m p e r a t u r e s , a g a i n r e s u l t i n g i n a 'cone-shaped' -.: c o n f i g u r a t i o n and l i a b i l i t y toward an h e x a g o n a l arrangment ( 1 5 ) . These m o l e c u l a r shape c o n s i d e r a t i o n s can a l s o be extended t o a c i d i c p h o s p h o l i p i d s f o r w h i c h the a r e a p e r m o l e c u l e a t the l i p i d - w a t e r i n t e r f a c e i s s e n s i t i v e t o t h e n e t charge i n the p o l a r r e g i o n (34). C a r d i o l i p i n , i s o l a t e d from m i t o c h o n d r i a , would be e x p e c t e d t o e x h i b i t a cone shaped s t r u c t u r e c o n s i d e r i n g the r e l a t i v e l y s m a l l headgroup r e g i o n and t h e l a r g e c r o s s - s e c t i o n a l a r e a o f f o u r ( u s u a l l y v e r y u n s a t u r a t e d ) a c y l c h a i n s (35). T h i s l i p i d a d o p t s a b i l a y e r c o n f i g u r a t i o n when h y d r a t e d , however, when i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s , charge n e u t r a l i z a t i o n o c c u r s and presumably r e d u c t i o n i n the e f f e c t i v e s i z e o f t h e headgroup a r e a , c o n s e q u e n t l y t r i g g e r i n g f o r m a t i o n o f h e x a g o n a l ( H I I ) phase ( 1 9 ) . -42-S i m i l a r l y , a t pH v a l u e s above 5, u n s a t u r a t e d p h o s p h a t i d y l -s e r i n e a d o p ts t h e b i l a y e r phase, b u t a pH=2.5 (below/the pK o f t h e c a r b o x y l group) t h e h e x a g o n a l phase i s o b s e r v e d (36 ) , w h i c h a g a i n may be a t t r i b u t e d t o reduced charge r e p u l s i o n e f f e c t s . A r e l a t i o n s h i p between a n a e s t h e t i c mechanism and the o b s e r v a t i o n s made i n t h i s s t u d y can be made i n terms o f t h e m o l e c u l a r shape o f t h e a n a e s t h e t i c s employed (30). E t h a n o l , f o r example, b e i n g r e l a t i v e l y p o l a r , would be e x p e c t e d t o r e s i d e a t t h e l i p i d - w a t e r i n t e r f a c e w i t h t h e hy d r o c a r b o n r e g i o n e x t e n d e d toward t h e i n t e r i o r o f t h e b i l a y e r . T h i s i n t e r a c t i o n w i t h PE would r e s u l t i n a ' c y l i n d e r ' shaped complex r e s u l t i n g i n an i n c r e a s e i n b i l a y e r s t a b i l i t y . I n c r e a s i n g t h e h y d r o c a r b o n c h a i n l e n g t h o f the a l c o h o l t o d e c a n o l produces a f u r t h e r i n t e g r a t i o n i n t o the a c y l c h a i n r e g i o n o f t h e p h o s p h o l i p i d s , e f f e c t i v e l y e x p a n d i n g t h i s r e g i o n and i n i t i a t i n g an h e x a g o n a l arrangment. S i m i l a r l y , c h l o r o f o r m would be e x p e c t e d t o r e s i d e i n t h e h y d r o p h o b i c r e g i o n o f the membrane p r o d u c i n g a s i m i l a r e f f e c t t o t h a t o f the l o n g e r c h a i n a l c o h o l s . T h i s e f f e c t may be i n t e r p r e t e d by t h e same model, however, i n t h e p r e s e n c e o f the a c i d i c PS, i t i s e x p e c t e d t h a t an i o n - i o n (charge n e u t r a l i z a t i o n ) o r i o n - d i p o l e i n t e r a c t i o n o f t h e l o c a l a n a e s t h e t i c w i t h a c i d i c p h o s p h o l i p i d o c c u r s . I t has been d e m o n s t r a t e d t h a t a d d i t i o n o f l o c a l a n a e s t h e t i c s t o an a c i d i c p h o s p h o l i p i d d i s p e r s i o n produces a d e c r e a s e i n b u l k pH, i n d i c a t i n g an i n t e r a c t i o n w i t h e i t h e r -43-phosphate o r c a r b o x y l groups (37). I n t h e mixed l i p i d s ystem s t u d i e d l o c a l a n a e s t h e t i c s may d i s p l a c e C a + + from s i t e s on t h e PS w h i c h i n i t i a l l y p roduces the h e x a g o n a l arrangment and c o n s e q u e n t l y r e f o r m a b i l a y e r s t r u c t u r e and a t t h e same ti m e produce t h e i r e f f e c t s by n e t e x p a n s i o n o f t h e headgroup r e g i o n o f t h i s m i x t u r e . To expand s l i g h t l y on t h e p r o p e r t i e s o f l o c a l a n a e s t h e i t c s , some common g e n e r a l i z a t i o n s a r e t h a t the u s e f u l l o c a l a n a e s t h e t i c s , w i t h few e x c e p t i o n s , a r e composed o f t h r e e c o n s t i t u e n t s : 1.) a c a r b o c y c l i c o r h e t e r o c y c l i c r i n g o f t h e a r o m a t i c t y p e ( l i p o p h i l i c p o r t i o n ) . 2.) an i n t e r m e d i a t e c h a i n , and 3.) an amino group ( h y d r o p h i l i c p o r t i o n ) ( 3 7 ) . I t i s f u r t h e r i n d i c a t e d t h a t t h e a r o m a t i c r i n g and i t s e l e c t r o n -d o n a t i n g s u b s t i t u e n t s c o n j u g a t e w i t h the a d j a c e n t c a r b o x y l group r e n d e r i n g i t more e l e c t r o n e g a t i v e and c a p a b l e o f f o r m i n g hydrogen bonds. The e l e c t r o n e g a t i v i t y o f the c a r b o n y l oxygen i s t h u s c o r r e l a t e d w i t h t h e i n t e n s i f i e d l o c a l a n a e s t h e t i c a c t i v i t y (38). T h i s t h e o r y r e a d i l y a c c o u n t s , f o r i n s t a n c e , f o r the l o c a l a n a e s t h e t i c a c t i v i t y o f p r o c a i n e (p-NI^ group) and t e t r a c a i n e (p-C^Hg-NH g r o u p ) , where the i n t r o d u c t i o n o f an e l e c t r o n - d o n a t i n g s u b s t i t u e n t r e s u l t s i n a marked i n t e n s i f i c a t i o n o f the weak e f f e c t o f the p a r e n t compound. I n g e n e r a l , t h e r e f o r e , compounds o f t h e t y p e i n w h i c h resonance e f f e c t s r e s u l t s i n an a c t i v e d i p o l e c a r b o n y l oxygen, and p a r t i c u l a r l y t h o s e i n w h i c h the n e g a t i v i t y o f t h e c a r b o n y l oxygen i s p a r t i a l l y i n t e n s i f i e d by e l e c t r o n - d o n a t i n g s u b s t i t -u e n t s , a r e p o t e n t l o c a l a n a e s t h e t i c s ( 3 8 ) . A c o r r e l a t i o n -44-between a n a e s t h e t i c p o t e n c y and t h e a b i l i t y t o s t a b i l i z e h y d r a t e d PE i n t o a b i l a y e r may be t h e r e s u l t o f an i o n - i o n , o r i o n - d i p o l e i n t e r a c t i o n o f the l o c a l a n a e s t h e t i c and t h e p h o s p h o l i p i d headgroup; such an a s s o c i a t i o n would produce a n e t e x p a n s i o n o f t h i s headgroup r e g i o n c o n s e q u e n t l y e f f e c t i n g b i l a y e r s t a b i l i t y w i t h g r e a t e r e x p a n s i o n o f t h i s r e g i o n b e i n g due t o g r e a t e r e l e c t r o n e g a t i v i t y o f the c a r b o n y l oxygen. G i v e n t h e above mechanism o f a n a e s t h e t i c e f f e c t s on l i p i d p o lymorphism t h e q u e s t i o n a r i s e s o f how t h i s r e l a t e s t o a b i o l o g i c a l system. N o n - b i l a y e r phases have n o t been proven t o e x i s t i n b i o l o g i c a l membranes however, s t r o n g e v i d e n c e e x i s t s t o s u p p o r t t h i s c o n t e n t i o n (see Ref. 3 0 ) . E r y t h r o c y t e g h o s t s , 31 f o r example, n o r m a l l y show P NMR s p e c t r a r e p r e s e n t i n g a b i l a y e r c o n f i g u r a t i o n even a f t e r t r e a t m e n t w i t h p h o s p h o l i p a s e s w h i c h produce a l a r g e p e r c e n t a g e o f n o n - b i l a y e r l i p i d s i n the membrane. The e r y t h r o c y t e membrane, t h e r e f o r e , e x h i b i t s a s t r o n g b i l a y e r s t a b i l i t y . I n t r o d u c t i o n o f o l e i c a c i d however ( a s o - c a l l e d 'fusogen') can cause a w h o l e s a l e d i s r u p t i o n o f b i l a y e r s t r u c t u r e , p r o m o t i n g f o r m a t i o n o f t h e h e x a g o n a l phase ( 2 3 ) . T h i s b e h a v i o r has been used t o suggest t h e i n v o l v e m e n t o f n o n - b i l a y e r phases as i n t e r m e d i a t e d u r i n g f u s i o n e v e n t s . A d d i t i o n a l l y , n o n - b i l a y e r phases may be p r e s e n t i n e n d o p l a s m i c r e t i c u l u m p r e p a r a t i o n s where i s o t r o p i c 31 m o t i o n has been o b s e r v e d w i t h P NMR i n r a t , b o v i n e , and -45-r a b b i t l i v e r e n d o p l a s m i c r e t i c u l u m (21, 2 2 ) . A l t h o u g h i t i s n o t c l e a r l y e s t a b l i s h e d what t h e s e i s o t r o p i c s p e c t r a r e p r e s e n t , t h e y d i m i n i s h below 37°C, such t h a t a g r e a t e r p e r c e n t a g e o f t h i s l i p i d c o n t r i b u t e s t o t h e b i l a y e r component o f t h e s p e c t r a : t h e same b e h a v i o r i s o b s e r v e d i n model systems where i t i s t h o u g h t t h a t the i s o t r o p i c m o t i o n a l a v e r a g i n g may r e p r e s e n t i n v e r t e d m i c e l l a r o r ( s h o r t ) c y l i n d r i c a l (H-j-j) arrangments o f l i p i d s i n s i d e the b i l a y e r 31 (30). P NMR e v i d e n c e i s now a v a i l a b l e w h i c h i n d i c a t e s t h a t the t e m p e r a t u r e dependent phase change o b s e r v e d i n model systems and e n d o p l a s m i c r e t i c u l u m p r e p a r a t i o n s a l s o o c c u r s i n t h e e n d o p l a s m i c r e t i c u l u m o f i n t a c t r a t l i v e r ( 4 1 ) . As t h i s i s o t r o p i c m o t i o n does n o t a r i s e from m i c r o s o m a l t u m b l i n g (21,22) i t i s t e m p t i n g t o a s c r i b e i t t o n o n - b i l a y e r l i p i d s t r u c t u r e s . However, the p o s s i b i l i t y t h a t l a t e r a l d i f f u s i o n i n t he b i l a y e r produces t h e o b s e r v e d a v e r a g i n g cannot be e x c l u d e d . S i m i l a r o b s e r v a t i o n s o f i s o t r o p i c m o t i o n have been o b t a i n e d f o r s a r c o p l a s m i c r e t i c u l u m membrane (39) as w e l l as i n n e r m i t o c h o n d r i a l membrane ( 4 0 ) . The e v i d e n c e s u g g e s t s t h a t n o n - b i l a y e r phases may e x i s t i n b i o l o g i c a l membranes and t h a t t h e s e phases may p l a y some f u n c t i o n a l p a r t i n membrane p r o c e s s e s . L i k e l y f u n c t i o n a l r o l e s f o r t h e s e phases may be membrane f u s i o n phenomena ( i n c l u d i n g r e l a t e d p r o c e s s e s such as exo- and e n d o c y t o s i s ) and t r a n s b i l a y e r t r a n s p o r t p r o c e s s e s ( i n c l u d i n g l i p i d ' f l i p - f l o p ' and f a c i l i t a t e d t r a n s p o r t ) . I t i s n o t u n l i k e l y t h a t p h o s p h o l i p i d s a r e i n v o l v e d i n - 4 6 -membrane t r a n s p o r t o f i o n s such as Ca"""1" (8) o r , i n d e e d , N a + and K + d u r i n g n e u r o n a l e x c i t a t i o n ( 1 7 ) . T h i s c o n t e n t i o n r e q u i r e s a t r a n s p o r t p r o c e s s whereby i o n s may be t r a n s p o r t e d i n a p o l a r environment t h r o u g h the h y d r o c a r b o n m a t r i x o f t h e membrane. T h i s c o u l d o c c u r by n o n - b i l a y e r phase f o r m a t i o n such as i n v e r t e d m i c e l l e s o r ( s h o r t ) h e x a g o n a l arrangements. T h i s would n ot be e x p e c t e d t o be a l o c a l i z e d phenomenon i n a nerve membrane where p r o p a g a t i o n o f t h e a c t i o n p o t e n t i a l r e q u i r e s v i r t u a l l y t he e n t i r e membrane s u r f a c e , however, the e f f e c t may be v e r y t r a n s i e n t and o c c u r i n a f a c i l i t a t e d f a s h i o n such as d e s t a b i l i z a t i o n o f n o n - b i l a y e r ('cone-shaped') l i p i d s by the temporary i n c r e a s e d b i n d i n g o f C a + + t o a c i d i c p h o s p h o l i p i d s d u r i n g e x c i t a t i o n . T h i s e f f e c t has been o b s e r v e d i n model systems and has a s t r o n g p o s s i b i l i t y o f o c c u r r i n g i n b i o l o g i c a l systems. I f , i n d e e d , t h i s were the case the mechanism o f a n a e s t h e t i c a c t i o n (based on the r e s u l t s o b t a i n e d here) would be one o f d i s r u p t i o n o f l i p i d polymorphism, e i t h e r by b i l a y e r s t a b i l i z a t i o n o r d e s t a b i l i z a t i o n . The a b i l i t y o f a n a e s t h e t i c m o l e c u l e s t o s t r o n g l y i n t e r a c t w i t h l i p i d s and t h e d e m o n s t r a t i o n t h a t t h e y have p r o f o u n d e f f e c t s on p o l y m o r p h i c phase b e h a v i o r would s u g g e s t t h a t t h e y c o u l d e a s i l y d i s r u p t a f u n c t i o n a l a s p e c t o f l i p i d s i n t h e membrane. An a l t e r n a t i v e model f o r a n a e s t h e t i c a c t i o n c o u l d r e g a r d t h e sodium c h a n n e l as h a v i n g a p r o t e i n c o n s t r u c t i o n r a t h e r t h a n b e i n g f a c i l i t a t e d t o t a l l y by l i p i d s . T h i s h y p o t h e s i s seems more r e a l i s t i c c o n s i d e r i n g t h e s p e c i f i c i t y o f b l o c k i n g a gents such as t e t r a o d o t o x i n and s a x i t o x i n f o r a -47-a f i n i t e number o f sodium c o n d u c t i o n s i t e s on axon membranes, p l u s t h e s e l e c t i v i t y o f t h e c h a n n e l f o r sodium i o n s . Such a p r o t e i n c h a n n e l may o b t a i n a l l , o r a p o r t i o n ^ , o f i t s s t a b i l i t y from s u r r o u n d i n g l i p i d s . As viewed by I s r a e l a c h v i l i (33) p r o t e i n s may have v a r y i n g shapes i n the b i l a y e r , r e q u i r i n g 'cone' o r ' i n v e r t e d - c o n e ' shaped l i p i d s t o p r o v i d e o p t i m a l p a c k i n g and s e a l i n g a t the p r o t e i n - l i p i d i n t e r f a c e . E v i d e n c e f o r such an i n t e r a c t i o n i s g i v e n by the r e c e n t o b s e r v a t i o n (41) t h a t r e c o n s t i t u t e d g l y c o p h o r i n d i o l e o y l p h o s p h a t i d y l -c h o l i n e membranes r e q u i r e t h e a d d i t i o n o f s m a l l q u a n t i t i e s o f cone shaped l i p i d i n o r d e r t o r e n d e r the membrane impermeable t o s h i f t r e a g e n t s . A n a e s t h e t i c m o l e c u l e s p a r t i t i o n i n g i n t o a c e l l membrane w i l l d i s r u p t a l l p r o t e i n f u n c t i o n s when p r e s e n t i n h i g h enough c o n c e n t r a t i o n s . The ' s e n s i t i v i t y ' t o p a r t i t i o n i n g a n a e s t h e t i c a g e n t s i n t h e r e g i o n o f t h e sodium c h a n n e l may be such t h a t an ' i n v e r t e d - c o n e ' shaped a l c o h o l , f o r example, would d i s r u p t t h e p a c k i n g around the p r o t e i n s u f f i c i e n t l y t o p r e v e n t i t s f u n c t i o n . The a b i l i t y o f a n a e s t h e t i c s t o a l t e r t h e phase p r o p e r t i e s o f s p e c i f i c l i p i d s has been c l e a r l y d emonstrated i n t h i s s t u d y ; d i s r u p t i o n o f the v i a b l e phase p r o p e r t i e s i n a b i o l o g i c a l system by a n a e s t h e t i c s would be e x p e c t e d t o markedly a f f e c t t h e membrane's f u n c t i o n . W i t h f u r t h e r u n d e r s t a n d i n g o f t h e r o l e s o f n o n - b i l a y e r l i p i d s i n b i o l o g i c a l membranes more e x a c t t e s t i n g o f a n a e s t h e t i c mechanisms can be made. T h i s p o s s i b i l i t y i s n e a r , c o n s i d e r i n g t h e v e r y r a p i d l y a c c u m u l a t i n g i n f o r m a t i o n i n t h i s f i e l d . A l r e a d y the f i n d i n g s -48-o f s t u d i e s d i s c u s s e d here have p r o v i d e d a new d i m e n s i o n t o membrane b i o l o g y and, a d d i t i o n a l l y , a n a e s t h e t i c mechanism. -49-BIBLIOGRAPHY -50-Seeman, P. (1972) P h a r m a c o l . Rev., 24, 583-655 Meyer, H.H. and G o t t l i e b , R. (1926) i n E x p e r i m e n t a l Pharmacology as a B a s i s f o r T h e r a p e u t i c s , 2nd ed., t r a n s l . by V.E. Henderson, J.B. L i p p i n c o t t Co., P h i l a d e l p h i a , p. 121 Meyer, K.H. (1937) T r a n s . Faraday S o c , 33, 1062-1068 Lee, A.G. (1976) N a t u r e , 262, 545-548 Zambrano, F., C e l l i n o , M., and C e n e s s a - F i s c h e r , M. (1971) J . Memb. B i o l . , 6, 289-303 Boggs, J.M., Yoong, J . , and H s i a , J.C. (1976) M o l . 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