Biochem. J. (1968) 109,909Print in Gret Britain

909

The Displacement of Calcium Ions from Phospholipid Monolayers byPharmacologically Active and other Organic Bases

BY H. HAUSER* AND R. M. C. DAWSONDepartment of Biochemi8try, Agricultural Research Council

Institute of Animal Physiology, Babraham, Cambridge

(Received 16 May 1968)

1. The binding of 45Ca2+ to a monolayer of phosphatidylinositol at the air-waterinterface wasmaxiimalwhen the separation ofthe phospholipidhead groups approxi-mated to the diameter ofahydrated Ca2+ ion. 2. The displacement ofCa2+ adsorbedon monomolecular films of phosphatidylinositol by a series of drugs (both narcoticand excitatory) and other organic bases was related to the ability of the bases topenetrate into the film. 3. With films of phosphatidylinositol at constant area, andat an initial surface pressure of 10dynes/cm., the displacement ofCa2+ by increasingconcentrations ofthe local anaesthetic, tetracaine, was linearly related to the changein surface pressure (A7r) caused by the penetration of the drug. 4. A7r and thedisplacement of Ca2+ showed a related fall when the initial surface pressure of thephosphatidylinositol film was increased from 4 to 40dynes/cm. both at a constantbulk tetracaine concentration and when this latter concentration was adjustedto keep it at a constant ratio to the surface density of phosphatidylinositol mole-cules. 5. The displacement of Ca2+ from phosphatidylinositol films by cetyltri-methylammonium ions was directly compared with the surface concentration ofthe base in the film, measured by using labelled base and a surface-radioactivitytechnique. 6. The ability of a series of straight-chain aliphatic amines to displaceCa2+ from phosphatidylinositol films increased with the number of carbon atomsup to C12. However, there was a marked jump in the displacing activity afterhexylamine, and this could probably be correlated with the carbon chain's being ofsufficient length to just reach the hydrophobic fatty acid chains of the orientatedphospholipid molecules with the charges on both substances in juxtaposition.

It is now generally assumed that calcium playsan important role in controlling the excitabilityof nervous tissue. Lowering the calcium concentra-tion results in an increase in the permeability ofbiological membranes and produces a spontaneousfiring of neurones (e.g. Frankenhaeuser & Hodgkin,1957; Ames, Tsukada & Nesbett, 1967).We have carried out an investigation ofthe mode

of binding of 45Ca2+ to monomolecular films of thecomplex lipids found in nerve-cell membranes(Hauser & Dawson, 1967). The acidic phospho-lipids readily bound Ca2+, the adsorption beinglargely independent of the chemical nature of thephospholipid, and controlled by coulombic forcesdirectly related to the net excess of negative chargeon the lipid molecule. The adsorbed Ca2+ on suchfilms could be displaced readily by relatively highconcentrations of univalent metal cations such asNa+ or K+. The starting point of this investigation

*Present address: Unilever Research Laboratories, TheFrythe, Welwyn, Herts.

was the observation that certain pharmacologicallyactive bases were several hundred times moreeffective at displacing Ca2+ from acidic phospholipidmonolayers than Na+ or K+. Because of the ideaoften put forward that these substances act byinterfering with or mimicking the effect of Ca2+ onthe nerve-cell membrane (e.g. Feinstein, 1964;Feinstein & Paimre, 1966; Rogeness, Krugman &Abood, 1966; Blaustein, 1967), the displacingeffect was investigated in some detail. The displace-ment of Ca2+ by organic bases at low concentrationis probably due to their ability to penetrate intothe phospholipid film so that their basic groupsare more effective in neutralizing the negative fieldaround the phospholipid polar groups than if theyare acting as simple gegenions.

EXPERIMENTALThe apparatus and techniques used for determining the

amount of 45Ca2+ adsorbed on a monolayer by surface-radioactivity measurements have already been described

H. HAUSER AND R. M. C. DAWSON

(Hauser & Dawson, 1967). However, a flow counter with a

large window (6.5cm. x 1-2 cm.) covered with a sheet of6,u-thick polyethyleneterephthalate plastic (Melinex; I.C.I.)gave better detection of the weak fl-emission from 45Ca2+(Dawson, 1968). As the phospholipid films used in thepresent work were mainly below collapse pressure, the slowloss of surface radioactivity after maximum adsorption of45Ca'+ was less of a problem than in our previous investi-gation (Hauser & Dawson, 1967). In general, a knownamount of phospholipid was spread as a monolayer on thetrough filled to the brim with doubly distilled water,pH5.5 (72ml.); 45CaC12 (0.0154,umole; 3 5,uc) (The Radio-chemical Centre, Amersham, Bucks.) was added to thetrough and the film was compressed with a barrier to thepressure required. After the surface radioactivity reachedequilibrium the displacing substance was added to thebulk phase and distributed throughout the trough bymagnetic stirring. When displacement was complete thesurface pressure was recorded, and another sample of thesolution of displacing agent was added to the trough. In thisway plots were constructed of the surface radioactivity andthe change in surface pressure (Axff) against concentration ofdisplacing substance, so that from these the amount of thedisplacing substance required to produce 50% displacementof CaO+ and the corresponding Air could be deduced.

Pho8pholipid8. Phosphatidic acid was prepared by themethod of Palmer & iDawson (1968), and phosphatidyl-inositol was prepared from frozen peas by a procedureevolved in this Laboratory by Dr A. Sheltawy and based onmethods described by Rouser, Kritchevsky, Heller & Lieber(1963) and Ansell & Hawthorne (1964). Thin-layerchromatography and alkaline degradation indicated thatboth were pure. Dicetylphosphoric acid was a commercialsample (Albright and Wilson Ltd., London, W. 1).

RESULTSBinding of Ca2+ to a pho8phatidylino8itol film at

variou8 8urface pre88ures. Fig. 1 shows the effect ofvarying the surface pressure of a phosphatidyl-inositol monolayer orientated at the air-waterinterface on the binding of Ca2+, measured by thesurface-radioactivity technique. With increasingsurface pressure there was a linear increase in thesurface concentration of phosphatidylinositol mole-cules. However, the surface concentration of Ca2+,[S]ca, was not proportional to the equivalentconcentration of phospholipid molecules, [S]PL(Fig. la). The ratio [S]PL/[S]ca reached a minimumof 3-7:1 between 20 and 30dynes/cm. pressure

(Fig. lb), the affinity of the Ca2+ for the film fallingoff at lower and higher pressures. However, theabsolute value of this ratio is fixed by the concen-

tration of Ca2+ in the subphase. In this instance,a saturating concentration of Ca2+ was not usedbecause of limitations in the surface-radioactivitytechnique (Hauser & Dawson, 1967). Each pointplotted in Fig. l(a) represents the surface concen-

tration of Ca2+ as determined by the amount ofCa2+ added to the system and the adsorptionisotherm for Ca2+ at the particular surface pressure

of phosphatidylinositol examined.

*

210

A1.5

-,.- 1-0O F

Z-5-50! 5.0;g-¢ 4-5* + 4 0co Cs

lo- 3-5w'-'o X

sa

1968

10 20 30 40 50Surface pressure (dynes/cm.)

1-

ol

co

Qx

~4

Fig. 1. Variation of adsorption of Ca2+ on a phosphatidyl-inositol film with surface pressure. (a) Surface concentrationof phosphatidylinositol (0) and Ca+ (0). (b) Ratio betweenthe surface concentration of phosphatidylinositol andCa2+ (A), and the calculated average spacing between thenegative charges on the phospholipid head groups (A).

Table 1. Compari8on of di8placement of Ca2+ frommonomolecular film8 of acidic phospholipid8 by Na+and by tetracaine

Displacing agents were added to the bulk phase in theLangmuir trough.

FilmDicetylphosphoric

TetracaineNa+ added addedfor 50% for 50%

Pressure displacement displacement(dynes/cm.) (uLg.ions) (1tmoles)

49*5 150 3-8acid

Phosphatidic acid 42-5Phosphatidyl- 42-5inositol

126 0 4390 0 30

At low pressures it is possible that the widespacing of the phospholipid molecules inhibitedtwo-point electrostatic attachment of Ca2+ (Hauser& Dawson, 1967) to the anionic sites on the film,and the close spacing at higher pressures mighthave prevented the penetration of the hydratedCa2+ ion between the anionic head groups of thephospholipid molecules. Thus calculation from theforce-area curves of phosphatidylinositol showedthat the separation ofnegative sites on the interfaceapproached the diameter of a hydrated Ca2+ ion(9.6 A) at a point when Ca2+ binding was maximal(Fig. lb).

Di8placement of Ca2+ from monolayer8. When

910

V1 Ca2+ DISPLACEMENT FROM PHOSPHOLIPIDS BY BASESTable 2. Displacement of Ca2+ from monolayer8 of

phosphatidylinositol by variouB base8

The films were at an initial pressure of 10dynes/em.Other details are given in the Experimental section.

SubstanceDrugs

Adrenaline hydrochlorideAmphetamine sulphateAtropine sulphateBrucine sulphateChlorpromazinehydrochloride

Cocaine hydrochlorideDibucaine hydrochlorideMeprobomateOrphenadrine hydrochlorideProcaine hydrochloridePromethazine hydrochlorideTetracaine hydrochlorideTyramine hydrochlorideStrychnine hydrochloride

Amino acidsPhenylalanineCysteineHistidine hydrochlorideArginine hydrochloride

Miscellaneous2-Aminoethylbenzenehydrochloride1-Aminoethylbenzenehydrochloride

TrimethylaminehydrochlorideTetramethylammoniumbromide

Amountrequired to

displace 50%of Ca2+(ttmoles)

3.551-12-040*480-027

0-710-022

32-40*1250-750 0750-11-920-92

Increase insurface pressure

(AxT) at 50%displacement(dynes/cm.)

1-73.79.31-58-3

4-44-84-09.11*95

11-83-51-14*2

23-789-04.47-8

2-93

3.9

40X6

48-7

Ca2+ was adsorbed on a monomolecular film of anacidic phospholipid it was found that many

pharmacologically active water-soluble bases were

far more effective agents that Na+ or K+ ions incausing a displacement of the bivalent ion. Thusthe local anaesthetic, tetracaine, displaced Ca2+from high-pressure monolayers of phosphatidic acidand phosphatidylinositol in concentrations (Table 1)that were several hundred times lower than thoseexpected if it were acting as a simple water-solubleunivalent gegenion such as Na+. Table 2 shows thatthere was considerable variation in the concentra-tions of a series of basic compounds required tocause a 50% displacement of Ca2+ from phospha-tidylinositol films. Many organic bases, includingboth anaesthetic and stimulatory drugs, and basicamino acids were effective. In nearly all instancesthe substances caused an increase in the surfacepressure (Air) of the film, suggesting that their

804

044. 603--00

40o20I

*.t 2N

+

20~~~~~~~~~~~

ov 0° 0 20 30 40Initial starting pressure (dynes/cm.)

Fig. 2. Displacement of Ca2+ (0) and increase in surfacepressure (AxT) (o) caused by adding tetracaine (0.1,umole)to the bulk phase below phosphatidylinositol monolayersat various surface pressures.

effectiveness in causing displacement of Ca2+ mightbe connected with their ability to penetrate intothe phospholipid film. However, as might beexpected from agents of such diverse chemicalstructure, there was no simple connexion betweendisplacement and penetration as judged by the Airvalue.The relationship between penetration and Ca2+

displacement was then examined in more detail in aseries of experiments with the local anaesthetictetracaine.

Displacement of Ca2+ with varying initial surfacepressure of the monolayer. Increasing the pressureof a phosphatidylinositol film caused a fall in thepercentage of Ca2+ displaced by adding a constantamount of tetracaine (0. 1 ,umole) to the bulk bufferphase (Fig. 2). At the same time the increase insurface pressure (A7r) on the addition of tetracainerose to a maximum at an initial film pressure of4-6 dynes/cm. and then fell steadily with increasingstarting pressure of the phosphatidylinositol film(Fig. 2). In Fig. 3 the displacement of Ca2+ from aphosphatidylinositol monolayer produced by addingtetracaine (0.1 ,umole) is plotted against the increasein surface pressure caused by penetration of thedrug. There wasanapproximately linearrelationshipbetween these two variables at starting filmpressures above 4dynes/em., but at lower startingpressures the relationship no longer held.

In the experiments with a constant amount oftetracaine (Figs. 2-3), the ratio between the bulkconcentration of tetracaine and the surface concen-tration of phosphatidylinositol decreased as thestarting pressure of the film was increased. Fig. 4shows an experiment in which the ratio of bulktetracaine concentration to the surface concentra-tion of phosphatidylinositol was kept constant.

Vol. 109 911

H. HAUSER AND R. M. C. DAWSON

80

60

40

100 r

*(3)(5)

*(9) 4)0

(20) (12)

w(30)

¢(40)20 p

0 1 2

Ax (dynes/cm.)3 4

Fig. 3. Increase in surface pressure (A7r) plotted againstthe displacement of Ca2+ produced by adding tetracaine(O1,umole) to the bulk phase below phosphatidylinositolmonolayers at various starting pressures (indicated, indynes/cm., by the numbers in parentheses).

660Cs

o .°

0- El

CsrCs Ca

elV8

1968

80 F

60 -

40F

20 F

0 2 4 6 8

AlT (dynes/lm.)Fig. 5. Plot of Ca2+ displacement against the increase insurface pressure (AlT) obtained when increasing amountsof tetracaine were introduced into the bulk phase belowphosphatidylinositol monolayers at a constant initialstarting pressure (10 dynes/cm.).

I;

00

;9

'4

0

Cs

Initial surface pressure (dynes/cm.)

Fig. 4. Displacement ofCa2+ (e) from phosphatidylinositolmonolayers at various initial starting pressures producedby adding tetracaine to the bulk phase. The amount oftetracaine added (0) was adjusted so that its concentrationwas approximately proportional to the surface concentra-tion of phosphatidylinositol molecules calculated from theforce-area curve.

qe 0-2

Cs0

0.I

0

Cs0

H- 0 1 2 3Tetracaine added (,umoles)

4

Fig. 6. Langmuir-type plot of the amount of tetracaineadded to the trough against this amount divided by theincrease in surface pressure (AxT) when the drug is introducedbelow a monolayer of phosphatidylinositol at lOdynes/cm.

There was a linear fall in the displacement of Ca2+as the initial starting pressure was increased.

Di8placement of Ca2+ at con8tant initial 8urfacepressure of the monolayer. In a further series ofexperiments the starting pressure ofa phosphatidyl-inositol film was kept constant and the amount oftetracaine added to the bulk phase was varied.The displacement of Ca2+ from films of phos-phatidylinositol at a starting pressure of aboutlOdynes/cm. was directly related to the increase

in surface pressure (A7r) on the addition of tetra-caine (Fig. 5). If Air is used as a measure of thesurface concentration of tetracaine, the pene-tration isotherms were all of the Langmuir formAir = (Kl[T]B)/(K2 +[T]B) where [T]B is the bulk

concentration of tetracaine (Pethica, 1955). Thusa plot of [T]B/AIr against [T]B is linear (Fig. 6).

912

0

c3

4 00+o0 X

_ v

4._.-4O'

0-(L+

egCa

100

.+5

6-.

0

6

+

0

c o

v)

Ca2+ DISPLACEMENT FROM PHOSPHOLIPIDS BY BASES

12

1-

ElC) 8

2

4

00 100

5 80_0 0

01 606

40Ca+ 40_

20

+ /ICs 0 4 8 12

10-13 x Surface concn. of CETA+ (molecules/cm.2)

Fig. 7. Displacement of Ca2+ (e) and the change in surfacepressure (Air) (o) caused by the penetration of cetyltri-methylammonium ions (CETA+) into a film ofphosphatidyl-inositol at an initial starting pressure of l0dynes/cm.A,Theoretical points for the displacement calculated from themass-equilibrium equation (see the Discussion section).

Di8placement of Ca2+ from pho8phatidylino8itolmonolayer8 with cetyltrimethylammonium bromide.The availability of [1-14C]cetyltrimethylammoniumbromide led us to investigate more directly therelationship between the displacement of Ca2+ froma phosphatidylinositol film and the concentrationof displacing agent in the surface monolayer ofphosphatidylinositol. The surface concentration ofcetyltrimethylammonium ions was computed for a

series of phosphatidylinositol films at pressures of9-12dynes/cm. by measuring the surface radio-activity after the addition ofvarious amounts of thebase to the bulk phase. Fig. 7 shows that, as thesurface concentration of cetyltrimethylammoniumions increased, A7r increased non-linearly, the valuesobtained in different experiments having acceptablereproducibility. Equivalent experiments were thenperformed in which Ca2+ was displaced from a

series of phosphatidylinositol monolayers byunlabelled cetyltrimethylammonium bromide.Thus a direct comparison of the Ca2+ displacement

4-i0

0kO

Ca10CaoO-)

C) v

0

0

.-

OV,

0

-_ _

-2

10

8

6 gg

4 z2 <I

0 2 4 6 8 10 12 14 16No. of C atoms in amine molecule

Fig. 8. Displacement of Ca2+ and surface-pressure changescaused by introducing a series of straight-chain aliphaticamines (hydrochlorides) into the bulk phase below phos-phatidylinositol films at initial pressures of l0dynes/cm.The logarithm of the concentration of amine required toproduce 50% displacement of Ca2+ (0) and the equivalentincrease in surface pressure (Ar) (o) are plotted againstthe number of carbon atoms in the amine molecule.

from phosphatidylinositol films and the surfaceconcentration of displacing agent was made (Fig. 7).

Di8placement of Ca2+ from pho8phatidylino8sitolmonolayer8 by homologou8 8erie8 of aliphatic amines.The effect on Ca2+ displacement from phosphatidyl-inositol monolayers of systematically increasingthe hydrophobic part of the displacing moleculewas investigated by using a homologous series ofstraight-chain aliphatic amines. The curve relatingthe change in surface pressure produced by addingsufficient amine to cause 50% displacement of Ca2+ions and the number ofcarbon atoms in the moleculeof the displacing agent (Fig. 8) showed a cleardiscontinuity between C6 and C7 (n-hexylamine andn-heptylamine). A plot of the logarithm of theconcentration of amine required to produce half-displacement of Ca2+ from the phospholipid mono-layer against amine carbon number produced aconstant slope between C2 and C12, with again aclear lateral shift between C6 and C7 (Fig. 8).Increasing the chain length of the amine beyondC12 did not increase its ability to displace calciumfrom the monolayer, but rather surprisingly theAir value for 50% Ca2+ displacement fell.

DISCUSSION

It has been shown that local anaesthetics such asprocaine and tetracaine can combine with phospho-lipids and prevent the lipid solubility that the

Vol. 109 913

H. HAUSER AND R. M. C. DAWSON

phospholipids confer on Ca2+ ions in a two-phasesolvent system (Feinstein, 1964; Blaustein, 1967).The present results show that very low concentra-tions of certain pharmacologically active bases candisplace Ca2+ ions adsorbed on monolayers of acidicphospholipids at the air-water interface. Thus,for example, chlorpromazine at a concentration of0-4/tM in the Langmuir trough displaces 50% ofthe calcium adsorbed on a monomolecular film ofphosphatidylinositol. Whether such films aresuitable models for the anionic binding sites ofcalcium on the nerve-cell membrane is a matter forconjecture. Certainly phosphatidylinositol and themetabolically related polyphosphoinositides arecomponents of these membranes (Eichberg,Whittaker & Dawson, 1964; Eichberg & Dawson,1965). It is likely that the negatively charged headgroups on the phospholipid molecules would beorientated on the surface of the organized lipidmicrostructures (e.g. dimolecular leaflets) thatprobably exist in membranes, and thus act ascentres of electrostatic interaction. However, theextent to which this potentiality for calcium bindingis modified by membrane protein is not known.There has recently been a great tendency to invokephospholipids as membrane models with scantconsideration for the role played by the proteincomponent.

All the base molecules that effectively displacecalcium from a phosphatidylinositol film possesstwo characteristics: first, their basic group or groupsare fully or at least partially ionized at the pH used;secondly, when introduced into the Langmuirtrough they cause an increase in the surfacepressure of the film.The most likely explanation of the rise of surface

pressure on addition of the displacing base is thatpart of the base molecule penetrates and physicallyoccupies the available spaces in the monolayer.If surface adsorption of the base molecules belowthe film occurred, this would tend to decrease boththe kinetic and the electrical energy of the systemand hence contract the film and decrease the surfacepressure (Phillips, 1955). This penetration need notnecessarily extend to the hydrophobic 'tail' regionof the phospholipid monolayer, and it is clear thatlimited penetration into the hydrophilic head-group region does occur with certain basic drugssuch as procaine (Gershfeld, 1962). As discussedbelow, this also applies to the short-chain aliphaticamines used in the present investigation.The ability of tetracaine to cause a displacement

of calcium from a phosphatidylinositol film seemsclearly related to its capacity to cause an increasein the surface pressure (L\r). Both at constant filmpressure (10dynes/cm.) with increasing concen-tration of tetracaine and at various film pressures(4-40dynes/cm.) with a constant concentration of

tetracaine there is a simple direct linear relationshipbetween calcium displacement and A7r. Belowinitial film pressures of 4dynes/cm. this relationshipis lost, the A7r curve for the concentration of tetra-caine used showing a maximum at 4dynes/cm. (Fig.2). At low initial starting pressures, the tetracainestill displaces Ca2+ ions from the film (Figs. 2 and3), but the penetration shows as a minimal incre-ment in the surface pressure compared with that ob-served at higher pressures. At these low pressuresthe film would be in the liquid expanded statewith plenty of space available between the widelyspaced phospholipid molecules. The penetration oftetracaine could occur without an appreciableincrease in the kinetic term of the equation of thestate of the monolayer, more space being madeavailable in the film by the hydrophobic 'tails'of the phosphatidylinositol passing to a morevertical orientation to the interface. At higherstarting pressures the tetracaine penetration wouldexert its full spacing potentialities, but the amountof penetration would fall as the pressure wasincreased owing to the increased energy barrierpreventing access to the more closely packedphospholipid film. The penetration of tetracainemay be based on a two-point electrostatic attach-ment of the two electropositive centres existing inthe molecule with two phospholipid head groups,as envisaged by Feinstein (1964). By making anassumption about the size of the tetracaine mole-cule, it is possible to calculate from a force-areacurve of a phosphatidylinositol film the surfaceconcentration of tetracaine representing a givenvalue ofA7T. Such a computation shows that at thesurface concentration oftetracaine required to causecomplete displacement of calcium the tetracaine/phospholipid surface concentration ratio is veryapproximately 1: 2, implying two-point electrostaticattraction ofthe drug. Withwidelyspacedphospho-lipid films this two-point attachment by itselfcould cause an electrostatic contraction of the film,so that A7T would be lower than that observed withmore closely packed films. It is doubtful if the C4hydrophobic chain of tetracaine would be longenough to penetrate to the hydrophobic fatty acidregion of the phospholipid film. Nevertheless itcould still have an affinity for the film as is shownby the increased penetration of aliphatic aminesinto the film as their chain length is increased fromC1 to C6, as is discussed below.In these experiments with tetracaine it was not

possible to measure directly the surface concentra-tion of tetracaine in the film, on which the displace-ment of calcium ultimately depends. Presumablythis concentration is related to A7T by the Gibbsadsorption isotherm (Pethica, 1955). A moredirect comparison was possible by the use oflabelled cetyltrimethylammonium ions, which

914 1968

Vol. 109 Ca2+ DISPLACEMENT FROM PHOSPHOLIPIDS BY BASES 915allowed the penetration to be measured in terms ofthe increment of surface radioactivity produced,assuming that such a long-chain amphiphile wouldentirely penetrate into the film rather than beingadsorbed on the surface without penetration. Thepenetration of cetyltrimethylammonium ions intothe film determined experimentally by this tech-nique agreed closely with the surface concentrationcalculated from the Gibbs adsorption isotherm(Pethica, 1955), even though no specific electrostaticterm was introduced into the equation.The experimental curve relating the displacement

of calcium to the surface concentration of cetyl-trimethylammonium ions (Fig. 7) shows thatcomplete displacement would occur when theconcentration of cetyltrimethylammonium ionsin the film approached that of the phosphatidyl-inositol (10.6 x 1013molecules/cm.2): in other words,on complete neutralization of the anionic sites ofthe film.The form of the curve relating calcium displace-

ment to the surface concentration of cetyltrimethyl-ammonium ion can in fact be calculated from themass-equilibrium equation:

K = [Ca]L/[L]f[Ca]r

where [Cahl iS the calcium combined with lipid,[Ca]f the free calcium and [L]f the free lipid on thesurface.

[L]f = [L]s-[CETA+]si.e. the surface concentration of phosphatidyl-inositol, [L]s, minus that of the cetyltrimethyl-ammonium ions, [CETA+]s. This curve, shown inFig. 7, closely approximates to that determinedexperimentally.The experiments measuring the displacement of

calcium by aliphatic amines clearly show that as thechain length of the amine is increased from C2to C6 there is an increased ability to displacecalcium. The logarithm of the concentration ofamine required to produce 50% displacement whenplotted against the number of carbon atoms in theamine shows a constant slope (Fig. 8). A linearrelationship is also often found in any homologousseries between the logarithm of the distributioncoefficients between two immiscible phases and thenumber ofcarbon atoms in the molecule. As pointedout by Ferguson (1939), this simply means thatthere is a constant increment for each successivehomologue in thepartialmolar free-energy differencebetween the standard states in the two phases.In the present experiments, the displacement plotshows a lateral shift between C6 and C7, indicatingthat heptylamine is more efficient at producingdisplacement of calcium than could be predictedfrom the behaviour of the C2-C6 amines. This is

reflected in discontinuity in the curve relating AlTat 50% calcium displacement to the number ofcarbon atoms in the amine. Calculations and thebuilding of molecular models show that, if it isassumed that the protonated amino group is at thelevel of the negatively charged phosphate on thephospholipid head group, the C7 chain atomswould just reach into the hydrophobic fatty acidchains of the phospholipids, assuming a verticalorientation of the head group. Presumably thismight assist the penetration of the amine into thefilm. Clearly, however, assisted penetration mustoccur with the shorter-chain amines without thistype of hydrophobic bonding. The curve relatingA7T at 50% calcium displacement to the number ofcarbon atoms is clearly a complex one and cannotbe explained adequately until more information isobtained about the surface concentration of amineat each chain length and the way the amines arepositioned in the interface.

It is perhaps premature to try to relate thepresent findings, obtained in a model system, to theaction of local anaesthetics in the intact animal.Certainly such anaesthetics would effectively liber-ate calcium bound to a phospholipid-water inter-face on a cell membrane, and it is known that theyincrease the calcium lost from excitable tissue suchas nerve fibre or muscle (Kuperman, Altura &Chezer, 1968). However, it is by no means apparentthat calcium and the local anaesthetics have asimilar site of action on the nerve cell (Shanes,Freygang, Grundfest & Amatniek, 1959). If, assuggested by Feinstein (1964), Feinstein & Paimre(1966) and Blaustein (1967), the displacement ofcalcium from phospholipid surfaces is of importancein the development of anaesthesia, it needs to beexplained why excitatory drugs such as brucine,strychnine or amphetamine or certain non-pharmacologically active bases are equally effectivein removing calcium from such interfaces.

H.H. is grateful for a grant from the European MolecularBiology Organisation.

REFERENCES

Ames, A., III, Tsukada, T. & Nesbett, F. B. (1967). J.Neurochem. 14, 145.

Ansell, G. B. & Hawthorne, J. N. (1964). Pho8pholipid8,p. 96. Amsterdam: Elsevier Publishing Co.

Blaustein, M. P. (1967). Biochim. biophy8. Acta, 135,653.Dawson, R. M. C. (1968). In Method8 in Enzymotogy. Ed.by Lowenstein, J. M. & Clayton, R. B. New York:Academic Press Inc. (in the Press).

Eichberg, J. & Dawson, R. M. C. (1965). Biochem. J.96, 644.Eichberg, J., Whittaker, V. P. & Dawson, R. M. C. (1964).

Biochem. J. 92, 91.Feinstein, M. B. (1964). J. gen. Phy8iol. 48,357.Feinstein, M. B. & Paimre, M. (1966). Biochim. biophys.

Acta, 115, 33.

916 H. HAUSER AND R. M. C. DAWSON 1968Ferguson, J. (1939). Proc. Roy. Soc. B, 127, 387.Frankenhaeuser, B. & Hodgkin, A. L. (1957). J. Physiol.

187,218.Gershfeld, N. L. (1962). J. phys. Chem. 66, 1923.Hauser, H. & Dawson, R. M. C: (1967). Europ. J. Biochem.

1,61.Kuperman, A. S., Altura, B. T. & Chezer, J. A. (1968).

Nature, Lond., 217, 673.Palmer, F. B. & Dawson, R. M. C. (1968). Biochem. J.

(in the Press).

Pethica, B. A. (1955). Trans. Faraday Soc. 51,.1402.

Phillips, J. N. (1955). Trans. Faraday Soc. 51,1726.

Rogeness, G. A., Krugman, L. G. & Abood, L. G. (1966).Biochim. biophy8. Ada, 125, 319.

Rouser, G., Kritchevsky, G., Heller, D. & Lieber, E. (1963).J. Amer. Oil Chem. Soc. 40, 425.

Shanes, A. M., Freygang, W. H., Grundfest, H. & Amatniek,E. (1959). J. gen. Physiol. 42, 793.