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Langmuir 1993,9, 228-232
An Epifluorescent Microscopy Study of the Effects of Procaine on Model Membrane Systems B. Asgharian,? D. A. Cadenhead,' and Maria Tomoaia-Cotiselt Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 Received January 7,1992. In Final Form: September 4,1992 Studiesof the effects of procaine substrateson monomolecular model membrane f h have been carried out over a range of procaine concentration (10""10-2M)and pH (pH 2-5.6). Filma of tetradecanoic acid and L-dipalmitoylphoephatidylcholinewere examined. Classicalsurfacepressureversus area per molecule isotherms were obtained in conjunction with an epifluorescent microscopy study and indicated that the procaine concentrates not only in the lipid phase but also at expanded/condensed lipid and lipid/protein interfaces. In addition to preferential penetration by neutral procaine, micelles created through procaine/ lipid charge interactions play a major role. The results indicate that procaine can both penetrate and electrostatically interact with charged membrane lipide and proteins, concentrating at both internal and external membrane interfaces.
Introduction Local anesthetics clearly act through the closure of the sodium channels of nerve membranes, thus preventing nerve signal propagation.' What is not clear is whether this blocking action takes place through direct action on the protein itselfZ or through a perturbation of the surrounding lipid m a t r i ~ .In ~ either case it is of interest to learn just how the anesthetic distributee itself throughout the membrane. Model membrane studies with liposomes indicate increasedfluidity and/or decreased order of the bilayer lipid hai in sf*^ Similar studies with monolayers at the air/water interface show film penetration resultingin an increasein the area/mdecule of monolayers maintained at constant pressure6J or an increase in the surface pressure of the film at constant area.798 When drug effects on isotherms are observed6J the increased area/molecule is also marked by a shift of the liquid expanded (LE)/liquid condensed (LC) phase transition to higher pressures accompanied by a gradual broadening and elimination. In this study we observed the (classical)surfacepressure (*)/area per molecule (A) isotherms for fatty acid and lecithinfilms on aqueousand aqueous-procaine substratee but report only the LE/LC transition shift data. We accompanied this with a simultaneous fluorescent micrascopy study of the transition region observingthe effects of aqueous Substrates, with and without procaine as an additive. The microscopic study necessitatedthe addition of a small amount (-2 mol %) of a selected fluorophore which dieeolved in the LE phase of the lipid but was excluded from the LC phase. Since most if not all of the LC phase domains were at least 2 Nm in size,these domains
* To whom all correspondenceshould be addressed.
+ Preaentaddreee:Alcon Laboratories,Inc., Fort Worth,TX 76116.
f Permanent address: Department of Physical Chemistry, University of Cluj-Napoca,3400 Cluj-Napoca, Romania. (1) Tmdell, J. R. In Molecular Mechniems of Anesthesia: Progress in, Anesthesiology; Fink, B. R., Ed.; Raven Press: New York, 1980;Vol.
2, pp 261-270. (2) Bog@, J. M.; b t h , S. M.;Yoong, T.; Wong, E.;Heia, J. C. Mol. Phrmacol. 1976,12,136. (3) Seeman, P. Prog. Aneshesiol. 1976,1, 243. (4) Turner, G. L.; Oldfield, E. Nature (London) 1979,277,669.
(6)Auger, M.;Smith, I. C. P.; Jarrell, H.C. In NMR Spectroscopy in Drug Research; Alfred Beneon Symp. 26; Jaroszewaki, J.W., Schaumberg, K., Kofod, H., Eds.;Mun~quaard:Copenhagen, 1988,pp 473-485. (6) Seelig, A. Biochim. Biophys. Acta 1987,899,196. (7) Cadenhead, D. A. In Structures andProperties of Cell Membranes; Benga, G., Ed.; CRC Prm: Boca Raton, FL, Vol. 111, Chapter 2,1985; Chapter 2, Vol. 111, pp 21-62. (8) Tomoaia-Cotieel, M.;Cadenhead, D. A. Langmuir 1991,7,964.
were readily detected when appropriate filter selection permitted excitation of the fluorophore.
Experimental Section The fluorescencemicroscopy setup used in these experimenta consisted of a modified Nikon Model L-Ke optical microscope positioned at one end of a f i i balance with the objective capable of being focused at the air/water interface. The objective stage was illuminated by a 75-W senon lamp and consisted of 40X objectivefitted with a slotted Teflon dam which ehnded below the interface to permit film access but prevent turbulence. The resultant fluorescence image was directed into an MTI SIT 66 camera, the camera image in turn being transmitted to a Sanyo VM4509 monitor, recorded by a Phillips VHS HQ VCR and/or printed via a Mitsubishi Model P61U video copy proceesor. The fluorescence probe l-palmitoyl-2-[6-[7-nitro-2-1-3-bemosadiazol-4-yl)aminolcaproyllphosphatidylcholiie(NBD-PC) was used in all experimentsand was purchased fromAvantiPolar Lipids Ltd. L-u-Dipalmitoylphosphatidylcholinewas obtained from Sigma. Tetradecanoic acid was purchased from Larodan Fine Chemicals, M h o , Sweden. Both fatty acid and phospholipid were spread from a l t 3M solution either in 9 1 (v/v) hexane/ethanolby volume or in chloroformand containing 2 mol % of fluorescence probe over a pure water substrate or over procaine hydrochloride containing substrates. Procaine hydrochloride was obtained from Sigma and was used as supplied. Regardless of the substrate pH and at concentrations of below M, procaine hydrochloride did not show detectable ( < l t l dyn/cm) surface activity at a clean aidwater interface based on the surfacepressure measured using a Wilhelmyplate suspended from a microbalance. Furthermore, when a f i i of pure fluorescent dye was spread over a procaine substrate (1VM), it failed to show any observableinteractions of procaine with the probe,asjudged by the absenceof any domain presenceat surface pressures above zero. Results and Discussion In the first series of experiments, the effect of changing substrate procaine hydrochloride concentration on tetradecanoic acid films was examined. All tetradecanoic acid films were compressed at a constant rate of 3 A2 molecule-' min-l in order to avoid possible rate variation of size and number of condensed domains due to differing compressional rates. In the absence of procaine, tetradecanoic acid at the air/water interface (pH 5.6) at 18.0 OC showed an LE/LC transition at about 11d y n / ~ m In . ~ the presence of a trace amount of fluorescent probe (2 mol %), below the transition a homogeneous phase was (9)hgharian, B.;Cadenhead, D. A. J. Colloid Interface Sci. ISSO, 134,622.
Q 1993 American Chemical Society
Effects of Procaine on Model Membrane Systems observed. Immediately above the transition, initially dendritic then, shortly, circular domains of the condensed fatty acid were obeerveed which increase in number with increasing film density (Figure 1). Even at relatively high surface pressures (-20 dyn/cm), large circular domains, typical of fatty acids, continued to be observed. The tetradecanoic acid film was then examined at 18.0 OC in the presence of procaine hydrochloride substrates of concentrations 1W2, lW3, and lo+ M at pH values ranging from 5.1to 5.3. Procaine addition to the substrate resulted in film expansion and a shift of the LE/LC transition (rd to higher pressures (Table I). Similar expansion effects were previously reported for octadecanoic acid on procaine substrates.8 The magnitude of the effect increaeed with increasing procaine concentrations. At a concentration of lo+ M procaine, the isotherm was only slightly different from that obtained on pure water. It is important to note that at pH 5.2, while the isotherm transition on a 1W2 M procaine substrate appears at 21 dyn/cm (Table I),the firat smallcondensed domainswere o b e e ~ e dat 8.0 dyn/cm. As the compression continued, the domainsincreased in size and number. However, they were still much smaller than those observed on a pure water substrateabove the transition. At the isotherm LE/ LC transition all that was observed was a discontinuous increase in the condensed particle number and size. Qualitatively the same effects were observed at lower procaine concentrations. However, the magnitude of the effecta was smaller and diminished with diminishing procaine concentration. At a lW3 M substrate concentration, the transition was observed at 15.5 dyn/cm, while nucleation again was detected at much lower pressures (9 dyn/cm). At pressures above the transition of the * / A isotherms, as before, slightly larger domains formed at the onset of the transition. Even a procaine concentration of lo-' M, despite the minimal effect on d A isotherm, showed similar effects but of a much smaller magnitude. Here, however, the domains and the LE/LC transition pressure were similar to those observed on pure water substrates. The effect of substrate pH was examined at a fired lO-9 M procaine substrate concentration. Since procaine is a tertiary amine, containing a primary amino group linked to an aromatic ring, it can exist as a neutral molecule, a monocation, or a dication.8 At pH 2 procaine can exist in either a dicationic (60% ) or monocationic (40% ) form. To achieve a pH 2 substrate, a M solution of procaine was pH adjusted with HN03. The resultant film of tetradecanoic acid showed very little effect and behaved in a very similar way to a film on water. In contrast, at pH 5.2 a procaine solution M) is essentially in a monocationic state.8 It is clear that the ionic interactions of procaine and the polar groups of fatty acids play a significant role, as is evident when the effects of procaine hydrochloride on tetradecanoic acid at pH 2 and pH -5.2 are compared. At pH 2 the acid is neutral, while at pH 5.2, assuming tetradecanoic and octadecanoic acid behave in a similar fashion, it is approximately half charged.8 One possible explanation is that at pH 2 the highly charged procaine is more soluble in the aqueous subphase and the neutral acid fails to interact electrostatically with it. It seems likely, therefore, that in addition to 'normal" domains, others, stabilized by electrostatic interactions, form at pH 6.2. Such domains would form preferentially at that pH since both entities are charged, but would not form at pH 2 where the acid is uncharged.
Langmuir, Vol. 9, No. 1, 1993 229
A previous stud9 had indicated that neutral procaine (pH -8) penetrates a octadecanoic acid fiilm better than do the cationic forma. We were not able to verify thiswith tetradecanoic acid because it proved too soluble at pH 8, the pH required to ensure that the procaine is neutral. For octadecanoic acid the charged form of procaine was thought to be additionally capable of interacting with either the charged or polar groups of the acid and this is clearly the m e for tetradecanoic acid at pH 5.2. Nevertheless, with tetradecanoic acid at pH 2 any penetration or interaction of procaine is greatly reduced and we see relatively little effect. It was of interest to see if procaine had similar effecta on films of more complex lipids. For this,L-u-dipalmitoylphosphatidylcholine (DPPC) was chosen since chiral DPPC has been shown to form asymmetric, windmii-like domainslo and the molecule constitutes a typical membrane component. In addition the polar head-group of DPPC is zwitterionic over a wide pH range. Although procaine has been shown to be only weakly bound to egg PC aqueous lamellar dispersions," effects of procaine on DPPC could be detected (Figure 2). At lW3 M procaine (pH -5.2),DPPC f h a t 20.0 OC showedverylittleeffect on the d A isotherm with only a slight shift in the LE/LC transition to higher pressure.ll This concentration of procaine also failed to induce any nucleation of condensed domains below the phase transition. However, the domains on a lW3 M procaine substratewere somewhat more rounded and smaller than those on a pure water substrate. As compression continued, the procaine-affectad film achieved a near equivalent appearance to that on a pure water substrate, but at much higher pressures. For either film the boundary regions of condensed and expanded lipid are stabilized in the presence of procaine. In accordance with a two-dimensional Gibbs adsorption isotherm assuming that concentration can be substituted for activity
where I'p is the procaine concentration at the condensed/ expanded line interface in mol/cm of free interface, TL is the conde&/expanded line tension, and cf is the LE film concentration, which is in turn determined by the lowering of the surface tension or increase in the surface pressure as indicated by Art (Table I). This means that procaine must produce this stabilization effect by concentrating itself at the expanded/condensed interfacial region. This type of behavior is not unique to procaine but is exhibited by other surface active (water/lipid interface) molecules including chole~terol.~~J3 The precise behavior, however, depends on the amphipathicity of the molecule, its bulk and film concentration and, most particularly, on potential charge interactions between the substrate additive and the host lipid. It appears that at pH 5.2, the effects of M procaine substrates are less on DPPC than on a fatty acid. It is also likely that monocationicprocaine would interact less with zwitterionic DPPC than it would with the anionic polar group of a fatty acid. The differing shapes of the condensed phases of tetradecanoicacid and DPPC reflect the differing chirality (10) Web, R. M.; McConnell, H. M. Nature (London) 1984,310,6972. (11) Rice, D. K.; Cadenhead, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1987,26,3206. (12) Heckl.W. M.;LBeche,P.M.:Cadenhead,D.A.: M6hwald.H.Eur. Biophys. J. 1986, 14, 11. (13) Heckl,W. M.; Cadenhead, D. A.; M6hwald, H. Langmuir 1988, 4, 1362.
230 Langmuir, VoZ.9, No.1,1993
Asgharian et al.
Figure 1. Fluorescent microscopy studies of tetradecanoic acid films on aqueous and aqueous-procaine substrates: (a, top left) Initially dendritic domains of LC phase (dark regions) shortly after the onset of the tetradecanoicacid LE/LC transition (11dyn/cm at 18 "C) on a pure aqueous subphase (pH -5.6). (b, top middle) As in part a but after allowing 1-2 min to elapse. (c, top right) LE/LC transition appears complete (-13 dyn/cm) based on the isotherms but fluid regions persist possibly due to enhanced probe concentrations in these regions. Condensed phase domain diameters range between 30 and 100 p M . (d, center left) In the LC state with bridging now apparent between the condensed domains (-20 dyn/cm). (e, center middle) Small (2-5 p M ) domains are readily M procaine substrates though the ?r/Aisotherm indicated the LE/LC transition has not been reached (-8 dyn/cm). detectable on (f, center right) As in part e. Condensed domains are more numerous and larger just above the transition (21.5 dyn/cm)when compared to part e. (g, bottom) LE/LC transition is apparently complete (-23 dyn/cm) but the size of the condensed domains remains much smaller on le2M procaine than on a pure aqueous substrate (c). There is also some indication of continuing dendritic character.
Effects of Procaine on Model Membrane' S'ystems
Langmuir, Vol. 9, No. 1, 1993 231
~~
I
Figure 2. Fluorescent microscopy studies of L-a-dipalmitoylphosphatidylcholinefilmson aqueous and aqueous-procaine substrates:
(a, top left) Windmill-like asymmetric domains of L-a-dipalmitoylphosphatidylcholine(DPPC) as observed on an aqueous substrate at pH 5.6 with the film in the LE/LC transition region (5.5 dyn/cm). (b, top middle) The same system as in part a but compressed into the LC state (-7 dyn/cm). (c, top right) The same system as in part a but at high pressures (-30 dyn/cm). (d, bottom left) Condensed phase domains of DPPC within the LE/LC transitions on a procaine substrate (pH 5.2,20 "C). Domains (2-5 pM)should be compared with those in part a. (e, bottom middle) The same system as in part d but compressed into the LC state. The domains should be compared with those in part b. (f, bottom right) The same system as in part d but at high pressures. The domains should be compared with those in part c. Isotherm pressures for the LE/LC transition for Figure 2d,e,f were about 1 dyn/cm above those for Figure 2a,b,c. Table I. Shift of zt as a Function of Procaine/Procaine Hydrochloride Substrate Concentration filmcomposition tetradecanoicacid tetradecanoic acid tetradecanoic acid tetradecanoicacid tetradecanoicacid
T (OC)
(a";a/ cm)
pH
substrate
drug concentration
~~~
DPPC
18 18 18 18 18 20
11 21 15.5 11.5 11 5.0
5.6 zero 5.1 10-2 M procaine hydrochloride 5.2 10-3 M procaine hydrochloride 5.4 10-4 M procaine hydrochloride 2 10-3 M procaine hydrochloride 5.6 zero
and charge structure of the two films. The nonchiral myristic acid, while initiallyformingsymmetricaldendritic particles, soon forms large (- 30-50 p M diameter) circular particles. Under the action of procaine the particles not only are smaller but also retain a greater degree of dendritic character. Both of these effects are results of diminished line tension. DPPC, in contrast, shows asymmetric windmill-like structures, not too dissimilar to the spiral shapes found at low cholesterol levels.12J3 The particles formed on 10-3 M procaine are only slightly smaller and
retain some asymmetry. Cholesterol addition (1-4 mol %) seems to promote, procaine addition to diminish, asymmetry. The difference between the two additives would seem to be in part due to the ability of the charged procaine to be retained in, or interact with, condensed phases at higher concentrations.8 The most surprising feature of this study was the revelation that for charged fatty acid f i i , prior to the LE/LC transition in the so-called LE state, smaU but detectable LC domains could be detected on pracaine substrates. The extent of this phenomenon increaseswith increasing procaine concentration. It would appear that procaine is capable of forming a unique typeof condensed domain in a fluid phase. Rather thanthe initialappearance of the LC phase at rt,we see only a discontinuous increase in the size and number of LC domains. The indications are that a new type of condensed domain is stabilized by acid/procaineelectrostatic interactions. Conclusions similar to these were reached by Winter et al.14 who studied the effects of a procaine analogue (tetracaine) on lecithin
232 Langmuir, Vol. 9, No.1, 1993
bilayers. At this time we estimate that this novel effect may woll be exhibited by a wide rmge of chargedsubstrate additives on charged lipid phase transitions, but further studieswill be required to establish the generality of this obrvation. Finally, prelimhwy studies with the addition of bovine eerumalbintoNBD-PC labeled DPPC expanded filme produced dark regions of aggregated protein in a light lipid background. On addition of sufficient procaine to (14) Winter, €2.; Chrietmann, M. H.; Bottner, M.;Thiyagarian, P.; Heenan, R. K.&r. Bunsen-Ges. Phys. Chem. 1991,96,811.
Asgharian et ai. raise the substrate level to 10-3 M,the dark protein region broke up indicating that procaine and/or lipid was penetrating the protein region. Sucha changeis consistent with the lipid interface results and indicates that the surface and line active procaine will also concentrate at lipid/protein interfaces.
Acknowledgment. B. Asgharian and D. Allan Cadenhead would like to acknowledge the financial support of the National Institutes of Health,Division of Reesarch Resources through the Biomedical Research Support Grant Program, and Grant BSRG SO7 RR 07066.
