Penetration of phospholipid monolayers by opioid peptides and opiate drugs (agonists and antagonists)

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Collotds und Surfuces 5 Biointerj&es. 1 ( 1993) 203-2 I I

203

Penetration of phospho~ipid monolayers by opioid peptides and opiate drugs (agonists and antagonists)

N. Bourhima,*, MS. Elkebbaj”, Y. Gargourib, P. Giraud”, J. van Rietschotend, C. Oliver”, R. Vergere

“Laboratoire de Biochimie, Biologic Cellulaire et Mol&xlaire, Facultt des Sciences Ain Chock, B.P. 5366, Casablanca, Morocco

bLaboratoire de Biochimie, Ecole Nutionale d’Ing&ieur de S’ax, Route de Soukra, Sfax 3000, Tunisia

“Laboratoire de Nearoendocr~no~ogie Exptrimentale, Facultk de Mgdecine Nord, UZ91. Boulevard Pierre

~r~~rnard. 13326 ~~arseil~e Cedex 1.5, France

‘Laboratoire de Bioc~timie, Fact&P de ~~~~es~ne Nord. Boulel~ard Pierre Dramard, 13326 ~~arse~~le Cedes 15, France

eCentre de Biorhimie et de Biologie Molt?culaire du CNRS, Chemin Joseph-Aiguier, B.P. II, 13402 Marseille Cedex 9, Frunce

(Received 4 November 1992; accepted 2 February 1993) Abstract

The monomolecular film technique was used to compare the specific mteractlon of opiold peptldes (Leu-enkephahn, Met-enkephalin, dynorphm A and P-endorphin) and opiate agonists (aikalolds such as levorphanol and ethylketocyclazocine) and opiate antagomsts (naloxone and dlprenorphlne) with varkous phosphoijpld films (phospha~~dylcho~~ne.

phospha~~dylethanoIam~ne, phosphatldylinositol. phosphatldylserlne and ~~oleoylphospha~~dylglyccrol). We were able to demonstrate the mteraction of optate drugs and oploid peptides (0.1 PM) wtth both zwztterlomc and negatively charged phosphoiiplds up to very high surface pressures. In the presence of a phospholipld monolayer. the surface activity of oplold peptldes became much greater than that observed at the an-water Interface. In the case of opiate drugs. the phenomenon was different and these drugs penetrate the monolayer films at a low pressure m comparison with opiold peptIdes. even at very high concentration. We have also evaluated the penetration of the antagonists naloxone and dlprenorphIne~ the mteraction of these was very low

Kep NY&.$ Monolayers: Opiate drugs: Oplcnd peptides; Phosphohplds

Introduction

Opiates are among the oldest pharmacological substances known to man; their analgesic, euphoric, and addictive effects have been tradi- tional focal points for opiate research. Based on pharmacological activities in animals and humans, Martin et al. [I] suggested the existence of multiple opiate receptors. They established two subtypes (p and K), according to their response to the

*Correspondmg author.

agonists morphine (ii) and ketocyclazocine (K).

After the discovery of endogenous opioid ligands (Methionine (Met-) and Leucine (Leu-) enkephalin, fi-endorphin and dynorphin) [2-41, further subtypes were identified and classified as 6-, p- and k--receptors [5,6]. Thus, Leu- and Met-enkephalin prefer 6 receptors and P-endorphin reacts with ,U receptors, while dynorphin A is a typical K agonist.

Opioid peptides offer excellent examples of receptor selectivity phenomena in nervous and endocrine regulation; they have a common N-terminal tetrapeptide sequence, Tyr--Gly-Gly-

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Phe, which is the “message” segment triggering the receptor responses but they have different C-terminal “address” segments. which are responsible for their receptor subtype preferences [7,8].

The attempts made so far to ascertain the structural characteristics of the opioid receptors have pro- duced data suggesting that proteins and phospholipids are probably functional components of the receptors [9.10]. The in~~olvement of phospholipids in the stereospecific binding of opiate ligands to the central and peripheral nervous systems has been suggested by receptor sensitivity to phospho- lipases [ 1 I-- 161. In general. opiate binding to the membrane can be diminished by prior treatment of the membrane with phospholipase A, and not at all. or slightly. with phospholipase C or D.

However, phosphatidyl serine (PS) decarboxylase, which converts PS to phosphatidylethanolamine (PE), inhibited bmding [ 173. In an attempt to purify the cc receptor, Cho and co-workers have isolated a protein from rat brain that shows selectivity for the lc-receptor but exhibits high binding activity only when the receptor is reconstituted with some lipids [9]. Different evidence is provided by the attempts made to solubilize and purify the opiate receptors. So far. these studies have met with limited success. Yet the data obtained with this approach substantially support the hypotheses of a protein-lipid structure for the opiate receptors [18,19].

More recently. high affinity binding of 6, /L and K agonists was reconstituted by polyethylene glycol (PEG) precipitation and resuspension of a 3-([3- cholamidopropyl]dimethylammonio)- 1 -propane- sulfonate (CHAPS) extract in the presence of NaCI.

Evidence was presented suggesting that this is the result of inclusion of receptors into liposomes [?O].

Another way to Investigate the characteristics of the opiate receptor environment is to study directly the interaction between opioids and lipids, using phospho~ipid monolayers as modei membrane [31]. The present report describes the use of the monomolecular film technique to estimate the specificity of the interaction between oproid peptides (Leu-enkephalin. Met-enkephalin, dynorphin and

fl-endorphin) and alkaloids : opiate agonists (levorphanol and ethylketocyclazocine) and antagonists (diprenorphine and naloxone), with phospholipids and in so doing to obtain information concerning the environment of opioid receptors to permit molecular selection of these receptors. To ascertain the specificity of the interaction, a comparative study was performed simultaneously with hydrolyzed peptides and/or their analogs.

Materials and methods

Egg phosphatidylcholine (PC) and L-x-dioleoyl phosphatidylglycerol (PG) were obtained from Dr.

D.E. Haas (Utrecht). L-x-phosphatidyl-L-serine (3- sn-phosphatidyl-r_-serine, PS) from bovine brain;

L-~-phosphatidylethanolamine~ (t-r-cephalin; 3-sn- phosphatidylethanolamine. PE) from sheep brain;

r_-x-phosphatrdylinositol ( I-(3-sn-phosphatidyl)-D- nr!~o-inositol, PI) from soybean were all from Sigma. The phospholipids were first dissolved in chloroform at a concentration of 2 mg ml

r .

Water which had been twice-distilled over potas- sium permanganate was used to prepare all solu- tions of opioid peptides, opiate drugs and buffers.

The buffer most often utilized was 10 mM N-Q- hydroxyethyl)-piperazine-N’-2-ethanesulfonic acid (HEPES).

The surface tension measurement device and the trough are housed in a metallic thermostated box (35 C). The position of the balance unit can be varied in both horizontal and vertical directions.

One of the two pans of the balance bears a small glass hook on which the Wilhelmy plate (platinum plate) is suspended. The vertical movement of the plate upon surface tension change IS counteracted

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N. Bourhm et ul.~Collo~ds Surfices B Biornterfaces I (1993) 203-211 205

by the efficient servo-mechanism of the electro- balance. This results in a rapid nulling of any force exerted on the plate. Furthermore, the fact that surface tension was constantly increasing mini- mized any possibility of contact angle change at the plate surface during the measurement. Any serious error in the surface tension measurement is also limited by avoiding measurements of decrease in surface tensions.

Pure PC, PG. PE, PS, or PI were gently depos- ited as monolayers at the air-water interface with a Hamilton syringe. A cylindrical Teflon trough (6.3 cm diameter x 1.4 cm) was used. The initial surface pressure (n,) was adjusted to the desired value and was kept constant for a period of 15 min.

Opioid peptides or opiate drugs were dissolved in water and small amounts of the peptides or alkaloids (stock solution, freshly prepared before use) were then injected into the buffer subphase. The buffer phase was stirred by tiny magnets to assure a homogeneous distribution of opioid peptides or opiate drugs. The insertion of these molecules into lipid monolayers was evaluated from the increase in the surface pressure (Art). Before each experiment, contaminating surface-active impurities were removed by simultaneous sweeping and suction of the interface. The troughs were thoroughly washed with ethanol after each experiment since proteins were adsorbed on the Teflon walls. The platinum plate was washed with sulfochromic acid. All components were abundantly rinsed with distilled water.

Opioid peptides

fl-Endorphin, Leu-enkephalin and Met- enkephalin were from Sigma; dynorphin A was from UCB (Belgium). The two peptides Phe-Gly- Gly-Phe-Leu-Arg-ArggIle-Arg-ProoLys-Leu-

Lys (Phenylalanine) and Leu-ArggArg-Ile-Arg- Pro- Lys-Leu-Lys (chromotryptic fragment) were ordered from Neosystem (Strasbourg, France) and analyzed by HPLC (purity was higher than 90%).

Opiate drugs

Morphine was from Francopia (Paris, France), naloxone was from Endo Laboratories (Garden City, New York, USA). Etorphine and diprenorphine were from Reckitt and Collman (UK); levorphanol was a gift of Hoffman La Roche (Basel, Switzerland).

cc-Chymotrypsin from bovine pancreas was from Sigma and all other chemicals were from Sigma or Serva.

Results

Kinetics of the udsorption of opiute drugs and opioid peptides to the air-water interface

Adsorption to the interface is reflected by the increase in surface pressure with time (Fig. l(A)).

I I I I I I I I I

0 5 10 15 20 25 30 35 40 45 50

Time (min)

Fig. I. (A) Adsorption kinettcs of dtfferent optotd peptides (dynorphm, fi-endorphtn, Leu- and Met-enkephalin). optate agomsts (levorphanol. ethylketocyclazocme) and optate antagonists (naloxone and dtprenorphtne) at a concentration of 0.1 uM at the atrrwater interface. (B) Kinettcs of surface pressure increase, related to optotd penetratton of a phospholiptd film, tn thta case penetration of dynorphtn A (0.1 PM) into a phosphatidylserme (PS) film at vartable tmttal surface pressure (n,). In the Insert, the surface pressure change (4x0 1s plotted as a functton of mtttal pressure (K,).

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206 A The time required to obtain a maximum surface pressure value depended on the particular drug or peptide. The opioid peptides were adsorbed more strongly than the opiate drugs. The maximum surface pressure observed was about 5 mN m ’ for opioid peptides and I mN m- ’ for opiate drugs.

Penetration of opioid peptides and opiate drugs irlto phospholipid monolayers

We first examined the concentration of peptides that gives a significant result. We found that 0.1 uM was the best for opioid peptides and opiate drugs (data not presented). Figure l(B) shows a typical experiment used in this study; the penetration of opioid peptide (dynorphin A) molecules into lipid monolayers of PS was accompanied by a notable increase of surface pressure. Thus increase was greater with lower initial surface pressure (n,) of the film. The observed kinetics was non-linear, reaching a plateau wrthin several minutes; the plot of drr vs. xc, is shown in the inset in Fig. l(B). This is in agreement with existing data on the penetration of protems into monolayer films [II]. This phenomenon was observed with all oproid peptides and opiate drugs used in this study.

Critical surf&e pressures (7~~) for opioid peptides and opiate drugs

The results in Fig. l(B) may be presented in another way. The maximum value of surface pressure increase can be plotted as a function of the initial film surface pressure at which the molecule was injected into the aqueous phase (Fig. l(B).

inset). A critical surface pressure (rr,) for penetration may thus be defined; it corresponds to the extrapo- lated value of initial pressure beyond which there is no increase in surface pressure. This was performed for different opioid peptides (dynorphin A, fl-endorphin, Leu-enkephalin and Met-enkephalin) and opiate drugs (levorphanol and ethylketocyclazocine), as well as for two antagonists (diprenorphine and naloxone) as shown in Figs. 2, 3 and 4 respectively.

rc, (mN/ml

Fig. 2 Change of surface pressure I-in) when IA) fl-endorphm and (B) dynorphm A were Injected beneath monolayers of 1.

PC. n . PE; 1. PG, 0, PS. and 1 :. PI spread at dtfferent uuttal surface pressures. Critrcal surface pressure In,) was determined by extrapolatton onto the mttlal surface pressure (n,) axts

5

n; j&N/m) ²⁵ ³⁵

Fig 3. Change of surface pressure (-lrr) when the opiate agonists (A) levorphanol and (B) ethylhetocyclazocme were mjected beneath monolayers of L. PC. a. PE. L. PC: 0. PS. and ‘I, PI spread at dtfferent uuttal surface pressures (n,)

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N. Bourhm et al /Co/loids Surfaces B Biointerfaces I (IYY3) 203.-211 207

10

A

El Q I

5 15 25

10

B

5 15 25

Fig 4 Change of surface pressure (Ax) when the opiate antagomsts. (A) naloxone and (B) diprenorphme were Injected beneath monolayers of L, PC; n , PE: Cl. PC: 0, PS: and G, PI spread at different nntlal surface pressures.

Opioid peptides Tyr-GlyyGly-PheeLeu-ArggArg-Ile

The behavior of opioid peptides varied depend- ing on which phospholipid formed the film.

P-Endorphin (Fig. 2(A)) was found to interact with all types of phospholipids tested but the higher interactions were observed with negatively charged molecules. The highest critical surface pressure of 37 mN m-l was observed with PI and the lowest with PC, 26 mN m - ‘. The same phenomenon was observed with dynorphin A but with a very high critical surface pressure of 50 mN mm ’ and the lowest value 35 mN m-i. In contrast, Leu- and Met-enkephalin, at 0.1 PM, do not interact signifi- cantly with the phospholipids tested in this study.

It is of interest to note that both P-endorphin and dynorphin A contain Met- and Leu-enkephalin peptides respectively in their N-terminal fragment.

Opiate agonists

Levorphanol (an agonist that interacts with 6, p and K receptors) penetrates all phospholipids in

the following order of potency PI > PS > PG >

PE > PC with critical surface pressures of 35 mN m- ’ for PI and 18 mN m ’ for PC (Fig. 3(A)).

Ethylketocyclazocine, a potent K agonist, also interacts with all the phospholipids tested. The critical surface pressures measured were essentially identical to those observed in the case of levorphanol; 35 mN m-l for PI and 16 mN mm1 foi (Fig. 3(B)).

PC

Opiate antagonists

The two antagonists tested (diprenorphine and naloxone) do not show significant interaction and the same critical surface pressure was observed (16 mN mm ‘) with all the phospholipids tested (Figs.

4(A) and 4(B)).

Specificity of the interaction of dynorphin A The sequence of dynorphin A contains one copy of Leu-enkephalin:

ArggPro-LyssLeu-Lys dynorphin A

Tyr-Gly-GlyyPhe-Leu Leu-enkephalin

Chymotrypsin can hydrolyze dynorphin A into two fragments (Gly-GlyyPhe and Leu-ArggArg- Ile-Arg-Pro-Lys-Leu-Lys) and tyrosine. As reported above, dynorphin A interacts strongly with phospholipids; a critical surface pressure of 50 mN m ~’ was observed for PI and PS, and Leu- enkephalin does not penetrate the phospholipid monolayers. To demonstrate that the interaction of dynorphin A was not due only to the presence of three arginine and two lysine residues in the structure of dynorphin A, the following experiments were done. (i) We have hydrolyzed dynorphin A with chymotrypsin and we have tested the penetration of monolayers by this hydrolyzed peptide. (ii) We have synthesized a modified peptide by substituting tyrosine, that is known to be essential for opiate receptor interaction [23], by phenylalanine and also the larger chymotryptic fragment.

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The interactions of these two synthetic peptides with phospholipids were measured under the same conditions as for opioid peptides.

The results are presented in Figs. 5 and 6. One can see that when dynorphin A was hydrolyzed by chymotrypsin the critical surface pressure decreased from 50 to 20 mN m- ’ (Fig. 5(C)). The control experiment indicated that chymotrypsin alone does not penetrate the phosphatidylserine fiims at the concentration used (Fig. 5(A)). Another experiment was done simultaneously, consisting of injecting the chymotrypsin when the dynorphin A

20 f

0 10 20 30 40 50 60 70

20, Time ( min )

O-0

0 ₀

1~. -._

₁₀ _~rL/m~” ⁴ ₄₀^I ₅₀

I

n,

Fig. 5 Kmetjcs of surface pressure Increase. (A) Penetration of PS film at II, -25 mN m- ’ for dynorphm A. dynorphm A hydrolyzed with an excess of chymotrypsm and for chymotrypsm alone (Bl Identical to (A) but chymotrypsm was injected at the time mdlcated. (C) Increase of maximum values of surface pressure (dn) as a function of mitral film pressure (n,) for Interaction of PS with 0, dynorphm A and ‘0. dynorphm A hydrolyzed by chymotrypsm.

.

s.., _

¹⁰l ^l^I. ²⁰^l

_

³⁰

_

^._

q(mN/m)

C

1

¹⁰

q f2L/rn )

30 ‘

Fig. 6 (A) AdsorptIon kmetlcs of modliicd dynorphm and synthetic chymotqptlc fragment (0.1 PM) at the air-water mterface (B) Change of surface pressure (2~) when the modified dynorphrn was Injected beneath monolayers of L. PC, n , PE:

!3. PG. @, PS. and ‘1. PI spread at different nntlal surface pressures (Cl Identical to (B) but m the presence of synthetic chymotryptlc fragment.

was in equilibrium with a PS film at an initial surface pressure of 25 mN m-r. The results indicated that the surface pressure decreased strongly and a plateau was observed at 4 mN m- ‘. suggesting that some fragment of dynorphin A penetrated the phospholipids irreversibly (Fig. 5(B)).

Figure 6 shows the interaction of the modified dynorphin A with all the phospholipids tested in this study. We can see that the substitution of tyrosine by phenylalanine causes a strong decrease in the critical surface pressure in comparison with

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N. Bourhm et al./Collods Surfaceb B Bminterfbces I / 1993) 303-211 209

dynorphin A; for example, for PS the critical surface pressure decreases from 50 to 28 mN m- ‘.

The synthetic chymotryptic fragment interacts moderately with phospholipids and we have found a critical surface pressure of 20 mN m _ ‘, suggesting that arginine and lysine residues are not exclusively responsible for the interaction of dynorphin A with phospholipids.

Discussion

Opioid receptors are heterogeneous, at least three different classes are present in the brain (a-, ,u- and k--receptors) and these differ in their ligand selectivity and in their pharmacological effects [6].

Most opioid ligands are not completely selective for a single type, thus it is difficult to isolate one type of opioid receptor. Several groups have recently reported a partial purification of opioid receptors [20,24-261. In most cases the isolated receptors have lower affinity toward opioid ligands than the membrane-bound form. These studies suggested that the loss of some lipid during frac- tionation could be responsible for the altered properties. This conclusion was also consistent with many studies documenting the role of lipids in opioid receptor binding [9,27,28].

A major problem encountered during our previous studies to identify opioid receptors is the possible difference in the mode of binding of alkaloids and peptide ligands. Previous work from our group and others has shown that these peptides are very sensitive to the action of peptidases and accurate results could not be obtained [ 15,16,29,30]. An alternative approach could be the detection of 6-, p- and k--receptors by the use of opioid alkaloids. The question arises whether opioid peptides and alkaloids interact in the same manner with membranes. The purpose of this study is to visualize directly the possible insertion of peptide ligands and alkaloids into the hydrophobic core of a model membrane, using the monolayer technique, and to obtain information about the interaction of alkaloids and opioid peptides at the molecular level.

Surfuce activity of opioid peptides and opiate drugs This surface activity reflects the power of opioid peptides or opiate drugs to adsorb and/or to penetrate an interface. It was determined by the variation of surface pressure activity at the air- water interface. This activity was low (2-5 mN m ‘) for opioid peptides and 1 mN m- ’ for opiate drugs (agonists and antagonists).

In the presence of phospholipid monolayers, the surface activity of opioid peptides and opiate drugs becomes much greater than that observed at the air-water interface (Fig. l(B)). This result indicates that there is an interaction of opioid peptides and opiate drugs with phospholipids but the interaction was different for each ligand and/or each phospholipid tested. P-Endorphin interacts with all phospholipids examined; the highest critical surface pressure observed was for PI and PS (40 mN m-l).

Concerning the conformation of /I-endorphin in the presence of phospholipids, it was demonstrated by circular dichroism that in ethanol or trifluoro- ethanol (a solvent known to mimic influences of membranes on peptide conformation) fi-endorphin exhibits considerable z-helical structure [31]. The high critical surface pressure observed may be due to this x-helical conformation of fi-endorphin; pre- sumably this conformation favors the insertion of fl-endorphin into the hydrophobic core of the phospholipids.

Dynorphin A, a potent x-receptor agonist, penetrated strongly into the phospholipid films with a critical surface pressure of 50 mN m- ‘. When dynorphin A was hydrolyzed by chymotrypsin, the critical surface pressure decreased from 50 to 20 mN m-l (Fig. 6(C)). The substitution of tyrosine, that is known to be essential for opioid receptor interaction [23], by phenylalanine abolished completely the conformation of dynorphin A.

Consequently, the interaction of modified dynorphin A with phospholipids was reduced and we have observed a critical surface pressure of 28 mN m-l as compared to the value of 50 mN m-l for non-modified dynorphin A, indicating that the interaction was specific and dependent on the

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conformation and/or orientation of the peptide.

The high interaction of dynorphin A with negatively charged phospholipids was not restricted to the presence of the arginine and lysine residues in the sequence of the peptide. Our conclusions were supported by the fact that the chymotryptic fragment (which contains three arginines and two lysines) does not interact strongly with negatively charged phospholipids. Concerning the conformation of dynorphin A, it was demonstrated by thermodynamic calculations that dynorphin A has very little order in water (random-coil conformation) [S]. On the zwitterionic phospholipids the peptide assumes an r-helical structure oriented perpendicularly to the surface. The N-terminal helical segment is inserted into the hydrophobic membrane layers, whereas the C-terminal strongly charged segment remains exposed to water as a random coil. The two domains are separated by the helix-breaking residue proline [8], suggesting that the conformation and orientation of dynorphin A are responsible for the high insertion of the peptide into the phospholipid monolayers.

The critical surface pressures for penetration into different types of phospholipid films were observed to be much higher in the case of negatively charged phospholipids, suggesting the impor- tance of electrostatic interaction [32] but hydrophobic interaction may play an important role because when dynorphin A-PS films were hydrolyzed with chymotrypsin, the surface pressure was decreased to 4 mN m ‘, suggesting that some fragment of dynorphin A penetrates the hydrophobic core of the phospholipid monolayers (Fig. 5(B)).

Surface actkity of opiate agonists

Neither levorphanol nor ethylketocyclazocine formed monolayers at the air-water interface, indicating the absence of surface activity; the same results have been reported for morphine [33].

Levorphanol is an opiate agonist that interacts with all types of opioid receptors (6, ,U and K) with the same affinity. This ligand presented no surface

activity at the air-water interface but in the presence of phospholipid monolayers, the surface activity becomes greater with a critical pressure of 35 mN mm1 for PI and PS.

The ti-receptor agonist ethylketocyclazocine presented the same properties, with a critical surface pressure of 30 mN m I. All these results indicate that alkaloids penetrate phospholipids moderately in comparison with opioid peptides. suggesting a different mode of interaction with the phospholipids. The ammonium and hydroxyl groups in the structure of alkaloids as well as in opioid peptides.

are known to be essential for binding activity.

Thus. in the absence of receptors these groups could interact with negatively charged phospholipids; this view was supported by the high interaction of the alkaloids with PI and PS (Fig. 3).

Surf&e actioity of opiate mltugoilists

Like agonists, the antagonists do not present surface activity at the air-water interface.

Diprenorphine and naloxone, the antagonists of all types of opioid receptors, present the lowest critical surface pressure observed in this study (17 mN mm ‘) (Fig. 4). This result is in agreement with the earlier observation where some lipids (ganglio- sides and lecithin,iganglioside mixtures) were expanded by both morphine (opiate agonist) and naloxone (opiate antagonist). The expansion of ganglioside-containing monolayers was greater with morphine (agonist) than with naloxone (antagonist) [33]. More recently, it was reported that antagonists of substance P (a neuropeptide which is widely distributed in both the central and peripheral nervous systems and acts as a neurotrans- mitter or neuromodulator) are much more surface active than substance P agonists, and they penetrate strongly into lipid membranes [34]. These discrepancies could be explained by the peptide nature of the substance P antagonists which may exist in a different three-dimensional structure that permits orientation and penetration into phospholipids.

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N. Bourhrm et al iCol1oid.s Surfaces B, Blolnterfaces I (lYY3) .?03-211 Conclusions

Our results indicate that opioid peptides are more surface active than alkaloids, with the unu- sual observation that dynorphin A interacts strongly with negatively charged phospholipids, giving a very high critical surface pressure of 50 mN m-l. The antagonists penetrate the monolayers moderately; we have observed a critical surface pressure of 17 mN mm ‘. All these results indicate that opioid peptides and alkaloids interact differentially with phospholipids. On the basis of penetration of phospholipid monolayers, it is difficult to conclude whether penetration of opioid peptides and alkaloids into lipid membranes is a necessary prerequisite for receptor binding or not.

To understand further the role of lipids on opioid receptors selection, we need to await the purification of opioid receptors. followed by reconstitution experiments with different endogenous and/or exogenous phospholipids.

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