Cell interactions of enkephalin/polypeptide conjugates

JOURNAL OF MOLECULAR RECOGNITION, VOL. 4,35-41(1991)

Cell Interactions of Enkephalin/Polypeptide Conjugates

Motohiko Tsukada, Yoshiko Sasaki-Yagi, Shunsaku Kimura and Yukio Imanishi* Department of Polymer Chemistry, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606, Japan

Enkephalin molecules were bound to poly(Lys) (ply-K) or poly(A1a-Lys-Ala-Leu) (ply-A) and their interactions with NG108-15 cells, platelets, erythrocytes and fibroblast cells were investigated. A fluorescent probe, rhodamine, also was bound to the conjugates for monitoring interactions with these cells. Observations by fluorescence microscopy revealed that NG108-15 cells, platelets, and fibroblast cells were labelled by the conjugates, whereas erythrocytes were not. Since polypeptides without enkephalin moieties were only weakly adsorbed on the cells, it was concluded that the enkephalin/polypeptide conjugates were bound specifically to receptors on the cell membrane. Interestingly, when the enkephalin/poly-K conjugate was bound to NG108-15 and fibroblast cells, fluorescent patches appeared on the membrane. Such patch formation was not clearly observed with an enkephalinhhodamine or enkephalin/poly-A conjugate. In the case of fibroblast cells, the fluorescence converged to a large cluster, which was ultimately internalized. The results suggest that clustering of the receptors in cell membranes is influenced by the carrier polymer presumably due to cross-linking of the receptors and/or the effect of the cationic polypeptides.

INTRODUCTION

When receptors in cell membrane are exposed to an agonist for a long time, cell response to the agonist is weakened (desensitization), and cells become adaptive only to a high local concentration of the agonist (Trig- gle, 1981). One of the mechanisms to explain such desensitization is reduction of the number of receptors on the cell surface (down regulation). This process consists of the formation of agonist/receptor complex and clustering the complexes leading to internalization of the complex into the cytosol (Sharma ef al., 1975b). On the other hand, as far as receptors for low density lipoprotein and transferrin are concerned, ligands internalizing into cytosol are thought to serve as intra- cellular signals (Anderson ef al., 1977, Dantry-Varsat et al., 1983). We therefore considered it very interesting to study the redistribution of receptors on cell membrane induced by agonist binding.

Several papers have reported the observation of clustering of receptors by using fluorescence microscopy. For example, the fluorescein-conjugated epider- mal growth factor and f3-melanotropin, and rhodamine/ enkephalin conjugate have been synthesized and the location of corresponding receptors on the cell membrane was investigated (Maxfield et al., 1978; Hazum et al., 1979). Furthermore, tobacco mosaic virus carrying about lo2 molecules of peptide hormone such as f3- melanotropin, enkephalin, angiotensin 11, or adreno- corticotropin as well as rhodamine was utilized as 'artificial fluorescent antibody' for mapping receptors for the peptide hormone (Schwyzer et al., 1981a, b; Kriwaczek et al., 1978). In addition, the peptide hormone/tobacco mosaic virus conjugates showed not only a high affinity for the receptor but also a superpo- tency (Eberle et al., 1981). We therefore were inter- ested in synthesizing agonist/polypeptide conjugates

carrying fluorescence probes and in investigating their interactions with cell membranes.

Poly(g1utamic acid) has been used frequently as a ligand carrier due to its biocompatibility and low antigenicity (Mlank et al., 1988). On the other hand, the diffusion of ligand and receptor in cell membranes is two-dimensional, which makes their association in membranes more favourable than that in solution, which is three-dimensional diffusion (Adam and Delbruck, 1968). This concept led us to use polypeptides such as poly(Lys) (poly-K) or poly(Ala-Lys-Ala- Leu) (poly-A) as ligand carriers, which are expected to exhibit a high affinity to the negatively-charged cell membrane (Fig. 1). The former polypeptide poly-K has been used as a drug carrier (Ryser, et al., 1982; ROOS, et al., 1984), and the latter polypeptide poly-A is designed to show a high affinity for the lipid membrane phase by taking an amphiphilic structure when it forms an a- helical conformation.

In addition, basic polypeptides are known to facili- tate the uptake of macromolecules by cells, which is the

Rhodamtne 6' Tyr-ala-Gly-Phe-Leu-Gly-Lys-NH2 ( RE )

-&

N CCH ,+,C-O Rhodamine

Figure 1. Moleculer structure of RE, poly-K-RE, and poly-A-RE. * Author to whom correspondence should be addressed.

0952-3499/91/010035-07 $05.00 @ 1991 by John Wiley & Sons, Ltd.

Receiued 19 June 1989 Accepted (reuised) 29 April 1991

36 M. TSUKADA E T A L .

result of enhanced endocytosis (Ryser and Hancock, 1965). Therefore, the receptor reorganization in membrane induced by ligand binding will be promoted by the effect of basic polypeptides. In the present investigation, [Leulenkephalin molecules were connected to the basic polypeptides and interactions of the conjugate with different types of cells such as NG108-15 cells, platelets, fibroblast cells and erythrocytes were investigated.

EXPERIMENTAL

Synthesis

The molecular structures of rhodamine/enkephalin conjugate (RE), poly(Lys)/rhodamine/enkephalin conjugate (poly-K-RE) and poly(A1a-Lys-Ala-Leu)/ rhodamine/enkephalin conjugate (poly-A-RE) are shown in Fig. 1. The synthesis of poly-A-RE is described in Fig. 2. The peptides were synthesized by the conventional liquid phase method. The handle connect- ing polypeptide to enkephalin was designed essentially according to Kriwaczek et al. (1978). The synthetic procedures are as follows.

RE. Boc-Tyr-ala-Gly-Phe-Leu-Gly-Lys-NH2 (53.8 mg) was dissolved in methanol + dichloromthane (15, v/v) and reacted with rhodamine isothiocyanate (25 mg) in the presence of triethylamine (3.5 mg) at 0°C for 2 h. After the addition of a few drops of acetic acid, the solution was evaporated. To the residue obtained was added anisole (0.1 mL) and trifluoroacetic acid (1 mL) at 0 "C. After 30 min, the reaction mixture was added

+Ala- Lys( HBd- Ala- Leu + +

5. C- N

a +Ala-Lys-Ala-Leu+~-+Ala-Lys-Ala-Leu~

lf Rhodamine

CH3CO-5-CH-C, P I 0

I I C- C H-S H I I 0 CHTCOOH

Rhodamine

TFA " > Anisole '

Rhodamine

COOH

Figure 2. Synthetic route to poly-A-RE.

to petroleum ether. The oil recovered was purified by eluting through a Bio-Gel P-2 column using acetic acid +water (l : l , v/v) as eluant.

Poly-A-RE. A solution of poly-A (average MI 25000, 46.4 nig) in phosphate buffer (pH 6.8, 0.75 mL) was diluted with Na,C03/NaHC03 buffer solution (pH 9.5, 6.75 mL), and reacted with rhodamine isothiocyanate (10.5 mg) in the presence of triethylamine (10 mg) at 37 "C for 30 min. Ammonia solution (1 M, 0.185 mL) was added and the product (poly-Ahhodamhe conjugate, poly-A-R) was purified by dialysis against water. The dialysed solution was reacted with S-acetylmercaptosuccinic anhydride (362 mg) under nitrogen atmosphere. During the reaction, the pH of the solution was adjusted to 6.8 by adding 0.1 N aqueous NaOH solution. The solution was dialysed against water for 1 week. The S-acetyl group was removed by the addition of hydroxylamine (0.01 M) at pH 7.4 under nitrogen atmosphere. The solution obtained was dialysed against water. Six thiol groups were found to be introduced to a chain of the polypeptides, which was determined by the mercury orange method (Sasaki, 1968).

On the other hand, enkephalin was extended by a handle segment as follows. N-(E- maleimidocaproy1oxy)succinimide (95 mg) was added to a solution of Boc-Tyr-ala-Gly-Phe-Leu-Gly-Lys-NH, (359 mg) in N,N-dimethylformamide (DMF) and reacted for 48 h. The product was purified by eluting through a Sephadex LH-20 column using DMF as eluant; yield 352 mg (86.3%), mp 196-197 "C. Found: C, 58.76; H, 7.19; N, 13.25%. Calcd for C52H74013N10:

A DMF solution of the enkephalin/handle obtained (78.8 mg) was reacted with thiol groups of poly-A-R (37.5 mg) in water (1 mL, pH 6 adjusted by acetic acid) for three days. Ether was added to precipitate the product (26.5 mg). The BOC protecting group of the N-terminal in the enkephalin moiety was removed by treating with trifluoroacetic acid (1 ml) and anisole (0.1 mL) at 0 "C. After 30 min, ether was added, and the precipitate was dissolved in acetic acid and dialysed against water to obtain poly-A-RE.

Poly-K-R and poly-K-RE were prepared by a similar procedure to those described respectively for poly-A-R and poly-A-RE. Poly(Lys) (poly-K) was purchased from Peptide Inst. Inc., Japan (Average MI 25 000).

The numbers of rhodamine and enkephalin molecules introduced per polypeptide chain were, respectively, 5 and 5 for poly-K-REd, 5 and 10 for poly-K-REc, and 9 and 5 for poly-A-RE.

C, 59.64; H, 7.12; N, 13.83%.

Interaction with NGlOS-15 cells

NG108-15 cells were kindly supplied from Dr H. Ueda of the Department of Pharmacology, Faculty of Pharmaceutical Science, Kyoto University. Cells were grown in Dulbecco's minimum essential medium (Gibco, USA) containing 10% fetal calf serum (Bio- product Co., USA) and 1% HAT (Flow Laboratories, USA) (Kosterlitz et al., 1985; Howard et al., 1985). Cells were cultured for 2 days on the Cell Desk (Sumilon, Sumitomo Bakelite Co., Ltd, Japan). After removing the medium, cells were incubated with 0.02 M

ENKEPHALIN/POLYPEPTIDE CONJUGATES 37

tris(hydroxymethy1)aminoethane (Tris-HC1) buffer (pH 7.4), containing enkephalin/polypeptide conjugate with or without 1 0 - 4 ~ naloxone for 10min at 37°C. The incubation was terminated by removing the incubation media, and adding ice-cold Tris buffer. Cells were kept in Tris buffer containing 1 0 m ~ NaN, in the ice bath. Cells attached to the Cell Desk were examined by optical and fluorescence microscopy.

Interaction with platelets and erythrocytes

Canine blood was treated with acid-citrate-dextrose (ACD) solution and centrifuged at 300 x g for 10 min. The precipitated pellet was washed with phosphate buffer solution and suspended again in 0.02 M Tris-HCI buffer (erythrocyte suspension). ACD solution (10 vol% of the supernatant) was added to the supernatant and the mixture was centrifuged at 1OOOXg for 10 min. The precipitate was washed with phosphate buffer and suspended again in 0.02 M Tris buffer (platelet suspension). After incubation with enkephalin conjugate at 37 "C for a requisite time in a polystyrene test tube, the reaction medium was removed by centrifuga- tion at 4"C, and the residue was washed three times with Tris-HC1 buffer. Cells were resuspended in Tris-HCI buffer, and examined by optical and fluorescence microscopy.

c-AMP assay (Kelly and Butcher, 1974)

Fibroblast cells were cultured in 60 mm Petri dishes for two days. The cells attached to the dish were rinsed twice in serum-free medium. Then, fresh serum-free medium (3 mL), opioid peptide, and prostaglandin El (PGE,) were added to the dish, which was incubated at 37°C for 20min. The incubation was terminated by removing the incubation media, and the cell sheet attached to the dish was immediately fixed by the addition of cold 5% trichloroacetic acid (TCA). Another portion of TCA was added, and the combined extracts were lyophilized. Cell residues were solubi- lized in 0 . 3 ~ aqueous KOH solution, and were sub- jected to the protein assay according to the modified method of Lowry et al. (1951) (BCA protein Assay Reagent, Pierce Chemical Company, USA).

The lyophilized sample was dissolved again in a small amount of water, and 10 pL of 1 M Tris-HCI (pH 7.4) was added. The solution was neutralized with aqueous NaOH solution, and the volume was adjusted to 1 mL. The neutralized sample was applied to a small column (0.5 x 4 cm) of 100-200 mesh 50W-X8 resin (Dowex, USA). After eluting 2mL of distilled water, another 4 mL of distilled water was added, and the eluant was collected and lyophilized. The residue was assayed by the Cyclic AMP Assay Kit (Amersham, UK).

Platelet aggregation (Born, 1962; Gryglewsaki et al., 1975)

ACD treated human blood was centrifuged at 500 x g for 20 min. The supernatant platelet-rich-plasma was tranferred, adjusted to pH 7.0 by addition of aqueous NaOH solution, and kept at room temperature until used. 2.5mL of the platelet suspension was pipetted into transparent plastic cuvettes at 37"C, set in to a JASCO UVIDEC-1 (Japan), and stirred vigorously. The change in light transmission at 600nm was moni- tored.

Interaction with fibroblast cells

Mouse fibroblast cells were kindly supplied from Professor M. Takeuchi of the Department of Biophysics, Faculty of Science, Kyoto University. Cells were grown in Dulbecco's minimum essential medium (Nissui Co., Japan) containing 10% fetal calf serum (Bioproduct Co., USA), 5.0 X wt% streptomycin (Sigma, USA, S-6501), and 3.1 X wt% penicillin (Sigma, USA, PEN-K). Cultured cells were treated with 0.2 wt% aqueous trypsin (bovine pancreatic, Sigma, USA, No. T8253, 11OOO unit mg-') solution in Ca2+ and Mg2+ free phosphate buffer solution (pH7.4). Cells were washed three times with phosphate buffer, and suspended again in 0.02 M Tris-HCI buffer. The experimental conditions for incubation with enkephalin conjugate and microscopic observation of these cells were similar to those for platelets and erythrocytes described above.

RESULTS

Characterization of polypeptide carrier. The elution pro- files of poly-K and poly-A through Sephadex G-50 column showed that the most frequently occurring molecular weight of both polypeptides is 25 OOO. Though poly-A was considered to aggregate in water due to its amphiphilic nature, only one peak appeared in the elution profile. Furthermore, the CD spectrum of poly-A was nearly independent of the concentration (Fig. 3).

Therefore, poly-A should be molecularly dispersed in a buffer solution. CD spectrum of poly-A showed two negative peaks at 203 nm (0 = -35 000) and 225 nm (0 = -13 OOO) (Fig. 3), indicating occurrence of a different conformation from the typical a-helix. However, the experimental facts, that the fluorescence intensity of 1-anilino-8-naphthalenesulfonic acid (ANS) in a buffer solution was enhanced and the maximum wavelength of emission shifted to shorter wavelength in the presence of poly-A (Fig. 4), indicate that the environ- ment provided by poly-A is hydrophobic. In addition, the fluorescence polarization of ANS increased from a value of 0.101 in a buffer solution to a value of 0.258 in the presence of poly-A. An induced positive Cotton effect of ANS was observed at 350 nm in the presence of poly-A. These experimental results indicate that ANS molecules should be tightly bound to the hydrophobic region of poly-A.

Interaction with NG108-15 cells. NG108-15 cells are known to possess 6-opioid receptors. The interaction of the conjugates with NG108-15 cells was studied by the fluorescence microscope. NG108-15 cells were labelled by poly-K-REc and fluorescence patches were observed on the cell surface (Fig. 5). Since such patch formation

38 M. TSUKADA E T A L

0

-10

0 -20 ' 0 X

K n a u -30

-40

- 50

h ( n m )

200 220 24 0 260 I

Figure 3. CD spectrum of poly-A in water. Assignments; -, 1.6 x 10-3 M; - - -, 3.2 x 1 0 - ~ M; --, 8.1 x 10-5 M.

was not detected in the presence of antagonist (Fig. 6), the enkephalin/poly-K conjugate is concluded to induce clustering of &receptors in NG108-15 cells. This observation is in contrast to the report by Hazum et al. (1979), that the rhodamine/enkephalin conjugate, Tyr-Ala-Gly-Phe-Leu-Lys-rhodamine (RE) was adsorbed on the cell surface of neuroblastoma cells without forming such fluorescent patches. Such micro- aggregation of receptors should be triggered by cross- linking of receptors with the polypeptide conjugate and/or the basic polypeptide carrier.

Interaction with platelets and erythrocytes. It has been reported that platelets possess opioid receptors, because the suppression by PGEl of the aggregation of platelets induced by ADP is inhibited by enkephalin (Born, 1962 and Gryglewsaki et al., 1975). This obser-

400 450 500 550 600

( nrn )

Figure 4. Change of fluorescence spectra of ANS in buffer with the addition of poly-A. [ANSI = 0.19 mM. The numerals in the Fig. indicate the value of [Lys residue in poly-AJIIANS].

Figure 5. Interaction of poly-K-REc ( lo-* M) with NG108-15 cell after incubation for 10 min. (a) Micrograph; (b) fluorescence micrograph.

Figure 6. Interaction of poly-K-REc ( l o - * M) with NG108-15 cell after incubation for 10 min in the presence of naloxone (0.1 mM). (a) Micrograph; (b) fluorescence micrograph.

vation can be interpreted to suggest that the increase of c-AMP level in cytosol caused by PGEl should be reduced by the action of enkephalin. On the other hand, no such evidence has been reported to suggest that erythrocytes possess opiate receptors.

When poly-K-REd was incubated with a mixture of platelets and erythrocytes, it was found by fluorescent microscopy that platelets were fluorescent because of labelling with poly-K-REd, whilst erythrocytes were not (Fig. 7). This result is consistent with the reports that the opioid receptors occur in platelets, but not in erythrocytes.

The physiological activity of the enkephalin unit in poly-K-REc was investigated in terms of the effect of poly-K-REc on platelet aggregation induced by ADP. The addition of poly-K-REc (the concentration in

Figure 7. Interaction of poly-K-REd with a mixture of platelets and erythrocytes after incubation for 10 min. (a) Mlcrograph; (b) fluorescence micrograph.

ENKEPHALIN/POLYPEPTIDE CONJUGATES 39

Time ( m t n )

Figure 8. Suppresion by poly-K-REc of inhibitory effect of PGE, on ADP-induced aggregation of human platelets. Poly-K-REc

M), PGE, (Sigma, final concentration of 8.5 nM) and ADP (Kohjin Co. Ltd., final concentration of 8 p ~ ) were added with intervals of 40 s. Assignments: - , +ADP; --, +PGE,+

enkephalin unit: 10-* M) partly antagonized the inhibitory effect of PGE, on the aggregation induced by ADP (Fig. 8). Therefore, it was confirmed that the biological activity of enkephalin was not impaired by conjugation with poly-K. However, poly-K-REc in higher concentration than lo-' M in enkephalin lost the antagonistic action against PGEl, because the effect of poly-K matrix in enhancing the aggregation of platelets exceeded the antagonistic effect of enkephalin.

ADP; - - - -, +poIy-K-RE + PGE,+ ADP.

Interaction with Fibroblast Cells. When the rhodamine/ enkephalin conjugate, RE, was incubated with fibroblast cells, the whole cell surface became fluorescent, indicating that fibroblast ce!ls might possess opioid receptors. However, patch fluorescence on the cell membranes was not observed clearly, and internaliza-

Figure 9. Interaction of RE with fibroblast cells after incubation for 90 min. (a) Micrograph; (b) fluorescence micrograph.

tion of agonist/receptor complex did not occur (Fig. 9). On the other hand, poly-K-REc was shown to be absorbed onto fibroblast cells forming patches, and further incubation at 37°C led to cap formation and finally internalization (Fig. 10). Since rhodamine- labelled poly-K (poly-K-R), which lacks the enkephalin moiety, did not induce such cell responses under the same experimental conditions (incubation medium washed three times with buffer), the cap formation and internalization of poly-K-RE were concluded to be caused by the specific interaction of enkephalin moieties in poly-K-RE with receptors on fibroblast cells. This interpretation is supported by the observation that poly-K-REd, which has half as many enkephalin moieties in a peptide chain as poly-K-REc, showed weaker interaction with fibroblast cells than poly-K-REc (data not shown). On the other hand, the binding of poly-A- RE to fibroblast cells did not induce the clustering of receptors so significantly as poly-K-RE (Fig. 11).

Figure 10. Interaction of poly-K-REc with fibroblast cells. (a, b) Incubation for 20 min; (c, d) incubation for 40 min; (e, f) incubation for 60 min; (9, h l incubation for 90 min. (a, c, e, g) Micrograph; (b, d, f, h) fluorescence micrograph.

40 M. TSUKADA E T A L

Figure 11. Interaction of poly-A-RE with fibroblast cells after incubation for 40 min. (a) Micrograph; (b) fluorescence micrograph.

DISCUSSION ~

Biologically active ligands have been conjugated to polymeric carriers in order to increase biocompatibility (or reduction of the antigenicity), resistance against biodegradation, site-specific delivery and pharmaceutical activity. In th present investigation, the receptor organization in cell membranes upon binding of agonist was shown to be influenced by conjugation of agonist with the carrier polymer. That is, enkephalin conjugated with poly-K (poly-K-RE) induced the clustering of receptors in NG108-15 and fibroblast cells, whilst enkephalin alone did not.

Although poly-K by itself possesses biological activi- ties, the observed clustering of receptors in NG108-15 cells cannot be explained only by the contribution of poly-K in the conjugate for the following reasons: (i) Poly-K-REc induced patch formation in NG108-15 cells, but poly-K-R did not at the same concentration of the conjugate; (ii) When poly-K-REd was incubated with the mixture of platelets and erythrocytes, only platelets were labelled by poly-K-REd, erythrocytes were not. The difference is consistent with the occurrence of opioid receptors in platelets, but not in erythrocytes. Therefore, the interaction of the conjugate with cells is attributed to the specific binding of the enkephalin moieties in the conjugate to receptors in the cell membrane. The specific binding of the conjugates is also supported by the following observations. Poly-K-REc antagonized the inhibitory effect of PGE, on the aggregation of platelets induced by ADP, which is ascribed to the activity of enkephalin units in the conjugate. The patch formation in NG108-15 cells induced by poly-K-REc was inhibited by naloxone which is an antagonist to opioid receptors.

It should be noted that the enkephalin/polypeptide conjugate (poly-K-RE) induced the clustering of the agonist/receptor complex on NG108-15 and fibroblast cells whilst enkephalin alone (RE) did not. It is well known that basic polypeptides stimulate the uptake of

macromolecules by mammalian cells (Ryser and Hancock, 1965). Although the mechanism responsible for stimulation of uptake of macromolecules is not known clearly, macromolecules were found in pinocy- totic vesicles (Ryser, 1968). This is obviously a different process from the clustering of the complexes observed here. On the other hand, it is reported that basic polypeptides by themselves were endocytosed into cytoplasmic vacuoles (Ryser 1968), but the endocytosis is not known to occur via the formation of the large cluster of the complex as observed with poly-K-RE. It is rather reasonable to attribute the clustering of the complex in membrane to the cross-linking of receptors by the conjugate, which resembles the case of the antigedantibody interaction in lymphocytes (Oliver et al. , 1980).

In the present investigation, poly-K-R was not taken up significantly by fibroblast cells. However, poly-K- RE showed a high affinity to fibroblast cells, which is ascribed to the specific interaction of enkephalin moieties in the conjugate with receptors in the membrane. Thereby, the conjugate/recptor complex formed the clusters on membranes mainly due to the cooperative binding of intramolecular enkephalin moieties in the conjugate to receptors. The succeeding cap formation and internalization should be ascribed additionally to the effect of the basic polypeptide. It is noteworthy that the amphiphilic polypeptides with less positive charges could not induce such clustering of the complex. This supports the theory that a dense distribution of positive charges along the molecule is needed for the observed receptor reorganization. Nonetheless, poly-K by itself does not induce the clustering. Therefore, it is likely that positive charges and cross-linking ability of the polymeric ligand are both required for changing receptor reorganization in the membrane.

Finally, the observation of receptor in fibroblast cells bears comment. There has been no report so far on the occurrence of opioid receptors in fibroblast cells. We could not detect the presence of opioid receptors in the membrane homogenate of NG108-15 cells (75 pg/ 150 pL) by competitive binding assay using [ 3H] [D-Ala2, MePhe4, Gly-ols]enkephalin (DAGO) and [ 3H] [D-Pen’, ~-Pen~]enkephal in (DPDPE). Opioid receptors are reported generally to couple with adenylate cyclase through G-protein (Brandt et al., 1980). In this respect, the effect of opioid peptides on the production of c-AMP in fibroblast cells was examined also. The amount of c-AMP in fibroblast cells was increased by the addition of PGE, as reported by Sharma et al. (1975a). However, neither enkephalin (6- selective), DAGO (p-selective), nor dynorphin ( K- selective) tolerated the effect of PGE1. The data, taken together, make it likely that the receptor found in fibroblast cells is different from opioid receptors found in NG108-15 cells. This receptor might be silent or coupled with other effector molecules such as Kf chan- nel. The nature of fibroblast receptor remains to be fully understood.

REFERENCES

Adam, G. and Delbruck, M. (1968). Reduction of dimensionality in biological diffusion process. In: StructuralChemistryand Molecular Biology, ed. by A. Rich, N. Davidson, pp. 198-

215, Freeman, San Francisco. Anderson, R. G. W., Goldstein, J. L. and Born, M. S. (1977). Role

of the coated endocytic vesicle in the uptake of receptor-

ENKEPHALINIPOLYPEPTIDE CONJUGATES 41

bound low density lipoprotein in human fibroblasts. Cell 10,

Born, G. V. R. (1962). Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature, 194,927-929.

Brandt, M., Buchen, C. and Hamprecht, B. (1980). Relationship between the action of calcium ions, opioids and prostaglandin El on the level of cyclic AMP in neuroblastoma x glioma hybrid cells. J. Neurochem. 34, 643-651.

Dantry-Varsat, A,, Ciechanver, A. and Lodish, H. F. (1983). pH and the recycling of transferrin during receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA, 80, 2258-2262.

Eberle, A. N., Kriwaczek, V. M. and Schulster, D. (1981). Corticotropin and melanotropin: organization and transmission of hormonal information. In Perspectives in Peptide Chemistry, ed. by A. N. Eberle. et a/.. pp. 407-422, Karger, Basel.

Gryglewsaki, R. J., Szczeklic, A. and Bieron, K. (1975). Morphine antagonizes prostaglandin Elmediated inhibition of human platelet aggregation. Nature 256, 56-57.

Hazum, E., Chang, K. J. and Cuatrecasas, P. (1979). Opiate (enkephalin) receptors of neuroblastoma cells: occurrence in clusters on the cell surface. Science, 206, 1077-1079.

Howard, A. D., de la Baume, S., Gioannini, T. L., Hiller, J. M. and Simon, E. J. (1985). Covalent labelling of opioid receptors with radioiodinated human @-endorphin. J. Biol. Chem. 260,

Kelly, L. and Butcher, R. W. (1974). The effects of epinephrine and prostaglandin El on cyclic adenosine 3',5'- monophosphate levels in WI-38 fibroblasts. J. Biol. Chem.

Kosterlitz, H. W. and Paterson, S. J. (1985). Types of opioid receptors; relation to antinociception. Phil. Trans. R. SOC.

Kriwaczek, V. M., Eberl e, A. N., Muller, M. and Schwyzer, R. (1978). Tobacco mosaic virus as a carrier for small molecules I: the preparation and characterization of a TMVI@-Melanotropin conjugate. Helv. Chim. Acta 61, 1232- 1240.

Lowry, 0. H. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275.

Mlank, R., Kala, H. and Strube, M. (1988). Darstellung unt testung von polymerpharmaka. Pharmazie 43, 1-4.

351-364.

10833-1 0839.

249,3098-3102.

Lond. 8308,291-297.

Maxfield, F. R., Schlessinger, J., Shechter, Y., Pastan, 1. and Willingham, M. C. (1978). Collection of insulin, EGF and y2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization. Cell 14,

Oliver, J. M., Gelfand, E. W., Pearson, C. B., Pfeiffer, J. R. and Dosch, H.-M. (1980). Microtubule assembly and concanava- lin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells. Proc. Natl Acad.

Roose, C. F., Matsumoto, S., Takakura, Y., Hashida, M. and Sezaki, H. (1984). Physicochemical and antitumor character- istics of some polyamino acid prodrugs of mitomycin C. lnt. J. Pharm. 22. 75-87.

Ryser, H. J.-P. and Hancock, R. (1965). Histones and basic polyamino acids stimulate the uptake of albumin by tumor cells in culture. Science 150. 501-503.

Ryser, H. J. -P. (1968). Uptake of protein by mammalian cells: an underdeveloped area. Science 159,390-396.

Ryser, H. J.-P., Drurnrnond, I. and Shen, W.-C. (1982). The cellular uptake of horseradish peroxidase and its poly(ly- sine) conjugate by cultured fibroblasts is qualitatively similar despite a 900-fold defference in rate. J. Cell. Phys. 113,

Sasaki, H. (1968). Quantitative microdetermination of total-SH groups in protein. Anal. Biochem. 26, 269-276.

Sharma, A. K., Nirenberg, M. and Klee, W. A. (1975a). Morphine receptors: regulators of adenylate cyclase activity. Proc. Natl Acad. Sci. USA 72, 590-594.

Sharma, A. K., Klee, W. A. and Nierenberg, M. (1975b). Dual regulation of adenylate cyclase accounts for narcotic depen- dence and tolerance. Proc. Natl Acad. Sci. USA 72, 3029- 3096.

Schwyzer, R. and Kriwaczek, V. M. (1981a). Tobacco mosaic virus as a carrier for small molecules. Biopolymers 20, 201 1-2020.

Schwyzer, R., Kriwaczek, V. M. and Wunderlin, R. (1981b). A method for mapping peptide receptors. Naturwissenschaften 68, 95-96.

Triggle, D. J. (1981 1. Desensitization. In Towards Understanding Receptors, ed. by J. W. Lamble, pp. 28-33, ElsevierINorth-Holland Biomedical Press, Amsterdam.

805-81 0.

Sci. USA 77, 3499-3503.

167-1 78.

Documents

Cell interactions of enkephalin/polypeptide conjugates