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Inhibitory mechanism of opioid binding to receptor by phospholipase A2
Gen. Pharmac. Vol. 19, No. 5, pp. 707-712, 1988 Printed in Great Britain. All rights reserved
0306-3623/88 $3.00+ 0.00 Copyright © 1988 PergamonPress plc
INHIBITORY MECHANISM OF OPIOID BINDING TO RECEPTOR BY PHOSPHOLIPASE A2 MIZUE MAKIMURA,* YOSHIHISA ITO and Yosnm MURAKOSHI Department of Pharmacy, College of Science and Technology, Nihon University, Kanda-surugadai 1-8, Chiyoda-ku, Tokyo I01, Japan (Received 2 December 1987)
Abstract--1. The inhibition of [3H]naloxone binding to the opioid receptor upon short-term incubation with phospholipase A2 (Plase A2) was abolished by treatment with BSA, but not after long-term incubation. 2. In contrast to the restorative effect of BSA on the strong inhibition occurring with Plase A2, BSA only partially abolished the inhibitory effect of arachidonic acid or lysophosphatides, even though the degree of inhibition was slight. 3. These results suggest that Plase A2 products only become associated with hydrophobic receptor sites or with phospholipids near to the receptor, thus reversibly inhibiting opioid binding, and that irreversible inhibition occurs through release of the phospholipid necessary for receptor binding.
INTRODUCTION It is well known that the properties of membrane receptors change according to local environmental conditions. It has also been shown that lipids in membranes play a role in the interaction of various ligands with their respective receptors (Loh and Law, 1980). In the case of the opioid receptor, Lowney et al. (1974) initially described it as consisting of proteolipids, and since then many investigators have proposed that the lipid is cerebroside sulfate (Loh et aL, 1974) and that the main lipid in the opioid receptor is phosphatidylserine (Abood and Hoss, 1975) and/or phosphatidylinositol (Wu et al., 1976). More recently, a considerable amount of evidence has suggested that lipids play a very significant role in ligand binding sites (Abood et al., 1978; Abood et al., 1980). Furthermore, it is believed that opioidreceptor proteins are associated with phospholipids, thereby maintaining their sterical structure within the membrane. In order to elucidate these possibilities, Pasternak and Snyder (1974) examined the influence of a variety of enzymes on opioid receptor binding and observed a remarkable reduction of opioid binding upon treatment with phospholipases. The reduction of receptor binding after pretreatment with phospholipase A 2 (Plase A2) and also that following mechanical disruption using methods such as freezethawing and sonication have also been shown to be abolished by the addition of phosphatidylserine and bovine serum albumin (BSA), the inhibition being abolished more strongly by BSA than by phosphatidylserine (Abood et al., 1978; Abood et al., 1980). Plase A2-induced inhibition of opioid receptor binding has also been shown to be reversible upon addition of albumin (Lin and Simon, 1978). It has been reported that Plase A2-induced inhibition of opioid *Author to whom correspondence should be addressed.
binding might be due to the products of phospholipolysis, i.e. unsaturated free fatty acids and lysophosphatides (Abood et al., 1977; Lin and Simon, 1978; Rfiegg et al., 1982). However, the details of the inhibitory mechanism of Plase A2 on opioid binding are not yet clear. In the present study, the inhibitory effect of Plase A2 and the properties of the opioid receptor were explored with special reference to the lipids existing in the membranes. The results are reported in this paper, together with a discussion of the possible physiological function of Plase A2. MATERIALS AND METHODS Chemicals
The following drugs were purchased from Sigma Chemical Co. (St. Louis, Mo): phospholipase A 2 (phosphatide 2acylhydrolase: EC 3.1.1.4., from bee venom), phospholipase C (phosphatidylcholine cholinephosphohydrolase: EC 3.1.4.3., from Bacillus cereus), phospholipase D (phosphatidylcholine phosphatidohydrolase: EC 3.1.4.4., from cabbage), oleic acid, linoleic acid, linolenic acid, arachidonic acid, lysolecithin, cardiolipin and fatty acid-free bovine serum albumin. [3H]naloxone, ([N-Allyl-2,3-3H]-, specific activity 80 Ci/mmol) was purchased from New England Nuclear (Boston, Mass). Prostaglandin (PG) E t , E2, F2~and 12 were generously donated by Onoyakuhin Kogyo Co. (Osaka, Japan). Other reagents (analytical grade) were obtained commercially from Wako Pure Chemicals (Tokyo, Japan). Receptor binding assay
Male Wistar rats (150-180g body wt) were killed by decapitation, the brains excised and the cerebella removed. Bullfrog brain membranes were prepared using whole brains as described previously (Ito et al., 1984). Isolated brains were homogenized in 20 vols of 50 mM Tris buffer (pH 7.4, Buffer A) with a polytron (Kinematica), at a power control setting of 6, for 30 see. The homogenate was centrifuged at 20,000 g for 20 rain. The pellet was washed once with Buffer A, and then twice with 50 mM Tris buffer containing 0.5 mM EGTA (Buffer B), using the above procedure. After 707
MIZUE MAK1MURAet al.
708
1OO,
_L
Treatment with unsaturated free fatty acids Various unsaturated free fatty acids were dissolved in chloroform and respective aliquots were taken into centrifuge tubes. The chloroform was then completely removed with a stream of N z gas. The fatty acids on the inner surface of the tube were homogenized in membrane solution (1 mg protein/ml) with a polytron, at a power control setting of 5, for 20 sec. The homogenate was centrifuged at 20,000g for 20 min. The pellets were resuspended in Buffer B and used as unsaturated free fatty acid-treated membrane preparations.
Do•• D• • • D•=• me% P•%
.E 75.
E: ¢D
It% •1% •e • • D• I•%
oo.
=•==
~.~
25-
Protein determination Protein concentration was measured by the method of Lowry et al. (1951) with BSA as the standard.
== • =
B-o I ,ti.• o• ¶nI o g =o=gl A2
C
D
Control. PhosphoLipose
Fig. 1. Effect of pretreatment with various phospholipases on [3H]naloxone binding to rat brain membranes. Brain membranes were preincubated with 0.05 unit of phospholipase (Plase) A 2, C or D/mg membrane protein at 37°C for 5 min in the presence of 2 mM CaCI2. After removal of enzymes by washing with buffer solution, determination of [3H]naloxone binding to the membranes was performed as described in Materials and Methods. Each value represents the m e a n + S E M obtained from 3 separate experiments examined in triplicate.
the final centrifugation, the resulting pellet was resuspended in 20 vols of Buffer B. Half-milliliter aliquots of the membranes were incubated with [3H]naloxone (1 nM) in the absence or presence of I # M levallorphane for 60min at 0°C. The final volume of the medium was 1 ml. After incubation, the reaction mixtures were vacuum-filtered through Whatman GF/B filters. The filters were then rinsed with 2.5 ml of cold Buffer A four times, and dried and counted in Triton X-100-toluene scintillation cocktail using a liquid scintillation counter. Specific binding was determined as the difference between the total binding and the binding in the presence of unlabelled levallorphane (1 # M). All procedures were carried out at 0-4°C.
Treatment with phospholipases The membrane preparations were diluted to a concentration of 1 mg protein/ml with Buffer B and aliquots were incubated with or without 0.05 unit of each phospholipase/ mg membrane protein in the presence of 2 mM CaC12 at 37°C (or 24°C in the case of bullfrog brain membrane). The reaction was then stopped by chilling the incubation mixture in an ice-bath, and the enzymes were removed by centrifugation at 20,000g for 20 min. The pellets were resuspended in the same volume of Buffer B and recentrifuged. Then, the resulting pellets were resuspended in the same buffer. These suspensions were used as the phospholipase-treated or control membrane preparations. Phospholipase-treated and control membranes were incubated with or without 0.1-0.5% BSA. After incubation at 37°C for 5 min, the membranes were chilled, centrifuged at 20,000 g for 20 min, resuspended in the same buffer and centrifuged again. Finally, the pellets were resuspended in the same volume of Buffer B and the levels of stereo-specific [3H]naloxone binding to the phospholipase-treated membranes and their controls were determined as described above. Treatment with detergents Various detergents were incubated with the membrane at 37°C for 5 min. After incubation, the membranes were treated in the same way as for the phospholipase experiment.
RESULTS
Effect o f phospholipase pretreatment on [3H]naloxone binding W h e n the m e m b r a n e s were treated with 0.05 unit p h o s p h o l i p a s e A2/mg m e m b r a n e protein at 37°C for 5 min, opioid receptor binding to [3H]naloxone was very sensitive to the action o f the enzyme a n d was drastically inhibited by between 10 a n d 20%. However, the receptor binding was considerably less sensitive to t r e a t m e n t with p h o s p h o l i p a s e C or D (Fig. 1). In order to clarify these inhibitory effects, we examined the action of added BSA. T h e a d d i t i o n o f 0.1% BSA simultaneously with Plase A 2 to the reaction m e d i u m a l m o s t completely prevented the inhibition o f opioid receptor activity (Fig. 2). Brain m e m b r a n e s treated with BSA at 37°C for 5 min, following Plase A 2 treatment, d e m o n s t r a t e d extensive recovery of opioid binding activity, a n d the potency o f recovery d e p e n d i n g o n the c o n c e n t r a t i o n o f BSA a d d e d (Fig. 2). Since 0.5% BSA p r o d u c e d almost complete recovery, we used this c o n c e n t r a t i o n o f BSA in s u b s e q u e n t experiments.
100.
"o~
[]
Phosphol.ipase
[]
PhosphoLipase A 2 + B S A
A2
[]
BSA
75.
50'
~
25.
0.0
0.1
r5
o.o o.1
O.Z 0.5
0.1
BSA IFraction'~;TI (%1 Co- incubation Subsequentincubation Fig. 2 Prevention and abolition of Plase A:-induced inhibition of [3H]naloxone binding with BSA. Coincubation: brain membranes were co-incubated with Plase A: (0.05 unit/rag protein) and 0.1% BSA. Subsequent incubation: brain membranes were incubated at 37°C for 5 rain with the indicated concentrations of BSA following Plase A 2 treatment, as described in legend to Fig. l. Each
value represents the mean + SEM from 3 separate experiments examined in triplicate.
Opioid binding and phospholipases
709
r--i Phosphotipose A 2 100.
~
~]
PhosphoLipose A 2 + BSA
7~.
~ ~. _q~~ o Z~
25-
0
0
5
20
30
90
PhosphoLipase A 2 treatment time (min)
Fig. 3. Time course of Plase A2-induced inhibition of [3H]naloxone binding and restorative effect of BSA. The conditions used were the same as for subsequent BSA treatment described in the legend to Fig. 2, except that the concentration of BSA was 0.5%. Each value represents the mean +SEM obtained from 3 separate experiments examined in triplicate.
After the membranes has been incubated with Plase A2 at 37°C for more than 20 min, the inhibitory effect of Plase A2 was slightly increased, and could not be abolished by BSA (Fig. 3). This result showed that the inhibition caused by short-term Plase A2 treatment could be reversibly abolished, but that treatment for a longer period produced an irreversible inhibitory state. In order to examine whether any fatty acid contaminants were present in the commericai BSA (Fraction V), the effect of fatty acid-free BSA in restoring receptor activity was compared with that of the BSA (V). The fatty acid-free BSA dosedependently elicited the same results as the BSA (V). To determine whether BSA was able to specifically abolish Plase A2-induced inhibition, the effects of 0.1-0.2% ovalbumin (egg albumin) and 0.05-0.5% human y-globulin were examined. However, no restoration of opioid binding activity by ovalbumin or y-globulin could be demonstrated (data not !
100 -
75-
]
BSA (-)
[]
BSA.(+)
shown), suggesting that the restorative effect was specific for BSA.
Effect of pretreatment with lysolecithin, Triton X-100 and digitonin It is well known that various detergents which are presumed to remove lipids from membranes, reduce opioid binding. Lysophosphatides, the products of Plase A2, also have a marked detergent-like action. Therefore, the effects of lysolecithin, Triton X-100 and digitonin on [3H]naloxone binding instead of Plase A2 were examined. After incubation of 0.02-0.05% lysolecithin at 37°C for 5 min with brain membranes, opioid binding activity decreased dose-dependently, but no restorative effect upon subsequent incubation with BSA could be demonstrated, even though the degree of inhibition was slight (Fig. 4). Other detergents, 0.02% Triton X-100 and 0.05% digitonin, also strikingly reduced [3H]naioxone binding. Although prevention of opioid binding was shown when the membranes were incubated with Triton X-100 in the presence of BSA, subsequent BSA treatment did not completely restore opioid binding. Similarly, digitonin-treated samples did not show any recovery of binding upon subsequent treatment with BSA (Fig. 5).
Influence of free fatty acids on opioid receptor binding Since the restorative effect of BSA was demonstrated only following short-term treatment with Plase A2, it seemed possible that the products of the enzymes might be responsible for reversible inhibi~ 25tion. However, the inhibitory effect of lysolecithin could not be abolished by BSA. Therefore, other products, i.e. unsaturated free fatty acids, were o ~ ~ 0.02 0.03 0.05 investigated with respect to receptor binding. When LyeoLecithin ( % ) the membranes were incubated with oleic acid (18: 1), linoleic acid (18:2), iinolenic acid (18:3) and Fig. 4. Effect of pretreatmcnt with lysolecithin on [3H]nal- arachidonic acid (20:4) as previously described, the oxone binding. Lysoleeithin treatment was essentially the same as described for Fig. 3 except that various concen- receptor binding was not influenced by oleic acid. trations of lysoleeithin were used instead of Plase A2 for However, the other fatty acids (0.1 mM), with more 5 rain. Each value represents the. mean+SEM obtained than two unsaturated bonds, inhibited receptor binding to 25-40% of the control levels (Fig. 6). The from 3 separate experiments examined in triplicate. 50.
710
MizuE MAKIMURAet al. 100.
I-1 ~s, ( , []BSA
.E
75-
o~ §
50.
"r 25. 0
100
(+)
~
nJ
N
\
§
~
50
m~
0
L./~l 0 10.5
C o - incubation Subsequent incubation Triton X - 1OO Digitonin
Fig. 5. Effect ofpretrcatment with Triton X-IOO or digitonin on [3H]naloxone binding. The tested concentrations o f T r i t o n X - t 0 0 and digitonin were 0.02% and 0.05%, respectively. These treatments were the same as for lyso-
°\o
\,
I 10.4 10.3 Concentration of orochidonic ccid (M)
Fig. 7. Inhibitory effect of arachidonic acid on [3H]naloxone binding. Various concentrations of arachidonic acid were preincubated with the membranes as described in Materials and Methods.
lecithin. 1984). We compared the effect of Plase A 2 treatment on bullfrog brain membranes with that produced by treatment with this enzyme of rat brain membranes in which [3H]naloxone was shown to bind mainly to the/~-receptor (Chang et al., 1981). The [3H]naloxone binding to bullfrog brain membranes was reduced by treatment with 0.05 unit Plase A2/mg protein and the short-term inhibitory effect of Plase A2 was abolished by subsequent treatment with BSA (Fig. 8). These results suggest that no differences may exist in the inhibitory effect of Piase A2 between p- and k-type receptors.
inhibitory effect of arachidonic acid, which is the main component of unsaturated fatty acids in the cell membrane, was demonstrated to be dose-dependent (Fig. 7). The restoration of binding with BSA after treatment with unsaturated fatty acids such as linoleic acid, linolenic acid and arachidonic acid was weak (Fig. 6). Cardiolipin (0.025%), which is a highly unsaturated lipid, diphosphatidylglycerol, reduced the degree of receptor binding to 10% of the control level, and its inhibition was partially abolished by BSA (Fig. 6). Effect o f Plase A 2 pretreatment on bullfrog brain membranes
DISCUSSION
Our previous report showed that k-type receptors were predominant among the opioid receptors present in bullfrog brain membranes, and that [3H]naloxone bound mainly to these receptors (Ito et al.,
It was demonstrated in the present study that inhibition of opioid binding by short-term Plase A2 treatment was abolished by subsequent treatment with
r=-]
BsA ( - )
[~
BSA (+)
100 -
m 50-
25-
O
OLeic acid (10 -4 M]
_inoLeic acid ( 10"4 M )
LinoLenic acid ( 10 -4 M )
Arachidonic acid
( 10 -4 M ]
( IO -3 M )
CordioLipin
{ 0.025 %)
Fig. 6. Effect of pretreatment with various unsaturated free fatty acids and cardiolipin on [3H]naloxone binding. These substances were preincubated with the membrane according to the description in Materials and Methods.
Opioid binding and phospholipases
/
200 ]
BSA(+)
g c 150 .c .~
o
,oo]t
eee e o, oee
z~ .:° ate
o Control
PhosphoLipase A2 BSA
Fig. 8. Plase A2-induced inhibition of [3H]naloxone binding to bullfrog brain membranes, and its abolition with BSA. Plase A 2 was incubated with the bullfrog brain membranes under the same conditions as described in the legend to Fig. 3 except that the enzyme reaction was for 5 min at 24°C.
BSA, whereas no abolition of inhibition occurred following long-term treatment (Fig. 3). Opioid binding to the membrane was markedly diminished by prior treatment of the membrane with Plase A 2 but only slightly with phospholipase C or D (Fig. 1). It was assumed that the inhibitory effect on receptor binding was dependent on iysophosphatides and unsaturated free fatty acids which were produced, and/or on a decrease in the level of phospholipids in the environment around the receptor by Plase A2. When these enzymatic products were presumably not excessive, their binding inhibition may have been reversibly removed by BSA, but not upon washing with buffer solution. It therefore appears that this inhibition is not due to the liberation of phospholipids required for binding activity. After the membranes had been treated with Plase A 2 for 5 min, 0.1% BSA was able to partially abolish the inhibition of opioid binding activity, and opioid binding was almost completely protected when the membranes were simultaneously incubated with Plase A z and BSA (Fig. 2). However, the inhibition observed under the same conditions as those mentioned above was excluded almost completely when the concentration of BSA was increased to 0.5% (Fig. 2). If the lipids required for binding had been removed during the enzymatic reaction or subsequent washing of the membrane, the degree of the observed restorative effect ought not to have been altered. In fact, the restoration of binding activity by BSA was demonstrated to be dose-dependent (Fig. 2). It appears that since these enzyme products have lipid solubility, they become bound to the hydrophobic site and/or the lipids on the receptor protein, and thus inhibit opioid binding until their removal by BSA. For these reasons, we postulate that a receptor in a state where it is temporarily unable to bind ligands shows reversible binding inhibition. This hypothesis would support the results of a previous study that endogenous products formed by an enzyme may exert a strong inhibitory effect on opioid receptors at much lower concentrations than those of exogenously added materials (Lin and Simon, 1978).
711
On the other hand, as shown in Fig. 3, the opioid binding activity did not completely disappear when the membranes were exposed to Plase A2 for longer that 20 min. In this case, the binding activity could no longer be restored, even when the concentration of BSA was increased to 0.5%. This irreversible inhibition suggests that the products of the enzyme increase to an extent sufficient for micelle formation, subsequently eliciting the liberation from the receptor of lipids which are essential for binding. In the case of exogenous treatment with lysolecithin and unsaturated free fatty acids (Figs 4 and 6), as the membranes were exposed to high concentrations of these substances in sonicated suspensions, the BSAinduced abolition of opioid binding inhibition was not complete, even though the degree of inhibition was slight. It seems that this inhibition was produced by liberation of lipids from the receptor by the same mechanism as that occurring with long-term enzymatic treatment; Triton X-100 and digitonin also produced inhibition in the same manner (Fig. 5). Our proposal that the liberation of phospholipids from the receptors results in irreversible inhibition of opioid binding, which is unaffected by addition of BSA, is in agreement with some reports that the receptors responsible for the binding of opioid require a number of lipids for the maintenance of steric structure (Cho et al., 1983; Farahbakhsh et al., 1986; Maruyama, 1987 and Remmers and Medzihradsky, 1987). Although it has been reported that [3H]naloxone binds mainly to/~-type receptors on rat brain membranes (Chang et al., 1981), we have already observed that [3H]naloxone binds chiefly to k-type receptors on bullfrog brain membranes (Ito et al., 1984). Plase A2 inhibits opioid binding to receptors of both types in almost the same manner. Therefore, such inhibitory actions are not considered to be due to differences in receptor type or species. Plase A 2 in the immediate vicinity of the opioid receptor is activated by some form of stimulation, producing lysophosphatides and unsaturated free fatty acids. The inhibitory effect on opioid binding by these products might induce a reduction in the pain threshold. On the basis of the data presented here, it is concluded that the reversible inhibition of opioid binding is dependent on the binding of a small amount of Plase A2 products either to the receptors or to structures in their immediate vicinity, resulting in a conformation which is inactive for receptor binding, whereas irreversible inhibition results from the liberation of phospholipids from receptors by Plase A 2 products and detergents.
REFERENCES
Abood L. G. and Hoss W. (1975) Stereospecific morphine adsorption to phosphatidyl serine and other membranous components of brain. Eur. J. Pharmac. 32, 66-75. Abood L. G. and Takeda F. (1976) Enhancement of stereospecific opiate binding to neural membrane by phosphatidyl serine. Eur. J. Pharmac. 39, 71-77. Abood L. G., Salem N., MacNeil M., Bloom L. and Abood M. E. (1977) Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by
712
MIZUE MAK1MURAet al.
other unsaturated lipids. Biochim. biophys. Acta 468(1), 51-62. Abood L. G., Salem N., MacNeil M. and Butler M. (1978) Phospholipid changes in synaptic membranes by lipolytic enzymes and subsequent restoration of opiate binding with phosphatidylserine. Biochim. biophys. Acta. 530(1), 35-46. Abood L. G., Butler M. and Reynolds D. (1980) Effect of calcium and physical state of neutral membranes on phosphatidylserine requirement for opiate binding. Molec. Pharmac. 17(3), 290-294. Chang K. J., Hazum E. and Cautrecasas P. (1981) Novel opiate binding sites selective for benzomorphan drugs. Proc. natn. Acad. Sci. U.S.A. 78, 4141-4145. Cho T. M., Ge B. L., Yamato C., Smith A. P. and Loh H. H. (1983) Isolation of opiate binding components by atfinity chromatography and re-constitution of binding activities. Proc. natn. Acad. Sci. U.S.A. 80, 5176-5180. Farahbakhsh Z. T., Deamer D. W., Lee N. M. and Loh H. H. (1986) Enzymatic reconstitution of brain membrane and membrane opiate receptors. J. Neurochem. 46, 953-962. Ito Y., Makimura M. and Murakoshi Y. (1984) Some properties of opioid receptors in membrane-bound and solubilized state. Neuropeptides 5, 193 196. Lin H. K. and Simon E. J. (1978) Phospholipase A inhibition of opiate receptor binding can be reversed by albumin. Nature 271, 383-384. Loh H. H. and Law P. Y. (1980) The role of membrane
lipids in receptor mechanisms. A. Rev. Pharmac. 20, 201-234. Loh H. H., Cho T. M., Wu Y. C. and Way E. L. (1974) Stereospecific binding of narcotics to brain cerebrosides. Life Sci. 14(11), 2231-2245. Lowney L. I., Schulz R., Lowry K. and Goldstein A. (1974) Partial purification of an opiate receptor from mouse brain. Science 183, 749 753. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Maruyama M., Sugino H., Akita K. and Hatanaka H. (1987) Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCI: the recovery of binding activity by dilution. Brain Res. 401, 14~22. Pasternak G. W. and Snyder S. H. (1974) Opiate receptor binding: effects of enzymatic treatments. Molec. Pharmac. 10, 183-193. Remmers A. E. and Medzihradsky F. (1987) Modulation of ligand binding to opioid receptors by membrane lipids. Abstr. Int. Narcotic Res Conf. in Adelaide. p. 64.
Riiegg U. T., Cuenoud S., Fulpius B. W. and Simon E. J. (1982) Inactivation and solubilization of opiate receptors by phospholipase A 2. Biochim. biophys. Acta 685, 241 248. Wu Y. C., Cho T. M., Loh H. H. and Way E. L. (1976) Binding of narcotics and narcotic antagonists to triphosphoinositide. Biochem. Pharmac. 25, 1551 1553.
0306-3623/88 $3.00+ 0.00 Copyright © 1988 PergamonPress plc
INHIBITORY MECHANISM OF OPIOID BINDING TO RECEPTOR BY PHOSPHOLIPASE A2 MIZUE MAKIMURA,* YOSHIHISA ITO and Yosnm MURAKOSHI Department of Pharmacy, College of Science and Technology, Nihon University, Kanda-surugadai 1-8, Chiyoda-ku, Tokyo I01, Japan (Received 2 December 1987)
Abstract--1. The inhibition of [3H]naloxone binding to the opioid receptor upon short-term incubation with phospholipase A2 (Plase A2) was abolished by treatment with BSA, but not after long-term incubation. 2. In contrast to the restorative effect of BSA on the strong inhibition occurring with Plase A2, BSA only partially abolished the inhibitory effect of arachidonic acid or lysophosphatides, even though the degree of inhibition was slight. 3. These results suggest that Plase A2 products only become associated with hydrophobic receptor sites or with phospholipids near to the receptor, thus reversibly inhibiting opioid binding, and that irreversible inhibition occurs through release of the phospholipid necessary for receptor binding.
INTRODUCTION It is well known that the properties of membrane receptors change according to local environmental conditions. It has also been shown that lipids in membranes play a role in the interaction of various ligands with their respective receptors (Loh and Law, 1980). In the case of the opioid receptor, Lowney et al. (1974) initially described it as consisting of proteolipids, and since then many investigators have proposed that the lipid is cerebroside sulfate (Loh et aL, 1974) and that the main lipid in the opioid receptor is phosphatidylserine (Abood and Hoss, 1975) and/or phosphatidylinositol (Wu et al., 1976). More recently, a considerable amount of evidence has suggested that lipids play a very significant role in ligand binding sites (Abood et al., 1978; Abood et al., 1980). Furthermore, it is believed that opioidreceptor proteins are associated with phospholipids, thereby maintaining their sterical structure within the membrane. In order to elucidate these possibilities, Pasternak and Snyder (1974) examined the influence of a variety of enzymes on opioid receptor binding and observed a remarkable reduction of opioid binding upon treatment with phospholipases. The reduction of receptor binding after pretreatment with phospholipase A 2 (Plase A2) and also that following mechanical disruption using methods such as freezethawing and sonication have also been shown to be abolished by the addition of phosphatidylserine and bovine serum albumin (BSA), the inhibition being abolished more strongly by BSA than by phosphatidylserine (Abood et al., 1978; Abood et al., 1980). Plase A2-induced inhibition of opioid receptor binding has also been shown to be reversible upon addition of albumin (Lin and Simon, 1978). It has been reported that Plase A2-induced inhibition of opioid *Author to whom correspondence should be addressed.
binding might be due to the products of phospholipolysis, i.e. unsaturated free fatty acids and lysophosphatides (Abood et al., 1977; Lin and Simon, 1978; Rfiegg et al., 1982). However, the details of the inhibitory mechanism of Plase A2 on opioid binding are not yet clear. In the present study, the inhibitory effect of Plase A2 and the properties of the opioid receptor were explored with special reference to the lipids existing in the membranes. The results are reported in this paper, together with a discussion of the possible physiological function of Plase A2. MATERIALS AND METHODS Chemicals
The following drugs were purchased from Sigma Chemical Co. (St. Louis, Mo): phospholipase A 2 (phosphatide 2acylhydrolase: EC 3.1.1.4., from bee venom), phospholipase C (phosphatidylcholine cholinephosphohydrolase: EC 3.1.4.3., from Bacillus cereus), phospholipase D (phosphatidylcholine phosphatidohydrolase: EC 3.1.4.4., from cabbage), oleic acid, linoleic acid, linolenic acid, arachidonic acid, lysolecithin, cardiolipin and fatty acid-free bovine serum albumin. [3H]naloxone, ([N-Allyl-2,3-3H]-, specific activity 80 Ci/mmol) was purchased from New England Nuclear (Boston, Mass). Prostaglandin (PG) E t , E2, F2~and 12 were generously donated by Onoyakuhin Kogyo Co. (Osaka, Japan). Other reagents (analytical grade) were obtained commercially from Wako Pure Chemicals (Tokyo, Japan). Receptor binding assay
Male Wistar rats (150-180g body wt) were killed by decapitation, the brains excised and the cerebella removed. Bullfrog brain membranes were prepared using whole brains as described previously (Ito et al., 1984). Isolated brains were homogenized in 20 vols of 50 mM Tris buffer (pH 7.4, Buffer A) with a polytron (Kinematica), at a power control setting of 6, for 30 see. The homogenate was centrifuged at 20,000 g for 20 rain. The pellet was washed once with Buffer A, and then twice with 50 mM Tris buffer containing 0.5 mM EGTA (Buffer B), using the above procedure. After 707
MIZUE MAK1MURAet al.
708
1OO,
_L
Treatment with unsaturated free fatty acids Various unsaturated free fatty acids were dissolved in chloroform and respective aliquots were taken into centrifuge tubes. The chloroform was then completely removed with a stream of N z gas. The fatty acids on the inner surface of the tube were homogenized in membrane solution (1 mg protein/ml) with a polytron, at a power control setting of 5, for 20 sec. The homogenate was centrifuged at 20,000g for 20 min. The pellets were resuspended in Buffer B and used as unsaturated free fatty acid-treated membrane preparations.
Do•• D• • • D•=• me% P•%
.E 75.
E: ¢D
It% •1% •e • • D• I•%
oo.
=•==
~.~
25-
Protein determination Protein concentration was measured by the method of Lowry et al. (1951) with BSA as the standard.
== • =
B-o I ,ti.• o• ¶nI o g =o=gl A2
C
D
Control. PhosphoLipose
Fig. 1. Effect of pretreatment with various phospholipases on [3H]naloxone binding to rat brain membranes. Brain membranes were preincubated with 0.05 unit of phospholipase (Plase) A 2, C or D/mg membrane protein at 37°C for 5 min in the presence of 2 mM CaCI2. After removal of enzymes by washing with buffer solution, determination of [3H]naloxone binding to the membranes was performed as described in Materials and Methods. Each value represents the m e a n + S E M obtained from 3 separate experiments examined in triplicate.
the final centrifugation, the resulting pellet was resuspended in 20 vols of Buffer B. Half-milliliter aliquots of the membranes were incubated with [3H]naloxone (1 nM) in the absence or presence of I # M levallorphane for 60min at 0°C. The final volume of the medium was 1 ml. After incubation, the reaction mixtures were vacuum-filtered through Whatman GF/B filters. The filters were then rinsed with 2.5 ml of cold Buffer A four times, and dried and counted in Triton X-100-toluene scintillation cocktail using a liquid scintillation counter. Specific binding was determined as the difference between the total binding and the binding in the presence of unlabelled levallorphane (1 # M). All procedures were carried out at 0-4°C.
Treatment with phospholipases The membrane preparations were diluted to a concentration of 1 mg protein/ml with Buffer B and aliquots were incubated with or without 0.05 unit of each phospholipase/ mg membrane protein in the presence of 2 mM CaC12 at 37°C (or 24°C in the case of bullfrog brain membrane). The reaction was then stopped by chilling the incubation mixture in an ice-bath, and the enzymes were removed by centrifugation at 20,000g for 20 min. The pellets were resuspended in the same volume of Buffer B and recentrifuged. Then, the resulting pellets were resuspended in the same buffer. These suspensions were used as the phospholipase-treated or control membrane preparations. Phospholipase-treated and control membranes were incubated with or without 0.1-0.5% BSA. After incubation at 37°C for 5 min, the membranes were chilled, centrifuged at 20,000 g for 20 min, resuspended in the same buffer and centrifuged again. Finally, the pellets were resuspended in the same volume of Buffer B and the levels of stereo-specific [3H]naloxone binding to the phospholipase-treated membranes and their controls were determined as described above. Treatment with detergents Various detergents were incubated with the membrane at 37°C for 5 min. After incubation, the membranes were treated in the same way as for the phospholipase experiment.
RESULTS
Effect o f phospholipase pretreatment on [3H]naloxone binding W h e n the m e m b r a n e s were treated with 0.05 unit p h o s p h o l i p a s e A2/mg m e m b r a n e protein at 37°C for 5 min, opioid receptor binding to [3H]naloxone was very sensitive to the action o f the enzyme a n d was drastically inhibited by between 10 a n d 20%. However, the receptor binding was considerably less sensitive to t r e a t m e n t with p h o s p h o l i p a s e C or D (Fig. 1). In order to clarify these inhibitory effects, we examined the action of added BSA. T h e a d d i t i o n o f 0.1% BSA simultaneously with Plase A 2 to the reaction m e d i u m a l m o s t completely prevented the inhibition o f opioid receptor activity (Fig. 2). Brain m e m b r a n e s treated with BSA at 37°C for 5 min, following Plase A 2 treatment, d e m o n s t r a t e d extensive recovery of opioid binding activity, a n d the potency o f recovery d e p e n d i n g o n the c o n c e n t r a t i o n o f BSA a d d e d (Fig. 2). Since 0.5% BSA p r o d u c e d almost complete recovery, we used this c o n c e n t r a t i o n o f BSA in s u b s e q u e n t experiments.
100.
"o~
[]
Phosphol.ipase
[]
PhosphoLipase A 2 + B S A
A2
[]
BSA
75.
50'
~
25.
0.0
0.1
r5
o.o o.1
O.Z 0.5
0.1
BSA IFraction'~;TI (%1 Co- incubation Subsequentincubation Fig. 2 Prevention and abolition of Plase A:-induced inhibition of [3H]naloxone binding with BSA. Coincubation: brain membranes were co-incubated with Plase A: (0.05 unit/rag protein) and 0.1% BSA. Subsequent incubation: brain membranes were incubated at 37°C for 5 rain with the indicated concentrations of BSA following Plase A 2 treatment, as described in legend to Fig. l. Each
value represents the mean + SEM from 3 separate experiments examined in triplicate.
Opioid binding and phospholipases
709
r--i Phosphotipose A 2 100.
~
~]
PhosphoLipose A 2 + BSA
7~.
~ ~. _q~~ o Z~
25-
0
0
5
20
30
90
PhosphoLipase A 2 treatment time (min)
Fig. 3. Time course of Plase A2-induced inhibition of [3H]naloxone binding and restorative effect of BSA. The conditions used were the same as for subsequent BSA treatment described in the legend to Fig. 2, except that the concentration of BSA was 0.5%. Each value represents the mean +SEM obtained from 3 separate experiments examined in triplicate.
After the membranes has been incubated with Plase A2 at 37°C for more than 20 min, the inhibitory effect of Plase A2 was slightly increased, and could not be abolished by BSA (Fig. 3). This result showed that the inhibition caused by short-term Plase A2 treatment could be reversibly abolished, but that treatment for a longer period produced an irreversible inhibitory state. In order to examine whether any fatty acid contaminants were present in the commericai BSA (Fraction V), the effect of fatty acid-free BSA in restoring receptor activity was compared with that of the BSA (V). The fatty acid-free BSA dosedependently elicited the same results as the BSA (V). To determine whether BSA was able to specifically abolish Plase A2-induced inhibition, the effects of 0.1-0.2% ovalbumin (egg albumin) and 0.05-0.5% human y-globulin were examined. However, no restoration of opioid binding activity by ovalbumin or y-globulin could be demonstrated (data not !
100 -
75-
]
BSA (-)
[]
BSA.(+)
shown), suggesting that the restorative effect was specific for BSA.
Effect of pretreatment with lysolecithin, Triton X-100 and digitonin It is well known that various detergents which are presumed to remove lipids from membranes, reduce opioid binding. Lysophosphatides, the products of Plase A2, also have a marked detergent-like action. Therefore, the effects of lysolecithin, Triton X-100 and digitonin on [3H]naloxone binding instead of Plase A2 were examined. After incubation of 0.02-0.05% lysolecithin at 37°C for 5 min with brain membranes, opioid binding activity decreased dose-dependently, but no restorative effect upon subsequent incubation with BSA could be demonstrated, even though the degree of inhibition was slight (Fig. 4). Other detergents, 0.02% Triton X-100 and 0.05% digitonin, also strikingly reduced [3H]naioxone binding. Although prevention of opioid binding was shown when the membranes were incubated with Triton X-100 in the presence of BSA, subsequent BSA treatment did not completely restore opioid binding. Similarly, digitonin-treated samples did not show any recovery of binding upon subsequent treatment with BSA (Fig. 5).
Influence of free fatty acids on opioid receptor binding Since the restorative effect of BSA was demonstrated only following short-term treatment with Plase A2, it seemed possible that the products of the enzymes might be responsible for reversible inhibi~ 25tion. However, the inhibitory effect of lysolecithin could not be abolished by BSA. Therefore, other products, i.e. unsaturated free fatty acids, were o ~ ~ 0.02 0.03 0.05 investigated with respect to receptor binding. When LyeoLecithin ( % ) the membranes were incubated with oleic acid (18: 1), linoleic acid (18:2), iinolenic acid (18:3) and Fig. 4. Effect of pretreatmcnt with lysolecithin on [3H]nal- arachidonic acid (20:4) as previously described, the oxone binding. Lysoleeithin treatment was essentially the same as described for Fig. 3 except that various concen- receptor binding was not influenced by oleic acid. trations of lysoleeithin were used instead of Plase A2 for However, the other fatty acids (0.1 mM), with more 5 rain. Each value represents the. mean+SEM obtained than two unsaturated bonds, inhibited receptor binding to 25-40% of the control levels (Fig. 6). The from 3 separate experiments examined in triplicate. 50.
710
MizuE MAKIMURAet al. 100.
I-1 ~s, ( , []BSA
.E
75-
o~ §
50.
"r 25. 0
100
(+)
~
nJ
N
\
§
~
50
m~
0
L./~l 0 10.5
C o - incubation Subsequent incubation Triton X - 1OO Digitonin
Fig. 5. Effect ofpretrcatment with Triton X-IOO or digitonin on [3H]naloxone binding. The tested concentrations o f T r i t o n X - t 0 0 and digitonin were 0.02% and 0.05%, respectively. These treatments were the same as for lyso-
°\o
\,
I 10.4 10.3 Concentration of orochidonic ccid (M)
Fig. 7. Inhibitory effect of arachidonic acid on [3H]naloxone binding. Various concentrations of arachidonic acid were preincubated with the membranes as described in Materials and Methods.
lecithin. 1984). We compared the effect of Plase A 2 treatment on bullfrog brain membranes with that produced by treatment with this enzyme of rat brain membranes in which [3H]naloxone was shown to bind mainly to the/~-receptor (Chang et al., 1981). The [3H]naloxone binding to bullfrog brain membranes was reduced by treatment with 0.05 unit Plase A2/mg protein and the short-term inhibitory effect of Plase A2 was abolished by subsequent treatment with BSA (Fig. 8). These results suggest that no differences may exist in the inhibitory effect of Piase A2 between p- and k-type receptors.
inhibitory effect of arachidonic acid, which is the main component of unsaturated fatty acids in the cell membrane, was demonstrated to be dose-dependent (Fig. 7). The restoration of binding with BSA after treatment with unsaturated fatty acids such as linoleic acid, linolenic acid and arachidonic acid was weak (Fig. 6). Cardiolipin (0.025%), which is a highly unsaturated lipid, diphosphatidylglycerol, reduced the degree of receptor binding to 10% of the control level, and its inhibition was partially abolished by BSA (Fig. 6). Effect o f Plase A 2 pretreatment on bullfrog brain membranes
DISCUSSION
Our previous report showed that k-type receptors were predominant among the opioid receptors present in bullfrog brain membranes, and that [3H]naloxone bound mainly to these receptors (Ito et al.,
It was demonstrated in the present study that inhibition of opioid binding by short-term Plase A2 treatment was abolished by subsequent treatment with
r=-]
BsA ( - )
[~
BSA (+)
100 -
m 50-
25-
O
OLeic acid (10 -4 M]
_inoLeic acid ( 10"4 M )
LinoLenic acid ( 10 -4 M )
Arachidonic acid
( 10 -4 M ]
( IO -3 M )
CordioLipin
{ 0.025 %)
Fig. 6. Effect of pretreatment with various unsaturated free fatty acids and cardiolipin on [3H]naloxone binding. These substances were preincubated with the membrane according to the description in Materials and Methods.
Opioid binding and phospholipases
/
200 ]
BSA(+)
g c 150 .c .~
o
,oo]t
eee e o, oee
z~ .:° ate
o Control
PhosphoLipase A2 BSA
Fig. 8. Plase A2-induced inhibition of [3H]naloxone binding to bullfrog brain membranes, and its abolition with BSA. Plase A 2 was incubated with the bullfrog brain membranes under the same conditions as described in the legend to Fig. 3 except that the enzyme reaction was for 5 min at 24°C.
BSA, whereas no abolition of inhibition occurred following long-term treatment (Fig. 3). Opioid binding to the membrane was markedly diminished by prior treatment of the membrane with Plase A 2 but only slightly with phospholipase C or D (Fig. 1). It was assumed that the inhibitory effect on receptor binding was dependent on iysophosphatides and unsaturated free fatty acids which were produced, and/or on a decrease in the level of phospholipids in the environment around the receptor by Plase A2. When these enzymatic products were presumably not excessive, their binding inhibition may have been reversibly removed by BSA, but not upon washing with buffer solution. It therefore appears that this inhibition is not due to the liberation of phospholipids required for binding activity. After the membranes had been treated with Plase A 2 for 5 min, 0.1% BSA was able to partially abolish the inhibition of opioid binding activity, and opioid binding was almost completely protected when the membranes were simultaneously incubated with Plase A z and BSA (Fig. 2). However, the inhibition observed under the same conditions as those mentioned above was excluded almost completely when the concentration of BSA was increased to 0.5% (Fig. 2). If the lipids required for binding had been removed during the enzymatic reaction or subsequent washing of the membrane, the degree of the observed restorative effect ought not to have been altered. In fact, the restoration of binding activity by BSA was demonstrated to be dose-dependent (Fig. 2). It appears that since these enzyme products have lipid solubility, they become bound to the hydrophobic site and/or the lipids on the receptor protein, and thus inhibit opioid binding until their removal by BSA. For these reasons, we postulate that a receptor in a state where it is temporarily unable to bind ligands shows reversible binding inhibition. This hypothesis would support the results of a previous study that endogenous products formed by an enzyme may exert a strong inhibitory effect on opioid receptors at much lower concentrations than those of exogenously added materials (Lin and Simon, 1978).
711
On the other hand, as shown in Fig. 3, the opioid binding activity did not completely disappear when the membranes were exposed to Plase A2 for longer that 20 min. In this case, the binding activity could no longer be restored, even when the concentration of BSA was increased to 0.5%. This irreversible inhibition suggests that the products of the enzyme increase to an extent sufficient for micelle formation, subsequently eliciting the liberation from the receptor of lipids which are essential for binding. In the case of exogenous treatment with lysolecithin and unsaturated free fatty acids (Figs 4 and 6), as the membranes were exposed to high concentrations of these substances in sonicated suspensions, the BSAinduced abolition of opioid binding inhibition was not complete, even though the degree of inhibition was slight. It seems that this inhibition was produced by liberation of lipids from the receptor by the same mechanism as that occurring with long-term enzymatic treatment; Triton X-100 and digitonin also produced inhibition in the same manner (Fig. 5). Our proposal that the liberation of phospholipids from the receptors results in irreversible inhibition of opioid binding, which is unaffected by addition of BSA, is in agreement with some reports that the receptors responsible for the binding of opioid require a number of lipids for the maintenance of steric structure (Cho et al., 1983; Farahbakhsh et al., 1986; Maruyama, 1987 and Remmers and Medzihradsky, 1987). Although it has been reported that [3H]naloxone binds mainly to/~-type receptors on rat brain membranes (Chang et al., 1981), we have already observed that [3H]naloxone binds chiefly to k-type receptors on bullfrog brain membranes (Ito et al., 1984). Plase A2 inhibits opioid binding to receptors of both types in almost the same manner. Therefore, such inhibitory actions are not considered to be due to differences in receptor type or species. Plase A 2 in the immediate vicinity of the opioid receptor is activated by some form of stimulation, producing lysophosphatides and unsaturated free fatty acids. The inhibitory effect on opioid binding by these products might induce a reduction in the pain threshold. On the basis of the data presented here, it is concluded that the reversible inhibition of opioid binding is dependent on the binding of a small amount of Plase A2 products either to the receptors or to structures in their immediate vicinity, resulting in a conformation which is inactive for receptor binding, whereas irreversible inhibition results from the liberation of phospholipids from receptors by Plase A 2 products and detergents.
REFERENCES
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712
MIZUE MAK1MURAet al.
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Riiegg U. T., Cuenoud S., Fulpius B. W. and Simon E. J. (1982) Inactivation and solubilization of opiate receptors by phospholipase A 2. Biochim. biophys. Acta 685, 241 248. Wu Y. C., Cho T. M., Loh H. H. and Way E. L. (1976) Binding of narcotics and narcotic antagonists to triphosphoinositide. Biochem. Pharmac. 25, 1551 1553.