Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 membrane

Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 membrane

European Journal of Pharmacology, 159 (1989) 83-88 83 Elsevier EJP 50569 Characterization of high affinity opioid binding sites in rat periaqueduct...

468KB Sizes 0 Downloads 54 Views

European Journal of Pharmacology, 159 (1989) 83-88

83

Elsevier EJP 50569

Characterization of high affinity opioid binding sites in rat periaqueductal gray P2 membrane J o s e p h P. F e d y n y s h y n *, G e o f f r e y K w i a t a n d N a n c y M. Lee Department of Pharmacology and Langley Porter Psychiatric Institute, University of California, San Francisco, CA 94143, U.S.A.

Received 11 July 1988, revised MS received 20 September 1988, accepted 27 September 1988

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation produced and opioid induced analgesia. In the present study the opioid binding characteristics of the PAG were examined with an in vitro radioligand binding technique. [3H]Ethylketocyclazocine (EKC), 2 nM, was used as a tracer ligand to nonselectively label #, 6, and • binding sites in PAG enriched P2 membrane. The/t selective ligand [D-Ala2,N-meth o ylPhe4,Glyol 5]enkephalin (DAGO) competed with [3H]EKC for more than one population of binding sites with both high and low affinity. In contrast the 6 selective ligand [D-PenZ,D-PenS]enkephalin (DPDPE) and the x selective ligand trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, methane sulfonate, hydrate (U50,488H) each competed with [3H]EKC for a single population of binding sites with low affinity. DPDPE and U50,488H also competed with 2 nM [3H]DAGO for a single population of binding sites with similar low affinity. DAGO and not DPDPE competed with 2 nM [3H][D-Ala2,D-LeuS]enkephalin (DADLE) with high affinity. 2 nM [3H]DPDPE did not substantially label PAG enriched P2 membrane, and 1 nM DAGO competed with all specific [3H]DPDPE binding which was observed. These binding data are consistent with the presence of a single population of/~ selective high affinity binding sites in PAG enriched P2 membrane to which 6 ligands and K ligands have low affinity, Periaqueductal gray; Opioid binding sites; Analgesia

1. Introduction

The periaqueductal gray (PAG) region of the midbrain has been implicated in both endogenous pain control and opioid induced analgesia. Focal electrical stimulation within the PAG results in a profound analgesia unaccompanied by general motor or behavioral depression (Mayer et al., 1971; Mayer and Liebeskind, 1974). Intracerebral microinjection of opioids into the PAG results in a similar analgesia (Yaksh et al., 1976), and intracerebroventricular injection of opioids into the

* To whom all correspondence should be addressed: Department of Pharmacology, Box 0450, University of California, San Francisco, CA 94143, U.S.A.

third ventricle and the vicinity of the P A G is now commonly used to activate the supraspinal components of antinociception. It appears that this opioid induced analgesia in the P A G is mediated by a opioid receptor in that it is stereoselective, dose dependent, and naloxone reversible (Yaksh and Rudy, 1978). It also appears that opioid agonism within the midbrain is important if not essential for the mediation of the analgesia observed after systemic administration of analgesic opioids. The analgesia produced by typical systemic doses of morphine can be reversible attenuated by intracerebroventricular microinjection of naloxone into the third ventricle in a dose dependent manner (Yeung and Rudy, 1980). Which of the proposed type or types of opioid receptors are responsible for the production of

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

84 this analgesia in the PAG? The heterogeneity of opioid receptors is now widely accepted, t~, •, o and 6 opioid receptors have been postulated on the basis of pharmacolgical data in the chronic spinal dog (Martin et al., 1976) and in in vitro guinea pig ileum and mouse vas deferens preparations (Lord et al., 1977). #, K, and 6 binding sites have subsequently been identified in brain tissue (Chang and Cuatrecasas, 1979; Chang et al., 1981; Kosterlitz et al., 1981), and respectively ~, ~ and 6 selective ligands have all been implicated in the production of analgesia (Tyers, 1980; Ward and Takemori, 1983). High affinity opioid binding sites have been identified in the P A G with in vivo autoradiographic localization techniques (Atweh and Kuhar, 1977). However in this initial study the high affinity opioid binding sites were defined with [3H]diprenorphine, a ligand which is relatively nonselective for proposed/x, ~ and 6 receptor types. A more recent in vitro autoradiographic study utilizing more selective tritiated opioid ligands and slide mounted brain sections has identified high affinity/x but not 6 or ~ binding sites in the PAG (Mansour et al., 1986). In contrast a second study utilizing a similar technique has identified both high affinity t~ and K binding sites in this brain region (Tempel and Zukin, 1987). It remains to be determined as to which opioid receptor types are actually present in PAG membrane and account for this reported high affinity opioid binding. It is the purpose of this qualitative study to more closely examine the opioid binding characteristics of the PAG. In particular the opioid binding activity of rat P A G enriched P2 membrane is directly assessed with in vitro radioligand binding techniques.

2. Materials and methods

2.1. Materials Tracer ligands were obtained from New England Nuclear Research Products and unlabelled opioid peptides from Penninsula Laboratories, Inc. Trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidin y l ) c y c l o h e x y l ] b e n z e n e a c e t a m ide, m e t h a n e

sulfonate, hydrate (U50,488H) was obtained from the U p j o h n C o m p a n y . Ethylketocyclazocine (EKC) was provided by the National Institute of Drug Abuse.

2.2. Membrane preparation Male Sprague-Dawley rats weighing 180-200 g were used in all experiments. Rats were killed by decapitation, and the brains minus cerebellum were immediately dissected over ice. The PAG region of the midbrain of each rat was initially isolated by three transverse cuts along the main cerebral axis, the first cut caudal to the corpus collasum, the second cut between the superior and inferior colliculi, and the third cut caudal to the inferior colliculi. PAG tissue was further concentrated by removing the white matter surrounding the clearly visible gray matter in the central region of the two inner transverse sections produced by the three previous transverse cuts. PAG tissue was washed in ice-cold 25 mM HEPES (Calbiochem)-0.32 M sucrose (Mallinckrodt) buffer (pH 7.7) and homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 1 000 x g for 10 min. The supernatant was saved and the pellet resuspended in HEPES-sucrose buffer and again centrifuged at 1 000 x g for 10 min. This pellet was discarded and the pooled supernatant from both 1 000 x g spins was centrifuged at 22500 x g for 20 min. The resulting pellet was resuspended in HEPES-sucrose buffer and again centrifuged at 22500 x g for 20 min. The final P2 pellet was resuspended in the HEPES-sucrose buffer and frozen in aliquots for future use. Typically the PAG enriched P2 membrane from 20 rats was resuspended in a final volume of 2 ml of buffer with an average protein concentration of 11.5 mg per ml as determined by Lowry assay (Lowry et al., 1951). Thawed PAG enriched P2 membrane was used once and never re frozen.

2.3. Radioligand binding assay Borosilicate glass tubes were utilized in all experiments. The incubation mixture of each radioligand competition binding assay consisted of 25

85 m M HEPES buffer (pH 7.7), various concentrations of unlabelled opioids, 2 nM tritiated tracer ligand, and 50/~1 of P A G enriched P2 m e m b r a n e to a total volume of 1 ml. The incubation mixture of each saturation binding assay consisted of 25 m M HEPES buffer (pH 7.7), either no or 10 m M D A G O , various concentrations of [3H]DAGO, and 50 ml of P A G enriched P2 membrane also to a total volume of 1 ml. Incubation was carried out for 1 h at a constant temperature of 25 ° C and a constant p H of 7.7. Samples were filtered under vacuum through glass fiber filters (Whatman G F / B ) after the incubation period. Filters were washed twice with 5 ml of ice-cold 5 m M HEPES buffer (pH 7.7) and placed in polyethylene counting vials. Scintilation cocktail (Fisher Scintiverse II), 9 ml, was added to each vial which was counted 48 h later. All assays were carried out in triplicate. Specific binding was determined as the difference between total bound and that binding remaining in the filter from samples containing both membrane and 10 m M excess unlabelled form of the tracer ligand.

3. Results 2 nM [3H]EKC was first used as a tracer ligand to nonselectively label all opioid binding sites in P A G enriched P2 membrane. Since previous studies have established that this nominal x ligand also binding to ~ and 8 opioid binding sites with high affinity (Chang et al., 1980; Snyder and Goodman, 1980), 2 nM [3H]EKC should nonspecifically label all three types of receptor binding sites which may be present in P A G membrane. This [3H]EKC tracer ligand was placed in competition with the relatively selective /~ ligand [DAla2,N-methylPhe4,Glyol 5]enkephalin ( D A G O ) (Kosterlitz and Paterson, 1980), 8 ligand [DPen2,D-PenS]enkephalin (DPDPE) (Mosberg et al., 1983) and x ligand U50,488H (Vonvoightlander et a l , 1983). Although these ligands are not specific for their respective opioid receptor subtype they are among the most selective kt, 8 and r ligands currently available. The results are shown in fig. 1. The shallowness and biphasic nature of the [3H]EKC and D A G O competition curve suggests

o.~ IOOl .c

~C nn

8c

u

4C

I

-9

-7

-5

--3

log [unlobelled ligond],M Fig. 1. Competition curves of DAGO (n), DPDPE (o) and U50,488H (zx)with 2 nM [3H]EKC binding; 10/~M unlabeled EKC was used to define nonspecific binding. Each data point is the mean of triplicate determinations. Specific [3H]EKC binding comprised 79.7% of total [3H]EKC binding measured. that [3H]EKC and D A G O share more than one population of c o m m o n binding sites. D A G O then competed with [3H]EKC for at least one population of binding sites with high affinity. In contrast both D P D P E and U50,488H each to compete with [3H]EKC for an apparent single population of binding sites with low affinity. 2 nM [ 3 H ] D A G O was next used as a tracer to selectively label only high affinity D A G O binding sites in P A G enriched P2 membrane in competition with D A G O , D P D P E and U50,488H. The results are shown in fig. 2. As expected D A G O competed with [ 3 H ] D A G O with high affinity. However both D P D P E and U50,488H competed with [ 3 H ] D A G O with low affinity in a manner very similar, if not identical, to their competition with [3H]EKC.

I00{ o, 8 0 ._~ -o 60 m 40 o 2.0' co o 1 -I O.

I

-9

-7

-5

-3

log [unlobelled ligond],M Fig. 2. Competition curves of DAGO (D), DPDPE (o) and U50,488H (zx) with 2 nM [3H]DAGO binding; 10 ~M unlabeled DAGO was used to define nonspecific binding. Each data point is the mean of triplicate determinations. Specific [3H]DAGO binding comprised 68.0% of total [3H]DAGO binding measured.

86 too

t~ 8 0 ¢:6 ,- 60

o [3

_u 40

o

20 Ct) 0 O) ¢:1.

-

I

,

I

log [unlobelled liga2dl, M

-3

-9

-7

Fig. 3. Competition curves of DADLE (~), DAGO ([3) and DPDPE (o) with 2 nM [3H]DADLE binding; 10 aM unlabeled DADLE was used to define nonspecific binding. Each data point is the mean of triplicate determinations. Specific [3H]DADLE binding comprised 37.2% of total [3H]DADLE binding measured.

2 nM [3H][D-Ala2,D-LeuS]enkephalin (DADLE), a partially selective 6 ligand, was also used as a tracer ligand in competition with D A D L E , D A G O and DPDPE. Since D A D L E has significant affinity for both 8 a n d / z opioid binding sites (Chang, 1984), 2 nM [3H]DADLE should label both high affinity ~ and ~t receptor binding sites which may be present in P A G membrane. The results are shown in fig. 3. D A G O competed with [ 3 H ] D A D L E in a manner similar to its competition with [3H]EKC shown in fig. 1 with a broad curve suggestive of both high and low. affinity

.~_

1

oo

8O

E

60

:~

40

C

a5

if)

o

i

1 o

21o

[3H-DAGO], nM

Fig. 4. Saturation binding curve of [3H]DAGO; 10 ,aM unlabeled DAGO was used to define nonspecific binding. Each data point is the mean of triplicate determinations. Specific [3H]DAGO comprised 60.4, 73.4, 67.5, 56.7, 46.5 and 31.5% of total [3H]DAGO binding measured respectivelyfor 0.2, 1, 2, 5, 10 an 20 nM concentrations of this tracer ligand.

binding sites. D P D P E however competed with [ 3 H ] D A D L E for a single population of sites with low affinity. There is no component to the [ 3 H ] D A D L E binding with which D P D P E competed with high affinity. Finally 2 nM [3H]DPDPE was used as a tracer ligand in competition with D P D P E and D A G O . Specific labelling of P A G enriched P2 membrane with 2 nM [3H]DPDPE was much less than that seen with identical concentrations of [3H]EKC, [3H]DAGO, and [ 3 H ] D A D L E in three separate binding experiments. 1 nM D A G O competed completely with [3H]DPDPE for the little specific binding of this tracer ligand which was detected (data not shown). Specific binding of [3H]DAGO in P A G enriched P2 m e m b r a n e is saturable. Specific binding of [ 3 H ] D A G O reached a maximum of 89 f m o l / m g m e m b r a n e protein at a concentration of 20 nM. The results are shown in fig. 4.

4. Discussion

It is now clear that multiple opioid receptor subtypes exist in the brain, but their relative involvement in the various pharmacological effects of opioids, particularly analgesia, remains to be elucidated. One approach to this problem is to determine the opioid receptor subtypes present in specific brain regions known to be associated with particular functions of opioids. The P A G was chosen for analysis as this region is known to play an important role in opioid analgesia. To determine the opioid receptor types actually present in this tissue a strategy of reversibly displacing a tracer ligand with unlabelled ligands that have been previously shown to be relatively selective for #, 6 and K opioid binding sites has been utilized. It is apparent from the radioligand binding data that only the /z selective ligand D A G O displaces 2 nM [3H]EKC from P A G enriched P2 membrane with high affinity; the 6 ligand D P D P E and the K ligand U50,488H displace both 2 nM [3H]EKC and 2 nM [ 3 H ] D A G O from PAG enriched P2 m e m b r a n e with very similar low affinities. These results are most parsimoniously explained by the existence of a single population of

87 primarily/~ selective high affinity opioid binding sites in the PAG which are labelled by both [3H]EKC and [3H]DAGO and to which 8 and x ligands are still capable of binding but with much lower affinity relative to the more # selective ligands. Neither high affinity 8 nor high affinity x binding was detected in PAG membrane. The absence of high affinity 8 binding sites in PAG enriched P2 membrane is further supported by both the [3H]DADLE and [3H]DPDPE binding data. D A G O competed with the 2 nM [3H] D A D L E more completely and with higher affinity than DPDPE. Similarly a lower 1 nM concentration of D A G O competed with the 2 nM [3H] D P D P E for all of the small amount of detectable binding of this tracer ligand. D A D L E also competed with [3H]DADLE in PAG P2 membrane with lower affinity than D A G O competed with its tritiated counterpart, [3H]DAGO. These results are consistent with both [ 3 H ] D A D L E and [3H]DPDPE primarily labelling only /~ selective high affinity binding sites in PAG membrane. It is reasonable to assume that the ~ selective high affinity opioid binding sites detected in the PAG enriched P2 membrane are associated with/~ opioid receptors. Specific high affinity [3H]DAGO binding in this tissue is saturable in a manner consistent with binding to physiologically relevant receptors. The nature of the low affinity D A G O displaceable [3H]EKC binding and the low affinity D A G O displaceable [3H]DADLE binding is less clear and requires further study. However these low affinity interactions could represent a second population of /t binding sites, consistent with evidence from several laboratories that at least two distinct # opioid binding sites exist (Loew et al., 1986; Nishimura et al., 1983; Wolozin and Pasternack, 1981). The existence of such a single population of high affinity # opioid receptors in the PAG is consistent with reported physiological data and the idea that supraspinal analgesia produced both by endogenous (Chaillet et al., 1983; 1984) and exogenous (Fang et al., 1986; Wood et al., 1981) opioids is mediated by the /~ opioid receptor. While both # and d selective opioid ligands produce analgesia after intracerebralventricular injection it appears that a single population of/~ recep-

tors is sufficient for mediating this effect. This conclusion is principally based on similar apparent pA2 values for naloxone antagonism of the analgesia produced by both /~ and 8 selective opioid drugs. It has also been reported that the observed increase in body temperature (Widdowson et al., 1983) and increase in hind-limb muscle tone (Widdowson et al., 1986) after microinjection of opioids into the PAG are both mediated by the /~ opioid receptor. The in vitro binding data is in agreement with in vivo pharmacological data. The data presented in this study while very suggestive of an absence of high affinity 6 and x opioid receptors in the PAG can not conclusively rule out the existence of these receptor subtypes in this brain region. It is still possible that a small number of 8 and • opioid receptors exist in the PAG below the detection limits of the radioligand binding assay, and thus small number of receptors may be physiologically important. It is also possible that 6 and x opioid receptors in the PAG are more sensitive to their environment relative to their/z receptor counterpart, and as such may not be functional and detectable under the in vitro binding conditions used in this study. However if, as the data indicates, only a single population of bt opioid receptors is present or significantly predominates in the PAG, PAG tissue much like the NG-108,15 cell line represents a system in which a single opioid receptor type may be characterized free from the confounding presence of other opioid receptor types.

Acknowledgements This research was supported in part by National Institute of Drug Abuse Grants DA 02643 and 1 F31 DA 05281-01(JPF).

References Atweh, S.F. and M.J. Kuhar, 1977, Autoradiographic localization of opiate receptors in rat brain. II. The brainstem, Brain Res. 29, l. Chaillet, P., A. Couland, M. Fournie-Zaluski, G. Gacel, B.P. Roques and J. Costentin, 1983, Pain control by endogenous enkephalins is mediated by mu opioid receptors, Life Sci. 33 Suppl. 1, 685.

88 Chaillet, P., A. Couland, J. Zajac, M. Fournie-Zaluski, J. Costentin and B.P. Roques, 1984, The /, rather than the 6 subtype of opioid receptors appears to be involved in enkephalin-induced analgesia, European J. Pharmacol. 101, 83. Chang, K. and P. Cuatrecasas, 1979, Multiple opiate receptors: enkephalin and morphine bind to receptors of different specificity, J. Biol. Chem. 254, 2610. Chang, K., E. Kazum and P. Cuatrecasas, 1980, Possible role of distinct morphine and enkephalin receptors in mediating actions of benzomorphan drugs (putative ~ and 6 agonists), Proc. Natl. Acad. Sci. 77, 4469. Chang, K., E. Hazum and P. Cuatrecasas, 1981, Novel opiate binding sites selective for benzomorphan drugs, Proc. Natl. Acad. Sci. 77, 4141. Chang, K., 1984, Opioid receptors: multiplicity and sequelae of ligand-receptor interactions, in: The Receptors, Vol. 1, ed. P.M. Corm (Academic Press, Orlando) p. 1. Fang, F.G., H.L. Fields and N.M. Lee, 1986, Action at the mu receptor is sufficient to explain analgesic effect of opiates, J. Pharmacol. Exp. Ther. 238, 1039. Kosterlitz, H.W. and S.J. Paterson, 1980, Tyr-D-AIa-GIyMePhe-NH(CH 2 )2 OH is a selective ligand for the/x-opiate binding site, Br. J. Pharmacol. 73, 299P. Kosterlitz, H.W., S.J. Paterson and L.E. Robson, 1981, Characterization of the K-subtype of the opiate receptor in the guinea-pig brain, Br. J. Pharmacol. 73, 939. Loew, G., C. Keys, B. Luke, W. Polgar and L. Toll, 1986, Structure-activity studies of morphiceptin analogs: receptor binding and molecular determinants of/*-affinity and selectivity, Mol. Pharmacol. 29, 546. Lord, J.A.H., A.A. Waterfield, J. Hughes and H.W. Kosterlitz, 1977, Endogenous opioid peptides: multiple agonist and receptors, Nature 267, 495. Lowry, O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265. Mansour. A., L. Lewis, H. Khachaturian, H. Akil and S.J. Watson, 1986, Pharmacological and anatomical evidence for selective ~t, 8 and ~ opioid receptor binding in rat brain, Brain Res. 399, 69. Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler and P.E. Gilbert, 1976, The effects of morphine and nalorphine-like drugs in the nondependent and morphinedependent chronic spinal dog, J. Pharmacol. Exp. Ther. 197, 17. Mayer, J.D., T.L. Wolfe, H. Akil, B. Carder and J.C. Liebeskind, 1971, Analgesia from electrical stimulation in the hrainstem of the rat, Sci, 174, 1351.

Mayer, J.D. and J.C. Liebeskind, 1974, Pain reduction by focal electrical stimulation of the brain, Brain Res. 68, 73. Mosberg, H.I., R. Hurst, V.J. Hruby, K. Gee, K. Akiyama, H.I. Yamamura, J.J. Galligan and T.F. Burkes, 1983, Cyclic pencillamine containing enkephalin analogs display profound delta receptor selectivities, Life Sci. 33 Suppl. I, 447. Nishimura, S.L., L.D. Recht and G.W. Pasternack, 1983, Biochemical characterization of high-affinity 3H-opioid binding: further evidence for mu 1 sites, Mol. Pharmacol. 25, 29. Snyder, S.H. and R.R. Goodman, 1980, Multiple neurotransmitter receptors, J. Neurochem. 35, 5. Tempel, A. and S. Zukin, 1987, Neuroanatomical patterns of the/z, 6, and ~ opioid receptors of rat brain as determined by quantitative in vitro autoradiography, Proc. Natl. Acad. Sci. 84, 4308. Tyers, M.B., 1980, A classification of opiate receptors that mediate antinociception in animals, Br. J. Pharmacol. 69, 503. Vonvoightlander, P.F., R.A. Lahti and J.H. Ludens, 1983, U50,488: a selective and structurally novel non-mu (kappa) opioid agonist, J. Pharmacol. Exp. Ther. 224, 7. Ward, S.J. and A.E. Takemori, 1983, Relative involvement of mu, kappa, and delta receptor mechanisms in opiate-mediated antinociception in mice, J. Pharmacol. Exp. Ther. 224, 525. Widdowson, P.S., E.C. Griffiths and P. Slater, 1983, Body temperature effects of opioids administered into the periaqueductal grey area of rat brain, Regul. Pept. 7, 259. Widdowson, P.S., E.C. Griffiths and P. Slater, 1986, The effects of opioids in the periaqueductal grey region of rat brain on hind-limb muscle tone, Neuropeptides 7, 251. Wolozin, B.L. and G.W. Pasternack, 1981, Classification of multiple morphine and enkephalin binding sites in the central nervous system, Proc. Natl. Acad. Sci. 78, 6181. Wood, P.L., A. Rackham and J. Richard, 1981, Spinal analgesia: comparison of the mu agonist morphine and the kappa agonist ethylketazocine, Life Sci. 28, 2119. Yaksh, T.L. and T.A. Rudy, 1978, Narcotic Analgetics: CNS sites and mechanisms of action as revealed by intracerebral injection techniques, Pain 4, 299. Yeung, J.C. and T.A. Rudy, 1980, Sites of antinociceptive action of systemically injected morphine: involvement of supraspinal loci as revealed by intracerebroventricular injection of naloxone, J. Pharmacol. Exp. Ther. 215, 626. Yaksh, T.L., J.C. Yeung and T.A. Rudy, 1976, Systematic examination in the rat of brain sites sensitive to the direct application of morphine: observations of differential effects within the periaqueductal gray, Brain Research 114, 83.