Modulation of ~-Mediated Antinociception by ~ Agonists: Characterization with Antagonists

European Journal of Pharmacology. 169 11989) 43- 52 43 Elsevier

EJP 50990

Modulation of ~-mediated antinociception by ~ agonists: characterization with antagonists

Julius S. Heyman 1, Qi Jiang 1, Richard B. Rothman 2 Henry I. Mosberg 3 and Frank Porreca I Department of PharmacoloL,~'. College of Medwine. Unit'erst(v of Arizona, Tucson, A Z 85724, " l~boratorv of Chnwal Science, NIMH. Bethesda. MD 20892. and s College of Pharmao', Unn'ersttv of Mlchtgan. Ann Arbor, MI 48109. U.S.A.

Received 6 March 1989, revised MS received 26 June 1989. accepted 18 July 1989

The functional interactions between supraspinal p, and 8 receptors were characterized in the mouse using p, receptor-selective antagonists. The effects of pretreatment with the p, opioid antagonists, /~-funaltrexamine (/'/-FNA) and naloxonazine on the modulation of morphine antinociception by the 8 agonists [D-PenZ,D-PenS]enkephalin (DPI)PE) and [D-AlaZ,MetS]enkephalinamide (DAMA) were studied. When co-administered in the same i.c.v. injection, a sub-antinociceptive dose of DPDPE consistently and significantly increased the antinociceptive potency of morphine in control animals, while a sub-effective dose of DAMA decreased morphine antinociception: both the respective increase and the decrease of morphine potency by DPDPE and DAMA had been previously shown to be blocked by ICI 174,864, a 8 antagonist. Pretreatment of mice with the non-equilibrium p. antagonist /'/-FNA 4 h prior to testing, a pretreatment which had no effect on i.c.v. DPDPE or DAMA antinociception, prevented the modulation of morphine antinociception by both DPDPE and DAMA. Pretreatment with the long acting pq antagonist naloxonazine, 24 h prior to testing, failed to affect the modulation of morphine antinociception by either DPDPE or DAMA: such a pretreatment had no effect on the antinociceptive effects of DPDPE or DAMA when given alone. These results provide further support for the concept of a functionally coupled p.-6 receptor complex which is sensitive to antagonism by fl-FNA, but not naloxonazine, and support the notion that subtypes of opioid p~ and 8 (i.e. complexed and non-complexed) receptors may exist.

Opioid antinociception: p, Opioid receptors; ~ Opioid receptors; fl-Funaltrexamine (fl-FNA): Naloxonazine; (Intracerebroventricular, Mouse)

!. Introduction focus of a great deal of research. Of the effects studied, opioid-induced antinociception remains The suggestion of multiple opioid receptor sub- at the forefront of interest and relevance. Recent types has been supported by a great deal of evi- investigations, which used heat as the noxious dencc obtained in vitro (Lord et al., 1977; Gioan- stimulus, have demonstrated that supraspinal nini et al., 1985; Cho et al., 1986) and in vivo opioid-induced antinociception in mice can be (Martin et al., 1976). Nevertheless, the correlation mediated by both 8 (Porreca et al., 1984: Heyman of opioid-induced effects with specific receptor et al., 1987: Mathiasen et al., 1987: Porreca et al.. subtypes has been difficult and continues to be the 1987: Takemori and Portoghese, 1987) and /x opioid receptors. In addition to the direct role played by the 6 Correspondence to: F. Porreca, Department of Pharmacology, receptor in production of antinociception in this University of Arizona, Health Sciences (:'enter, Tucson, AZ species, data also exist which suggest that [LeuS]- 85724, U.S.A. and [Met ~ ]enkephalin, endogenous 6 ligands (Lord

0014-2999/89/$03.50 , 1989 Elsevier Science Publishers B.V. (Biomedical Division) 44 et al., 1977), can indirectly modulate p.-mediated [YH]DADI.E from the lower affinity site. ('hang effects through actions at the 8 receptor. In- and Cuatrecasas (1979) identified it as att binding tracerebroventricular (i.c.v.) administration of site. However, a number of experimental manipu- sub-antimx:iceptive doses of [LeuY]enkephalin, as lations demonstrate differences between tt binding well as [LeuS]enkephalin analogs, have been shown sites and the lower affinity [~H]DADI.E binding to increase i.c.v, morphine antinociceptive potency site. These include: (a) the ionic composition of in the mouse (Vaught and Takemori, 1979: Barrett the assay medium (Bowen et al., 1981: Rothman and Vaught. 1982: Vaught et al., 1982). Con- et al.. 1984b): (by the ability of the site directed versely, sub-antinociceptive doses of [MetS]en - acylating agent FIT (N-phenyl-N-[l-(2-(p-isothio- kephalin and [MetS]enkephalin analogs signifi- cyanato)phenylethyl-4-piperadinyl]propanamide)- cantly decrease i.c.v, morphine antinociceptive H('I to unmask lower affinity [~H]I)ADLE bind- potency (l.ee et al.. 1980: Vaught et al., 1982). ing sites but not p. sites (Rothman et al.. 1985b): Evidence that these modulatory effects are media- (c) the observation that the i.c.v, administration of ted via a 8 receptor includes the recent observa- fl-funaltrexamine (fi-ENA, Portoghcse et al.. 1980) tions that the 6-selective antagonist ICI 174.864 to rats 24 h prior to preparation of brain mere- (Cotton et al.. 1984) prevented both the increase branes results in an approximately 6()~ decrease (Heyman et al.. 1986b; 1989) and the decrease in the B...... of the lower affinity' [~H]DADI.f- (Heyman et al.. 1986b) of morphine antinocicep- binding site without any alteration in [>H][D- tion produced by sub-agonist doses of the a-selec- Ala-'.NMPhe'*.Gly-ol]enkephalin ([ ~HIDAG()) tire [D-PenZ,D-PenY]enkephalin (DPDPE) (Mos- (Handa el al.. 1981 ) binding parameters (Rothman berg et al.. 1983: Porreca et al.. 1984: James and et al.. 1984a: 1986: 1989) and (d) the finding that Goldstein, 1984) and [D-AlaZ.MetS]enkepha - tt ligands are non-competitive inhibitors of a IL linamide (DAMA), respectively: ICI 174,864 had binding site labelled bv ['H]naloxone (Rothman no direct effect on morphine antimueiception et al.. 1985c)and [~H]17-cych)propylmethyl-3.14- (Heyman et al., 1987). dihydroxy-4,5-ct-epoxy-6-/~-fluoro-morphinan- The ability of 8 agonists to produce antinoci- ([~H]cycloFOXY) (Rothman et al.. 1987h). ception directly, as well as to modulate (i.e. in- These reciprocal non-co,npetitivc interactions crease or decrease) g-mediated antinociceptive between p. and 6 receptors led to the suggestion potency, may imply the existence of subclasses of that (a) the lower affinity [~I-i]DAI)LE binding 8 and p. receptors. Early' ligand binding studies site is the a binding site of an opioid receptor demonstrating apparent non-competitive interac- complex (termed ,$, to indicate 'in the complex') tions between p. and ~ binding sites (Rothman and that the non-competitive interaction of p and Westfall, 1982a.b) led to the hypothesis that ligands on the lower affinity [~H]DADI.E binding the modulatory effects of 8 agonists on p.-medi- site is mediated through an adjacent # binding site ated antinociception occur through a 8 binding (termed /,~, to indicate in the complex): (by the site of an opioid receptor complex made up of higher affinity ['I1]DAI)LE binding site is a 6 distinct, yet interacting /, and /} binding sites binding site which is not part of the receptor (Vaught et al.. 1982). More recent ligand binding complex (termed ,$.... to indicate "not in the corn- studies have refined this hypothesis and have plex'), and (c) that p.-mediated antinociception shown that the 8 agonist, [~H][D-AlaZ,D-l.euY]en - occurs via binding of p. ligands to the #~, binding kephalin ([~HIDADLF.) labels two binding sites in site while the modulatory effects of ,~ agonists on vitro which are distinguished by the inhibitory p.-mediated antinociception is mediated via bind- mechanism of tt ligands (Rothman et al., 1985a.c); ing to the ,8 binding site of the opioid receptor while p. ligands are weak. competitive inhibitors at complex ( 8, ). the higher affinity [3H]DADLE binding site, corn- The present investigation was designed to fur- monly identified as 8. they are potent, non-compe- ther elucidate the possible functional interaction titive inhibitors at the lower affinity [~H]DAI)LE of p. and 8 receptors in the production of anti- binding site. Based on the potent displacement of nociception and to test the hypothesis that the 45 modulatory effects of 8 agonists on morphine ~00 antinociception are mediated via actions at the 8~ 80 receptor. Recent studies in the rat have suggested 4- that the irreversible ~ antagonist, fl-FNA may ~ 60 1 compoundPr°videa means to test this hypothesis, aSrecep_this ~ "[1 ;~[~T~[ has been shown to alkylate the 40 .._.___ tor complex (Rothman et al., 1986; 1989). Thus, ~ , 1~ , the ~a modulatory effects of DPDPE and DAMA, ~ 20 8 agonists previously shown to potentiate and ] antagonize morphine antinociception, respectively, o o ~o 20 30 40 so were studied in the presence of the selective opioid r,me (~,n) antagonist, ,8-FNA (Portoghese et al., 1980). Ad- Fig. 1. Time-response curve for i.c.v. DAMA (1.7 nmol) anti- ditionally, in an attempt to gain further insight nociception in the mouse. Data are means and S.E. into the nature of the ~t.~ and ~,~ sites, naloxonazine (Hahn et al., 1982), an antagonist of the putative ~ receptor (Pasternak et al., 1980) was (vehicle) or fl-FNA (18 nmol) 4 h prior to agonist studied, administration, similar to the procedure described by Ward et al. (1982). In studies with naloxonazine, each mouse received a single subcutaneous 2. Materials and methods (s.c.) injection of distilled water (vehicle) or naloxonazine HCI (35 mg/kg) 24 h prior to testing as 2.1. Animals described by Ling et al. (1986).

Male, ICR mice (20-30 g, Harlan, Indianapolis, 2.4. Antinociceptwe testing IN) were used for all experiments. Animals were kept in groups of five in a temperature-controlled Antinociceptive responses were determined us- room with a standard 12 h light/dark cycle (lights ing warm (55°C) water as the nociceptive stim- on 07:00 h). Food and water were continuously ulus where the latency to tail withdrawal was available, taken as the endpoint (Janssen et al., 1963). Prior to agonist administration, the tail of each mouse 2.2. h!jection techniques was immersed in the water and the latency to a rapid flick recorded. Animals not flicking their Compounds were delivered into the lateral tails within 5 s were eliminated from the study. cerebral ventricle using a modification of the This procedure was repeated 20 rain after i.c.v. method of Haley and McCormick (1957) as previ- administration of morphine, DPDPE and DAMA; ously described (Porreca et al., 1984). Briefly, the this was the time of peak agonist effect as de- mice were lightly anesthetized with ether, an inci- termined from time-response curves (morphine and sion was made in the scalp and bregma located. DPDPE, Heyman et al., 1986a; DAMA, fig. 1). The injections were made 2 mm caudal and 2 mm Animals not flicking their tails within 15 s were lateral to bregma at a depth of 3 mm using a removed from the nociceptive stimulus and as- Hamilton (Reno, NV) microliter syringe with a 26 signed a maximal score of 100% in order to avoid gauge needle. All i.c.v, injections were made in a tissue damage. Antinociception was expressed as: volume of 5 v,l. % antinociception = 100 × (test latency - control latency)/(15 s- control latency). All testing was 2.3. Antagonist pretreatment done in unanesthetized mice. Following the determination of the i.c.v, dose- In studies employing fl-FNA, each mouse re- response curves for DPDPE, morphine and ceived a single i.c.v, injection of distilled water DAMA in control (DPDPE and morphine, Hey- 46

": : 2.5. Chemicals

;52 - -" DPDPE and DAMA (Peninsula I,aboratories, ~:: .... - Inc.. San Carlos, ('A) were dissolved in distilled ~'f water, frozen in aliquots and lyophilized, and te- a - . dissolved immediately before use. Morphine sul- .... fate (Mallinckrodt Inc., St. Louis. M(-)), fl-I-NA >:: -'g " (Research Biochemicals Inc.. Wavland, MA)and -/ i naloxonazine HCI w.ere dissolved in distilled water :..7 ".: ~1 '" :' just prior to administration. Naloxonazine was a :':~ c,~'u~ "~-o~ ~ " generous gift of Dr. Diane DeHaven (Nova Phar- Fig 2. Dose-response relationship of i.c.v. DAMA antmocicep- maceutical ('o., Baltimorc, MD). tion m the mouse ",.,,'hen tested at 20 rain post-injection. Data are means and S.E. 2.6. Statistics

The doses of DPDPE and DAMA chosen for man et al., 1986a: DAMA, fig. 2), fl-FNA (DPDPE the modulation of morphine antinociccption were and morphine, Heyman ct al.. 1987; DAMA, fig. extrapolated from regression lines determined by 3) and naloxonazine (DPDPE and morphine, Hey- plotting each individual point using the computer man et al., 1988: DAMA, fig. 3), pretreated mice, program of Tallarida and Murray (1986) (proce- doses of DPDPE and DAMA which produced dure 8) in control, fl-FNA and naloxonazinc pre- barely detectable antinociception (0-5%) in the treated mice. A minimum of 10 mice were studied respective groups were chosen by downward ex- at each dose level. Modulatory effects of DPDPE and DAMA on morphine antinociception were trapolation of the dose-response line. In order to determine if DPDPE and/or DAMA were capa- identified using a Student's t-test for grouped data. ble of modulating i.c.v, morphine antinociception The data are presented as the mean and the error in animals pretreated with ,8-FNA or naloxona- bars arc the S.E. zine, the 6 agonists were co-administered in the same i.c.v, injection with morphine as previously described by Vaught el al. (1982). Testing took 3. Results place 20 rain after injection. 3.1. Studies in mice pretreated with fl-FNA

,0o ! The first study examined the effects of/3-FNA 901 pretreatment (18 nmol i.c.v., at -4 h)on the

~3 80 respective ability of DPDPE to increase, and -~ ,o DAMA to decrease, i.c.v, morphine antinocicep- .g_ so tive potency. As the/3-FNA pretreatment used in 5o the current study had no significant effect on the •~, ,o direct antinociceptive effects of i.c.v. DPDPE .~, 3o (Heyman et al., 1987; present study) or DAMA ,, 2o (fig. 3), the modulatory doses for each 8 agonist ,0 (1.6 nmol DPDPE and 0.17 nmol DAMA) re- o o., ~ 3 ~o 2o mained the same. As expected (Ward et al., 1982: Dose DAMA (nmol, i.c.v.) Ward and Takemori, 1983; Heyman et al., 1987), Fig. 3. Effects of pretreatment with /3-FNA (18 nmol i.c.v, at however, morphine antinociception was signifi- -4 h) (A) or naloxonazine (35 mg/kg s.c. at -24 h) (I) on cantly antagonized by ,8-FNA pretreatment: thus, i.c.v. DAMA (e) antinociception. Data are means and S.E the doses of morphine used in ,8-FNA pretreated 47

Control p- F'NA Pretreoted

100 ~k

80 [ U3 + N$

~" 2o

o -- 1.6 0 1.6 sol 1.6 0 1.6 DPDPE 0 3 3 0 9 9 Morphine Don (nmol, i.c.v.) Fig. 4. Effects of /~-FNA pretreatment (18 nmol at -4 h) on DPl)PE-induced m~xtulation of morphine antint~iception. /~-FNA pretrealment abolishes the increase of morphine antincx:iceptivepotency produced by DPDPE. Data shov,n are means and S.E. and asterisks indicate a significant difference from control (P < 0.05, Student's t-test). mice were increased by 10-fold (3 and 30 nmol, 3.2. Studies in mice pretreated with naloxona:ine respectively, in naive and fl-FNA groups shown in figs. 4 and 5). Pretreatment with the p, antagonist Naloxonazine pretreatment (35 mg/kg s.c. at ,8-FNA blocked the modulatory effect of DPDPE - 24 h) had no effect on the direct antinociceptive (fig. 4) and that of DAMA (fig. 5) when compared effect produced by i.c.v. DPDPE (fig. 6), in agree- to control (5 p,l distilled water i.c.v., at -4 h) ment with previous results (Heyman et al., 1988), groups. ,8-FNA had no antinociceptive effect alone or DAMA (fig. 3), thus, no changes were required in this test. in the modulatory doses of these 6 agonists. As

100

j 4O "jIi o r-"-n 0.17 0 0.17 ~1 0.17 0 0.17 ~ 0 3 3 0 I • (nm~. Lr.v.) Fig. 5. Effects of fl-FNA pretreatment (18 nmol at -4 h) on DAMA-induced modulation of morphine antin(x:iception, fl-FNA pretreatment abolishes the attenuation of morphine antinociception by DAMA. Data shown are means and S.E. and asterisks indicate a significant difference from control (P < 0.05, Student's t-test). 48

Control Noloxonazine Pretreated

100 *

~ 80 ÷ c •

'~ 40 .E ~ 20

0 -- 0 3 3 sol O 9 9 30 30 Morphine 1.6 0 1.6 1.6 0 1.6 0 1.6 DPDPE Dose (nrnol, i.c.v.) Fig. 6. Effects of naloxonazine pretreatment (35 mg/kg s.c. at - 24 h) on DPDPE-induced modulation of morphine antint~iception. Naloxonazine pretreatment does not affect the increase in potency of morphine resulting from DPDPE. Data are means and S.E. and asterisks indicate a significant difference from controls (P < 0.05, Student's t-test).

expected (Ling et al., 1986: Heyman et al.. 1988), DAMA to modulate (fig. 7) morphine antinoci- pretreatment with naloxonazine effectively antag- ception. onized i.c.v, morphine antinociception (figs. 6 and 7) and so the doses of morphine used were in- 4. Discussion creased by 10-fold. Unlike the effects of pretreatment with t~-FNA, naloxonazine pretreatment did The present study attempted to further our not alter the ability of DPDPE (fig. 6) or of understanding of #-8 interactions in the produc-

Control Noloxonozlne Pret~eoted ,k

I O0 - r'-"'/~" ,

+ I ~g 60 i ,u i

~q 20'

0 ~ -" 0 3 ,3 sol 0 30 30 90 90 Morphine 0.17 0 0.17 0.17 0 0.17 0 0.17 DAMA Dose (flmol, i.C.V.) Fig. 7. Effects of naloxonazine pretreatment (35 mg/kg s.c. at - 24 h) on DAMA-induced modulation of morphine antinociception. Naloxonazine pretreatment does not affect the attenuation of morphine antin(x:iception produced by DAMA. Data are means and S.E. and asterisks indicate a significant difference from controls (P < 0.005, Student's t-test). 49 tion of antinociception using receptor selective formulation of the following testable prediction: if antagonists. Doses of the 8 agonists DPDPE and the modulatory effects of sub-antinociceptive do- DAMA which produced no significant antinoci- ses of 6 agonists on morphine antinociception are ception when given alone were found to increase mediated via an opioid receptor complex, then and decrease the potency of i.c.v, morphine in pretreatment with ,8-FNA should prevent the abil- producing antinociception, respectively, in agree- ity of 8 agonists to modulate morphine antinoci- ment with previous studies (Lee et al., 1980: ception, Vaught et al., 1982; Porreca et al., 1987; Heyman The present study demonstrates that pretreat- et al., 1986b: 1989). These modulatory effects have ment with/3-FNA abolishes both the increase and previously been shown to be due to an interaction decrease of morphine potency associated with of the agonists with the 8 receptors as the 8-selec- DPDPE and DAMA. respectively, a finding which tive antagonist ICI 174,864 (Cotton et al., 1984) might be interpreted as supporting the concept of prevented both the increase (Heyman et al., 1986b; functional uncoupling of the /~-8 complex given 1988b) and decrease (Heyman et al., 1986b) of the observation of disruption of the opioid recep- morphine potency produced by DPDPE and tor complex by J3-FNA in vivo (Rothman et al., DAMA, respectively: IC1 174.864 did not directly 1986: 1989). An alternative explanation seems antagonize morphine antinociception (Heyman et possible, however, as previous studies have shown al.. 1987), that after blockade of available kt receptors with Reasoning, therefore, that morphine antinoci- fl-FNA, morphine produces its antinociception ception may be modulated in part via a hypothe- at a non-p, site (Heyman et al., 1987; Takemori sized ~-8 complex, attempts to disrupt this inter- and Portoghese, 1987). Takemori and Portoghese action were made using the non-equilibrium #- (1987) have shown that the naloxone apparent selective antagonist, ~-FNA (Portoghese et al., pA 2 value against morphine in the mouse abdomi- 1980; Ward and Takemori, 1983). Previous work nal stretch test changes significantly after/3-FNA has shown that /3-FNA pretreatment prevents the treatment suggesting that morphine interacts with ability of ICI 154,129, a 8-selective antagonist non-,a (8 and x) receptors to produce antinocicep- (Shaw et al., 1982) to reverse endotoxic shock tion. Additionally, Heyman et al. (1987) and (Holaday and D'Amato, 1983; D'Amato and Takemori and Portoghese (1987) have shown that Holaday, 1984). Reasonable p, selectivity has been the selective 6 antagonists ICI 174,864 (Cotton et demonstrated for/3-FNA, however, as this antag- al., 1984) and ICI 154.129 (Shaw et al., 1982), onist does not inhibit 6.... binding in rat brain respectively, compounds which do not directly membrane preparations (Rothman et al., 1984a; antagonize morphine tail withdrawal antinocicep- 1987a,b), nor does it block the antinociceptive tion in control mice, significantly antagonize effects of 8 agonists given i.c.v, in mice (Heyman morphine antinociception in ,8-FNA-pretreated et al., 1987). Thus, the ability of/3-FNA to pre- mice. Modulation by 6 agonists, therefore, would vent the effects of ICI 154,129 was suggested to be not be possible if both agonists were acting at the the result of an alteration in the p,-6 receptor same receptor (6,,(,x). Furthermore, the competi- complex (Holaday and D'Amato, 1983; D'Amato tive interactions of p, and 6 ligands at this binding and Holaday, 1984). Additionally, i.c.v, adminis- site (6,,,.,) in vitro are consistent with the observa- tration of/3-FNA to rats 18-24 h prior to preparations in vivo which demonstrate that DPDPE and tion of brain membranes has been demonstrated DAMA do not modulate morphine antinocicep- to lead to a selective alkylation of the opioid tion when morphine acts at the 6,~ site. receptor complex which was reflected by a sub- Further attempts to characterize the /.t-6 func- stantial decrease in the Bma~ of the 6~,~ binding site, tional interaction were made using the long-lasting while the binding of [3H]DADLE to the 6r,~x site proposed ,% antagonist naloxonazine. Naloxona- was not significantly altered (Rothman et al.. 1986; zine pretreatment had no effect on i.c.v. DPDPE 1989). This apparent selective effect of ,8-FNA on antinociception in agreement with previous studies the opioid receptor complex in vivo allowed the where this antagonist neither blocked i.c.v. DPDPE 5O antinociception in the mouse (Heyman et al., 1988) DAGO antinociception by DPDPE, therefore, is or rat (Ling el a[,, 1986), nor altered binding of not directly in accord with the concept that the p.~., DPDPE in rat brain preparations (Clark et al., receptor is the sole site for the mediation of p. 1986). Similarly, naloxona~,.ine pretreatment had antinociception. It is also important to note that no effect on i.c.v. DAMA antinociception (present while naloxonazine pretreatment has previously study). In contrast to the lack of effect of nalo- been shown to antagonize both i.c,v, morphine xonazine on DPDPE and DAMA antinociception, and DAGO antinociception (Heyman et al,. 1988), this antagonist exhibited the expected long-lasting this antagonist had no effect on the ability of blockade of i.c.v, morphine antinociception in the DPDPE or I)AMA to modulate morphine anti- mouse in agreement with previous studies in this nociception. The lack of effect of naloxonazine oq species (Heyman et al., 1988) as well as in the rat the modulation of morphine antinociception cou- (Ling et al.. 1986). Potentiation and attenuation of pied with the antagonism of both morphine and morphine antinociception by DPDPE and DAMA. DAGO antinociception by naloxonazine again respectively, were still evident in mice that re- suggests a discrepancy with the concept that anti- ceived naloxonazine treatment prior to testing, nociception is mediated solely at thc p., site. Various interpretations might be made from these Although reasons which might account for these findings. For example, naloxonazine might alter discrepancies can be formulated, it seems clear the conformation of the receptor complex in such that further experiments are needed to resolve the a way that morphine binds with lower affinity, inconsistencies noted above. Although incon- thus reducing the potency, but leaving the mod- sistencies do exist as to the identity of the specific ulatory mechanisms intact. As the mechanism of site where p. agonists act to produce their anti- action for the long-lasting antagonism associated nociceptive effects, it is important to note that the with naloxonazine is not well understood, con- differential antagonism of the modulation ob- tinued action of morphine may be due to incom- served with /~-FNA and naloxonazine, neverthe- plete blockade of ~t~ receptors. Alternatively, it is less continues to support the concept of p. receptor also possible that the p, receptors in the receptor subtypes. complex are naloxonazine insensitivity (i.e.. p,:). Although the existence of such a complex re- As is often the case, the present findings in vivo mains tentative, p,-8 interactions have been dem- are not in complete agreement with hypotheses onstrated for other endpoints from studies in vivo drawn from findings in vitro. Based on evidence and in vitro. In addition to antinociception, p.-8 from binding studies with fl-FNA, one would interactions have also been observed in vivo in the predict that all # agonists act at the P-c, receptor reversal of endotoxic shock (Holaday and to produce their antinociception (Rothman et al., D'Amato, 1983" D'Amato and Holaday, 1984; 1986; 1987a: 1989). It has been shown previously Holaday et al., 1985, 1986) and the elevation of (Heyman et al., 1989), however, that while mor- flurothyl seizure threshold (Holaday et al., 1985). phine antinociception is potentiated by DPDPE, The finding that ,8-FNA, but not naloxonazine, the antinociceptive effects of the p, agonist DAGO affected the 6 modulation of g, antinociception (Handa et al., 1981) are not. a finding which provides support in vivo for the concept that suggests that morphine and DAGO may act at morphine antinociception is mediated through this two distinct p, receptors (p,~.~ and p, ..... respec- complex, that 8 modulation of morphine anti- tively) to produce their antinociceptive effects, nociception occurs through this complex and that Although morphine and DAGO antinociception the p, site in the receptor complex is naloxonazine are both antagonized by pretreatment with/3-FNA insensitive. Thus. in addition to playing a direct (Heyman et al., 1987), a finding in agreement with role in the production of antinociception, the en- binding studies and the above prediction, only kephalins may also play a modulatory role in morphine antinociception is modulated by DPDPE antinociception by acting in an opioid (p~-~) recep- (Heyman et al., 1989). The lack of modulation of tor complex. 51

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