Meptazinol: Unusual in vivo opioid receptor activity at supraspinal and spinal sites

Newo&wnacak?gy Vol. 25, No. 4, pp. 343-349, 1986 Printed in Great Britain. All rights nservtd

Copyright 0

0028-3908/86 S3.00 + 0.00 1986 Pergamon Press Ltd

MEPTAZINOL: UNUSUAL IN ‘VW0 OPIOID RECEPTOR ACTIVITY AT SUPRASPINAL AND SPINAL SITES A. DRAY*, L. NUNAN and W. WIRE Department of Pharmacology, University of Arizona, Health Sciences Center, Tucson, AZ 85724, U.S.A. (Accepted 5 July 1985) Smmnary-Systemic (l-10 mg/kg, s.c.), intracerebroventncular (i.c.v. 2u-80 fig) and spinal intrathecal (i.t., 5-2Opg) administration of meptaxinol hydrochloride produced dose-related inhibition of reflex contractions of the urinary bladder, recorded isometrically in urethane-anesthetized rats. The effects of meptaxinol were reversed by naloxone administered by the same route. Indeed, this was achieved with jn~~rebrovent~c~ar or intrathecal a~inis~tion of naloxone (2&g), which also selectively antagonized the p-receptor ligand [D-Ah’, MePhe”, ~l~ol)~~nk~h~in (DAGO). However ICI 174,864 (3 ~8, i.c.v. or it.), a &opioid receptor antagonist, did not affect the actions of meptaxinol given intracerebroventricularly or intrathecally though it consistently abolished the equieffective actions ofa selective B-receptor ligand (2.n-penicillamine, fi+penicillamine) enkephalin (DPLPE). Naloxonaxine (5 fig, i.c.v. or i.t.), an irreversible p,-opioid receptor antagonist, produced prolonged antagonism of the effects of DPLPE and meptaxinol. The effects of DPLPE partially or completely recovered by 24 hr, indicatinlg that naloxonaxine produced prolonged antagonism of d-opioid receptors. The effects of maptazinol however only recovered after 72 hr, suggesting that antagonism by naloxonazine of this ligand was irreversible and was mediated through a unique opioid receptor interaction. Subthreshold doses of meptaxinol (lOpg, i.c.v.; 3 pg, i.t.) consistently antagonized the effects of morphine &en in~a~brov~t~~riy or intmth~lly but not the equieffective doses of DPLPE or DAGO. These observations suggest that meptaxinol inhibited reflex contractions of the bladder by supraspinal and spinal p-opioid receptor activation. Furthermore, its agonistic effect and its antagonistic actions were compatible with interactions at a subpopulation of opioid receptors, possibly p,-receptors. These observations support the view that meptaxinol is an opioid with an unusual profile of activity. Key words: contractions,

meptaxinol,

supraspinal/spinal

activity, opioid antagonists,

Multiple populations of opioid receptors, designated as p, 6 or K, have been proposed to mediate the specific effects of opioids in the central nervous

system (Martin, Eades, Thompson, Huppler and Gilbert, 1976; Lord, Waterfield, Hughes and Kosterlitz, 1977; Paterson, Robson and Kosterlitz, 1983; Ling and Pasternak, 1983; Pastemak, Gintzler, Houghten, Ling, Goodman, Spiegel, Nishimura, Johnson and Recht, 1983; Porreca, Mosberg, Hurst, Hruby and Burks, 1984). Amongst these actions inhibition of reflex contractions of the bladder in the rat (Dray and Metsch, 1984a, b) and cat (Hisamitsu and de Groat, 1984) have been shown to be mediated by p and 6 opioid receptors at both supraspinal and spinal sites. In order to further characterize the central effects of opioids on the activity of the bladder the opioid meptazinol [m-(Eethyl-l-methyl-hexahydro-l-Hazepin-3-yl) phenol hydrochloride] has been utilized. This compound is a potent analgesic with an unusual profile of pharmacological activity. Like other *Present address: Sandoz Institute for Medical Research, Gower Place. London WCIE 6BN. U.K.

reflex urinary

bladder

opioids analgesia induced by meptazinol may be reversed by naloxone, but meptazinol produces relatively little respiratory or cardiovascular depression (Robson, 1983; Stephens, Waterfall and Franklin, 1978) and does not inhibit contractions of the guinea pig ileum (Bill, Cowlrick, Fox, Todd, Ward, Wood and Wyllie, 1981). Moreover meptazinol behaves as a mixed ago~st/~tagonist but with properties unlike other opioids of this type, such as pentazocine (Stephens et al., 1978; Goode, Rhodes and Waterfall, 1979; Paymaster, 1977; Rashid and Waterfall, 1979). More recently, it has been suggested that meptazinol interacts with a high affinity opioid receptor, the p, receptor (Pastemak, Childers and Snyder, 1980; Wolozin and Pastemak, 1981; Hahn, Carroll-Buatti and Pastemak, 1982; Pastemak et al., 1983; Nishimura, Recht and Pastemak, 1984) and such a cellular interaction has been suggested to be in keeping with the unique pharmacological profile of activity (Spiegel and Pasternak, 1984). In the present in vi& study it has been demonstrated that meptazinol inhibits reflex contractions of the bladder at spinal and supraspinal sites by an unusual opioid receptor interaction.

A. DRAYer al.

344 METHODS

Female Sprague-Dawley rats (200-220 g, Division of Animal Resources, University of Arizona) were anesthetized with urethane (1.2 g/kg, i.p.) throughout the experiment. Body temperature was maintained at 37°C by means of a warm water blanket. The urinary bladder was catheterized via the urethra using PE50 polyethylene tubing. The intravesicular pressure was measured using a physiological pressure transducer and displayed continuously on a chart recorder. The bladder was filled with warm physiological saline via the recording catheter until spontaneous contractions occurred as a result of central reflex activity. Contractions were then recorded isometrically and occurred rhythmically and reproducibly for many hours. Substances were administered into the CNS at sites. Intracerebrosupraspinal and spinal ventricularly (i.c.v.) administrations were made into a lateral ventricle via a burr hole in the skull (coordinates 2 mm posterior to Bregma, 2 mm lateral to the midline and 4mm deep from the skull surface). The head of the animal was fixed in a stereotaxic apparatus throughout this procedure and the Hamilton syringe (26 gauge needle) which was used for injections was held in a micromanipulator. Spinal intrathecal (it.) injections were made between the intravertebral space (usually between L, and L4) with a microsyringe handheld during the procedure. All substances were administered in a volume of 1-4~1. Microinjections of physiological saline were used as a control. These injections rarely affected the activity of the bladder. For threshold (greater than 50% change in amplitude and frequency of contraction) and potency comparisons, one dose of meptazinol was tested per animal to avoid the possibility of tachyphylaxis or tolerance from repeated administration of drug. When repeated administrations were required, as in the antagonist studies, approx. 60 min was allowed between the recovery from the effect of one dose and the administration of another. In the antagonist studies microinjections of a submaximal dose of meptazinol were alternated with an equipotent dose of either the selective p-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)‘]enkephalin (DAGO; Handa. Lane, Lord, Morgan, Rance and Smith, 1981) or the selective a-receptor agonist (2-D-penicillamine, 5-t_-penicillamine) enkephalin (DPLPE; Mosberg, Hurst, Hruby, Gee, Yamamura, Galligan and Burks, 1983). The opioid antagonists used in this study included naloxone, the novel d-receptor selective compound ICI 174,864 (NJ-diallyl-TyriAib-Aib-Phe-Leu-OH: Aib = aaminoisobutyric acid; Cotton, Giles Miller, Shaw and Timms, 1984; Dray and Nunan, 1984a; Corbett, Gillan, Kosterlitz, McKnight, Paterson and Robson. 1984) and the irreversible opioid antagonist naloxonazine (Hahn er al., 1982). In the studies with

naloxonazine, the effects of rneptazinol were tested approx. 30 min after the naloxonazine was administered. and at various times (3-6 hr and 24 or 72 hr) afterwards in the same animals. A 50-100% reduction of a submaximal response to meptazinol was regarded as a significant antagonism. Only one antagonist was tested in an individual animal. In other experiments the opioid antagonistic effects of meptazinol were assessed using morphine. DAGO or DPLPE as opioid receptor ligands. Unless otherwise indicated, all substances were dissolved in sterile physiological saline. Concentrations of drugs are expressed as those of the salt. The following compounds were used in this study: DPLPE (Bachem, Bubendorf, Switzerland); DAGO (Peninsula Laboratories Inc., California, U.S.A.); naloxone hydrochloride (Endo Laboratories). The ICI 174,864 was a gift from Dr R. Cotton (ICI Pharmaceutical Divison, Macclesfield, England): naloxonazine was a gift from Dr G. Pasternak (Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, U.S.A.); meptazinol hydrochloride was a gift from Dr D. Green (Wyeth Research, U.K.). RESULTS

Meptazinol hydrochloride administered subcutaneously, produced a dose-related inhibition of the activity of the bladder (1 mg/kg = 27 k 6 min; 2 mg/kg = 38 f 7min; 5 mg/kg = 58 k 9min; IOmg/kg = 75 f 14 min, n = 6-8 for each determination). The threshold subcutaneous dose was 0.2-0.4 mg/kg (n = 6). Inhibition of the activity of the bladder occurred after a delay of several minutes (mean 3.9 +_2.1 min, n = 26) and could be consistently (6 of 6 experiments) blocked by a prior administration of naloxone (0.5mg/kg, s.c., n =6; Fig. 1). Meptazinol also depressed the reflex contractions bladder after either intraof the urinary cerebroventricular or intrathecal injections. The onset of these effects was relatively rapid (35-125 set) and was characterized at threshold doses (i.c.v. = 5-10 pg; it. = 2-5 pg) by a gradual reduction in both the amplitude and frequency of contractions. At suprathreshold doses, this pattern of activity rapidly culminated in the complete cessation of contractions of the bladder or activity of the bladder stopped abruptly (20-100 set) after the injection (Fig. 2). The period of quiescence of the bladder was used as an index of activity of the drug and in this respect the depressant effect of meptazinol was dose-related (Table I). Intrathecal administrations of meptazinol appeared somewhat more active than the intracerebroventricular administrations (Table 1). This difference in activity has been observed with other opioids (Dray and Metsch, 1984a, b) and seems likely to have resulted from the more limited intrathecal distribution of drug, inferred from administrations of dye (Dray, 1985).

Meptazinol and central opioid activity

N

I-

I

E

0

Q

*

Meptazlnol

min

In

1.0 mghg

x.c.

.Mmptorinol

l

Noloxonr 0.5 mg/

345

kg SC.

A1 180min

Ie

” minay-L 0

Meptozinol

Fig. 1. inhibition of reflex contractions of the bladder by systemic injection of meptazinol and antagonism by naloxone. The top trace shows inhibition of bladder activity after a systemic administration of meptazinol hydrochloride (1 mg/kg, SC.). Note the delay before contractions of the bladder were inhibited and compare with subsequent figures showing the more rapid effects of central administration of meptazinol. The middle trace shows antagonism of the effect of meptazinol by a prior administration of naloxone hydrochloride (0.5 mg/kg, s.c.). Some 180 min after the administration of naloxone, partial recovery of the effect of meptazinol could be observed (bottom trace). All traces are taken from the same experiment. Calibration bars are 6Ocm of water and 1min.

Antagonist

studies

The submaximal effect of meptazinol (40 pg, i.c.v.) and DAGO (0.5 pg, i.c.v.) were consistently attenuated or abolished when tested some IO-36 (17 + 10, n = 7) min after the administration of a small (2 pg, i.c.v.) dose of naloxone (7 of 8 experiments; Fig. 2). This effect was reversible and 50-100% recovery of the response to meptazinol was observed some

/bJ

I< .

11 min

Moptazinol

0

n

Mn

t-------‘“-L

y\-

L

’

n

96-280 min after the administration of naloxone (Fig. 2). Similar antagonism of the effects of meptazinol (1O~g) and DAGO (0.01 pg) was observed in the spinal cord (11 of 11 experiments) when these agents were tested some 5-28 min (10 + 7, n = 11) after the intrathecal administration of naloxone (2 pg, i.t.). As before 50-100% recovery of the agonist effects was observed some 99-198 (147 + 31 min, n = 1I) after

40 pg

i.c.v.

OAGO0.5

PLp 1.C.v.

Noioxona

2.0

0 PLQ i.C.V.

Mptarinol

0

OAGO

At 2lOmin

cemin,nn

0

Meptarinol

Fig. 2. lntracerebroventricular (i.c.v.) injection of naloxone abolished the inhibition of contractions of the bladder by meptazinol hydrochloride and DAGO given intracerebroventriculariy. The top two traces show control responses lo meptazinol (40 pg. i.c.v.) and DAGO (0.5 pg, i.c.v.). After the administration of naloxone (2 pg. i.c.v.) responses to both meptazinol and DAGO were completely abolished. This effect was reversible and recovery of the effect of meptazinol was observed some 210 min after the injection of naloxone (bottom trace). Calibration bars are 60cm of water and I min.

A. DRAY er

346

Table I, Meptazinol hydrochloride produced dose-related inhibition of contraction of the bladder after intracerebroventricular (kv.) br spinal intrathecal (i.t.) administration Intracerebroventricular (i.c.v.)

Intrathecal (i.t.)

lJB

nmol

Effect (min)

Fg

nmoi

Effect (min)

20 40 80

74 148 296

II *3 21 +7 35211

5 IO 20

18 37 74

1553 29 k 5 51 *II

Doses are expressed in ~(8 and nmol per rat and the effect is the duration (min) of complete suppression of contractions of the bladder. n = 6-8 for each determination.

the administration of naloxone. This dose of naloxone given intracerebroventricularly or intrathecally was not seen to produce any significant change in the frequency of contractions of the bladder. Small doses of ICI 174,864 (3/1g, i.c.v. or i.t.), previously shown to selectively antagonize the in vivo effects of a highly selective d-receptor ligand (Dray and Nunan, 1984a), produced weak and inconsistent effects against inhibition of the bladder by meptazinol, administered by the intracerebroventricular (2040 pg, 1 of 6 experiments) or intrathecal (5-10 pg, 2 of 8 experiments, reduced to approx. 60% of control) route. However the equieffective inhibition produced by DPLPE was consistently abolished by ICI 174,864 in the same experiment. This dose of ICI 174,864 did not itself produce changes in the activity of the bladder when administered by either route. Intracerebroventricular administration of naloxonazine hydrochloride (5 pg, i.c.v.), produced an increase in the frequency of contraction of the bladder shortly after its administration (Fig. 3) and blocked the effects of DPLPE (8 pg, 14 k 3 min, n = 12), as well as those of meptazinol (40 pug, 16+9min, n = 12), when these agents were tested 3041 min afterwards (12 of 12 experiments). The effects of both agents were still blocked when they were administered 190-360 min (268 f 64, n = 12) after the initial administration of naloxonazine (Fig. 3). On repeating these tests in the same animals 24 hr later, the effects of DPLPE usually recovered (70-100% of control), whereas those of meptazinol were still blocked (Fig. 3). In the final series of experime’nts (n = 6), meptazinol and DPLPE were retested 72 hr after the initial dose of naloxonazine (5 pg, i.c.v.). Recovery of the effects of both DPLPE and meptazinol were observed in each animal. In the studies with intrathecal administration, conducted in parallel with the intracerebroventricular studies, naloxonazine (5 pg, i.t.) also consistently increased the frequency of contraction of the bladder and blocked (15 of 15 experiments) the effects of DPLPE (1 pg i.t. = 38 &-12 min) and meptazinol (10 pg = 24.f 4 min), when these agents were tested 24-40 and 164-240 min later. In two further series of

al.

experiments the effects of DPLPE recovered (5 of 7 animals), whereas those of meptazinol were still blocked (7 of 7 experiments) 24 hr after administration of naloxonazine. However, by 72 hr after administration of naloxonazine the effects of both meptazinol and DPLPE were observed to be completely recovered. It was notable that inaccurately placed injections of naloxonazine (2 experiments, i.t.) produced neither an increase in the frequency of bladder contraction, nor did they affect the responses to intrathecal administrations of meptazinol or DPLPE. Control intracerebroventricular (n = 2) or intrathecal (n = 2) injections of saline vehicle did not affect contractions of the bladder nor did they change the activity of the opioid agonists. When meptazinol was tested for antagonistic activity, doses were chosen which produced little direct change in bladder activity. Thus, a submaximal effect of morphine (1 pg, i.c.v.) was consistently blocked (6 of 6 experiments) when tested 15 min after a 10 pg (i.c.v.) dose of meptazinol. Recovery of the effect of morphine was observed following further tests some 210-316min later (Fig. 4). Meptazinol given intracerebroventricularly did not significantly change the response to DPLPE (8 pg, i.c.v. control 23 + 4 min: post-meptazinol 24 + 3 min, n = 6) or DAGO

A

’

I

I

‘6min-

Mcptozinol 10 ~9 i.t. 22 min i,

’ DPLPE

I-

1.0 rcg i.t.

* Naloxonozinr

5.0 co i.t.

At 3 kr

A( 24 hr

II

lMopta2inol

‘DPLPE

Fig. 3. Prolonged antagonism of the effects of meptazinol by intrathecal (i.t.) administration of naloxonazine. The top traces show control submaximal effects of meptazinol (10 pg, i.t.) and DPLPE (I pg, i.t.). After the administration of naloxonazine (5 pg. i.t.) an increase in the frequency of contraction of the bladder may be noted. When tested 3 hr later neither meptazinol nor DPLPE was effective. However, when testing was repeated 24 hr later, the effect of DPLPE had largely recovered, whereas that of meptazinol was still blocked. All traces were obtained from the same animal. Calibration bars are 60cm of water and 1 min.

Meptazinol and central opioid activity

u

&

,13mlnt

-

347

0 Morphine

1.0 ~0

i.c.v.

0 Meptozinol

10~9

0

ic.v.

m

/5

Morphine

min&

Morphinr

Fig. 4. Meptazinol blocked the inhibition of contractions of the bladder produced by morphine (i.c.v.). The top trace shows the control response to morphine (1 pg, i.c.v.). A subthreshold dose of meptazinol hydrochloride (10 pg, i.c.v.) produced little direct effect on the frequency of contraction of the bladder but blocked the effect of a subsequent administration of morphine (1 pg, i.c.v., middle trace). The effect of morphine recovered some 215 min after the administration of meptazinol. Calibration bars are 60 cm of water and 1 min.

(0.4pg, i.c.v. control 19 +4min: after meptazinol 23 + 3 min, n = 8). Essentially similar observations were made following intrathecal administrations of drug. Thus, the effect of morphine (0.2pg i.t.) was consistently attenuated or blocked (8 of 8 experiments) when tested I l-20 min after the intrathecal administration of meptazinol (3 pg, i.t.). Partial (50%) or complete recovery of the response to morphine was observed 116-290min later. As before meptazinol (3 pg, i.t.) did not affect responses to DPLPE (4 of 4 experiments, 1 pg, control = 25 + 5 min: after meptazinol 25 k 7 min) or DAGG (0.01 pg, control = 36 &-4 min: after meptazinol = 34 &-6 min, n = 6). DISCUSSION

Like other opioids, meptazinol produced a doserelated, naloxone-reversible, inhibition of reflex contractions of the bladder after either systemic, intracerebroventricular or intrathecal routes of ad(Dray and Metsch, 1984a, b, c; ministration Hisamitsu and de Groat, 1984; Jubelin, Galeano, Ladouceur, Lemaires and Elhilali, 1984). Indeed inhibition of the activity of the bladder was produced at systemic doses comparable to those used in other studies of analgesia induced by meptazinol (Stephens et al., 1978; Goode et al., 1979; Spiegel and Pasternak, 1984). The effects of direct central administration of meptazinol have not been described. Thus, the present observations after intracerebroventricular

or intrathecal administration suggest that important actions of this opioid, with respect to changes in the activity of the bladder, were exerted at both supraspinal and spinal sites. This conclusion is supported by previous studies of inhibition of the activity of the bladder mediated by opioids (Dray and Metsch, 1984a, b, c; Dray, 1985; Dray and Nunan, 1985). However, in comparison with morphine for example, meptazinol appeared to be considerably less active (about 50 times) after both intracerebroventricular and intrathecal injection, although, as previously noted with other opioids (Dray and Metsch, 1984a, b), the effects by the intrathecal route were consistently more prolonged than those with the intracerebroventricular one. This may be explained most conveniently by the more limited distribution of substances within the spinal cord following direct intrathecal administration (Dray, 1985). The mechanism of inhibition of reflex contractions of the bladder mediated by opioids is poorly understood. However, the rapidity of onset of the effects and their relatively limited distribution after either intracerebroventricular or intrathecal administration have suggested possible loci of action close to the site of injection. Thus, opioids given intracistemally may affect structures similar to those implicated in opioidinduced analgesia, for example the periventricular and periaqueductal regions and closely associated areas (Yaksh and Rudy, 1978). In addition, it is possible that centers in the brain stem mediating micturition may be involved (Satoh, Shimizu,

348

A. DRAY e! al

Tohyama and Maeda, 1978). These supraspinal effects of opioids appear however to be mediated indirectly through descending monoamine mechanisms (Dray and Nunan, 1985). On the other hand opioid effects in the spinal cord appear to be exerted more directly (Dray and Nunan, 1985) and may mimic inhibitory spinal enkephalinergic mechanisms. Thus, enkephalinergic fibers arise from the sacral parasympathetic nucleus and via the pelvic nerve innervate parasympathetic bladder ganglia (Glazer and Basbaum, 1980; de Groat, Kawatani, Hisamitsu, Lowe, Morgan, Roppolo, Booth, Nadelhaft, Kuo and Thor, 1983). In support of this hypothesis are observations showing that naloxone increased bladder motility and pelvic nerve activity, resulting possibly from the antagonism of an endogenous inhibitory opioid mechanism (Roppolo, Booth and de Groat, 1983; Thor, Roppolo and de Groat, 1983; Dray and Metsch, 1984a, b, c; Hisamitsu and de Groat, 1984). The involvement of p-opioid receptors in the central effects of meptazinol was supported by the fact that its actions were reversed by small doses of naloxone which antagonized the actions of selective p but not 6-ligands (Dray and Metsch, 1984a, b). Furthermore, the lack of effect of the 6-selective antagonist ICI 174,864 (Cotton et al., 1984; Dray and Nunan, 1984a; Corbett et al., 1984), excluded the participation of 6-opioid receptors. Since the activity of the bladder has been shown to be unchanged by K-receptor ligands given either intracerebroventricularly or intrathecally (Dray and Metsch, 1984a, b) this suggested rather specific interactions between meptazinol and p-opioid receptors. The effects of meptazinol given both intracerebroventricularly and intrathecally could further be antagonized by naloxonazine with recovery of the effect of meptazinol occurring some 72 hr afterwards. This time course appears to be compatible with that of an irreversible antagonism, previously described with naloxonazine and related compounds (Pasternak et al., 1980; Hahn et al., 1982; Pasternak et al.. 1983). In vitro studies have shown naloxonazine to selectively inhibit a high affinity opioid binding site, a subpopulation of sites designated as p, opioid receptors (Hahn et al., 1982; Pasternak et al., 1980; Pasternak et al., 1983). In vivo, p, opioid receptors have been implicated in supraspinal actions of opioids (Pasternak et al., 1983) but not in spinal analgesia (Ling and Pasternak, 1983) or in opioid-induced respiratory depression (Ling, Spiegel, Nishimura and Pasternak, 1983). Meptazinol has been shown to bind to a high affinity opioid site and its analgesic effects were attenuated by pretreatment with naloxonazine (Spiegel and Pasternak, 1984). Thus, meptazinol would appear to be a p, selective opioid agonist. The present observations, depending largely on the time course of antagonism of the effects of meptazinol by naloxonazine, would support this conclusion. In addition, the data with intrathecal injection also suggest that these receptors exist at spinal sites to mediate

inhibition of reflex contractions of the bladder. .As in the previous study (Dray and Nunan. 1984b). naloxonazine also produced long-lasting antagonism 01 a d-receptor agonist, but this effect was reversed after 24 hr. These findings are compatible with the view that naloxonazine. predicted to act as a multifunctional molecule (Hahn et ~1.. 1982). also has antagonistic properties at d-receptors. This is supported by the following. A number of studies have confirmed the d-receptor selectivity of DPLPE and other conformationally-restricted analogues of enkephalin (Mosberg et al., 1983: Corbett et al.. 1984: Cotton, Kosterlitz, Paterson, Rance and Traynor. 1985). Recovery of the effects of a-ligands from naloxonazine (Dray and Nunan. 1984b) occurred more rapidly than predicted from previous studies with irreversible opioid antagonists (Pasternak er al.. 1980; Hahn et al., 1982; Pasternak ef al.. 1983). This differential time course of antagonism also supports a fundamental difference between the actions of meptazinol and DPLPE. It should be noted however that a number of p-receptor agents, including morphine, DAGO and not-morphine, have recently been reported to displace [3H] (2-D-penicillamine. 5-Dpenicillamine) enkephalin from a high affinity binding site (Cotton et al., 1985). The significance of this observation for determining the receptor subtypes which mediate particular functional changes produced by opioids requires further examination. Finally in keeping with previous in cico observations (Stephens et al., 1978) meptazinol was shown to antagonize the actions of morphine, administered intraventricularly and intrathecally. Since this effect was observed after a subthreshold dose of meptazinol, it seemed unlikely to have resulted from acute opioid tolerance. Indeed, the antagonism was quite selective since neither the effects of DPLPE nor DAGO were changed. It is possible that meptazinol may have indeed caused an acute tolerance or receptor blockade of a high affinity p, opioid site activated by morphine (Wolozin and Pasternak, 198 I; Pasternak et al., 1983). On the other hand, the antagonism may have occurred at a stage secondary to receptor activation. Further studies are required to elucidate these possibilities. In conclusion, meptazinol hydrochloride inhibited reflex contractions at the bladder by supraspinal and spinal mechanisms which appeared to involve p rather than d-receptors. Furthermore, prolonged antagonism of its effects with naloxonazine suggested properties compatible with activation of p, opioid receptors. Finally, meptazinol exhibited selective antagonistic activity, also possibly involving p, receptors. This spectrum of pharmacological properties supports the view that meptazinol is an unusual opioid. REFERENCES

Bill D.. Cowlrick

I. S.. Fox J.. Todd

M. H.. Ward

P. J..

Meptazinol and central opioid activity Wood M. D. and Wyllie M. G. (1981) Does meptazinol bind to opiate receptors? Er. J. Pharmac. 74: 866P. Corbett A.- D.. Gillan M. G. C., Kosterlitz H. W., McKniaht A. T.. Paterson S. J. and Robson L. E. (1984) SelectivTties of opioid peptide analogues as agonists and antagonists of the &receptor. Er. J. Pharmac. 83: 271-279. Cotton R., Giles M. G., Miller L., Shaw J. S. and Timms D. (1984) ICI 174,864: a highly selective antagonist for the opioid 6-receptor. Eur. J. Pharmoc. 9? 331-332. Cotton R., Kosterlitx H. W., Paterson S. J.. Rance M. J. and Traynor J. R. (1985) The use of [“HI-[n-Pen*, o-Pen51 enkephalin as a highly selective ligand for the b-binding site. Br. J. Phormoc. 84: 927-932. Dray A. (1985) The rat urinary bladder, a novel preparation for investigating central opioid activity in uivo. J. Pharmoe. Med. 13: 157-165. Dray A. and Metsch R. (1984a) Inhibition of urinary bladder contractions by a spinal action of morphine and other opioids. J. Phormac. exp. Ther. 231: 254-260. Dray A. and Metsch R. (1984b) Opioid receptor subtypes involved in the central inhibition of urinary bladder motility. Eur. J. Phurmoc. 104: 47-53. Dray A. and Metsch R. (1984c) Morphine and the centrallymediated inhibition of urinary bladder motility in the rat. Brain Res. 297: 191-195.

Dray A. and Nunan L. (1984a) Selective d-opioid receptor antagonism by ICI 174,864 in the central nervous system. Peptides 5: 1015-1016.

Dray A. and Nunan L. (1984b) Evidence that naloxonaxine produces prolonged antagonism of central delta opioid receptor activity in viuo. Brain Res. 323: 123-127. Dray A. and Nunan L. (1985) Onioid inhibition of retlex urinary bladder contractions: _Dissociation of supraspinal and spinal mechanisms. Brain Res. 33% 142-145. Glazer E. L. and Basbaum A. 1. (1980) Leucine enkephalin: Localixation in and axoplasmic transport by sacral parasympathetic preganglionic neurons. Science 2@& 1479-1481. Goode P. G., Rhodes K. F. and Waterfall J. F. (1979) The analgesic and respiratory effects of meptazinol, morphine and pentazocine in the rat. J. Phnrm. Pharmuc. 31: 793-795.

Groat W. C. de, Kawatani M., Hisamitsu A. M., Lowe T., Morgan C., Roppolo J., Booth A. M., Nadelhaft I., Kuo D. and Thor K. (1983) The role of neuropeptides in the sacral autonomic reflex pathways of the cat. J. Auton. New. Syst. 7: 339-350.

Hahn E. F., Carroll-Buatti M. and Pastemak G. W. (1982) Irreversible opiate agonists and antagonists: the 14-hydroxydihydromorphinone azines. J. Neurosci. 2: 572-576.

Handa B. K., Lane A. C., Lord J. A. H., Morgan B. A., Rance M. J. and Smith C. F. C. (1981) Analogs of P-LPH 61-64 possessing selective agonist activity of p-opiate receptors. Eur. J. Pharmoc. 70: 531-540. Hisamitsu T. and Groat W. C. de (1984) The inhibitory effect of opioid peptides and morphine applied intrathecally and intracerebroventricularly on the micturition reflex in the cat. Bruin Res. 298: 51-65. Jubelin B.. Galeano C., Ladouceur D., Lemaires S. and Elhilali M. M. (1984) Effects of enkephalin on the micturition cycle of the rat. Life. Sci. 34: 2015-2025. Ling G. S. F. and Pastemak G. W. (1983) Spinal and supraspinal opioid analgesia in the mouse: role of subpopulations of opioid binding sites. Brain Res. 271: 152-156.

Ling G. S. F.. Spiegel K., Nishimura

S. and Pastemak

349

G. W. (1983) Dissociation of morphine’s analgesic and respiratory depressant actions. Eur. J. Pharmoc. 86: 487488.

Lord J. A. H., Watertield A. A., Hughes J. and Kosterlitz H. W. (1977) Endogenous opioid peptides: multiple agonists and receptors. Nature, Land. 267: 295-299. Martin W. R., Eades C. G., Thompson J. A., Huppler R. E. and Gilbert P. E. (1976) The etfects of motphineand nalorphine-like drugs in nondependent and morphine-dependent chronic spinal dogs. J. Phormoc. exp. Ther. 197: 517-532.

Mosberg H. I., Hurst R., Hruby V. J., Gee K., Yamamura H. I., Galligan J. J. and Burks T. F. (1983) Bispenicillamine enkephalins possess highly improved specificity towards delta opioid receptors. Proc. notn. Acad. Sci. U.S.A. 80: 5871-5874.

Nishimura S. L., Recht L. D. and Pastemak G. W. (1984) Biochemical characterization of high-affinity 3H-opioid binding: further evidence for mu, sites. Mofec. Phormuc. 25: 29-37.

Pastemak G. W., Childen S. R. and Snyder S. H. (1980) Opiate analgesia: evidence for mediation by a subpopulation of opiate receptors. Science #)8: 514-516. Pastemak G. W., Gintxler A. R., Ho&ten R. A., Ling G. S. F., Goodman R. R., Spiegal K., Nishimura S., Johnson N. and Recht L. D. (1983) Biochemical and pharmacological evidence for opioid r&ePtor multiplicity in the central nervous system. Life Sci. 31: 167-173. Paterson S. J., Robson L. E. and Kosterlitx H. W. (1983) Classification of opioid receptors. Br. med. Bull. 39: 31-36. Paymaster N. J. (1977) Analgesia after operation. A controlled comparison of meptazinol, pentaxocine and pethidine. Br. j. Anaesth. 49: -1139-l 146. Porreca F.. Mosbern H. I.. Hurst R.. Hrubv V. J. and Burks T. F. (1984) R&s of mu. delm and kappa opioids receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse. J. Pharmoc. exp. Ther. 230~ 341-348. Rashid S. and Waterfall J. F. (1979) Cardiovascular action of meptazinol in comparison with pentazocine and morphine. Gen. Phormac. 10: 459-464. Robson P. J. (1983) Clinical review of parenteral mep tazinol. Postgrad. med. J. 59: 85-92. Roppolo J. R., Booth A. M. and Groat W. C. de (1983) The effects of naloxone on the neural control of the urinary bladder of the cat. Brain Res. 264~ 355-358. Satoh K.. Shimixu N.. Tohvama M. and Maeda T. (1978) Locali;ation of the’ mic&ition reflex center at dorso: lateral pontine tegmentum of the rat. Neurosci. Lett. 8: 27-33.

Spiegel K. and Pastemak G. W. (1984) Meptazinol: a novel mu-l selective opioid analgesic. J. Phurmac. exp. Ther. 228: 414-419.

Stephens R. J., Waterfall J. F. and Franklin R. A. (1978) A review of the biological properties and metabolic disposition of the new analgesic agent, meptaxinol. Gen. Phormat. 9: 73-78.

Thor K. B., Roppolo J. R. and Groat W. C. de (1983) Naloxone induced micturition in unanesthetized paraplegic cats. J. Ural. 129: 202-205. Wolozin B. L. and Pastemak G. W. (1981) Classification of multiple morphine and enkephalin binding sites in the central nervous system. Proc. natn. Acad. Sci. U.S.A. ‘18: 61816185.

Yaksh T. and Rudy T. A. (1978) Narcotic analgesics: CNS sites and mechanisms of actions as revealed by intracerebral injection techniques. Pain 4: 299-359.