Structure-constrained endomorphin analogs display differential antinociceptive mechanisms in mice after spinal administration.

We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the µ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and ß-funaltrexamine (ß-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP. Antinociception induced by analog 2 through 4 was not reversed by δ opioid receptor (DOP) or κ opioid receptor (KOP) antagonist; antibodies against dynorphin-A (1-17), dynorphin-B (1-13), and Leu5/Met5-enkephalin had no impact on the antinociceptive effects of these analogs. In contrast, antinociceptive effects induced by a spinal injection of the fluorine substituted analog 1 were significantly reversed by KOP antagonism. Furthermore, intrathecal pretreatment with antibodies against dynorphin-B (1-13) attenuated the antinociceptive effect of analog 1. These results indicate that the antinociceptive activity exerted by intrathecally-administered analog 1 is mediated, in part, through KOP with increased release of dynorphin-B (1-13). The chemical modifications used in the present study may serve as a useful tool to gain insight into the mechanisms of endomorphins activity.

Intrathecal morphine-3-glucuronide-induced nociceptive behavior via Delta-2 opioid receptors in the spinal cord.

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces severe hindlimb scratching followed by biting and licking in mice. The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with an antisera against dynorphin. However, the selective κ-opioid receptor antagonist, nor-BNI did not prevent the M3G-induced behavioral response. Dynorphin is rapidly degraded by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with the antisera against Leu-ENK. We also showed that M3G co-administered with Leu-ENK-converting enzyme inhibitors, phosphoramidon and bestatin produced much stronger behavioral responses than M3G alone. Furthermore, the M3G-induced behavioral responses were inhibited dose-dependently by i.t. co-administration of the non-selective δ-opioid receptor antagonist, naltrindole or the selective δ2-opioid receptor antagonist, naltriben, whereas the selective δ1-opioid receptor antagonist, BNTX had no effect. An i.t. injection of M3G also produced a definite activation of ERK in the lumbar dorsal spinal cord. Western blotting analysis revealed that antisera against dynorphin, antisera against Leu-ENK, naltrindole or naltriben resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that M3G-induced nociceptive responses and ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK, which is formed from dynorphin in the spinal cord.

Opioid and non-opioid receptor-mediated immunoregulatory role of leucine-enkephalin in teleost Channa punctatus.

Leucine-enkephalin (Leu-enk) is an endogenous opioid peptide and highly conserved throughout the vertebrates. Despite its conserved nature, the immunoregulatory property of Leu-enk is explored only in mammals. The present study describes the immunomodulatory role of Leu-enk in a lower vertebrate, spotted murrel Channa punctatus. Leu-enk increased the percentage phagocytosis and phagocytic index, though its stimulatory effect on phagocytosis markedly decreased at concentrations higher than 10(-9) M. Moreover, it had bell-shaped stimulatory effect also on the superoxide production by phagocytes. On the other hand, Leu-enk showed bimodal effects on nitrite release. The lower concentrations of Leu-enk produced inhibitory effect, while higher concentrations had stimulatory effect on nitrite release. Interestingly, the Leu-enk-induced increase in nitrite release was unaltered by non-selective opioid receptor antagonist though the same completely antagonized the inhibitory effect of Leu-enk on nitrite release and the stimulatory effect on phagocytosis and superoxide production. This suggests that the stimulatory effect of Leu-enk on nitrite production is mediated by the non-opioid receptor. Further, delta-opioid receptor was precisely seen involved in mediating the stimulatory effect of Leu-enk on phagocytosis and superoxide production, or inhibitory effect on nitrite release. It can be concluded that Leu-enk regulates the innate immune response of splenic phagocytes acting via both opioid and non-opioid receptor in the fish C. punctatus.

Plasma met-enkephalin, beta-endorphin and leu-enkephalin levels in human hepatic encephalopathy.

To address the role of the opioid system in the pathogenesis of hepatic encephalopathy (HE) we measured plasma met-enkephalin, beta-endorphin and leu-enkephalin in patients with different grades of HE compared to control subjects and patients with cirrhosis. Plasma met-enkephalin levels were significantly higher in patients with cirrhosis and all grades of HE than controls. Plasma beta-endorphin levels were similar in the 3 groups. Plasma leu-enkephalin levels were significantly higher in HE grades II, III and IV than in controls, patients with cirrhosis and HE grade I patients. Our results support data on the involvement of met-enkephalin and leu-enkephalin in the pathogenesis of HE and provide a rationale for the use of opioid receptor antagonists in the treatment of HE.

The role of leucine-enkephalin on insulin and glucagon secretion from pancreatic tissue fragments of normal and diabetic rats.

Leucine-enkephalin (Leu-Enk) has been shown to be present in endocrine cells of the rat pancreas and may play a role in the modulation of hormone secretion from the islets of Langerhans. Since little is known about the effect of Leu-Enk on insulin and glucagon secretion, it was the aim of this study to determine the role of Leu-Enk on insulin and glucagon secretion from the isolated pancreatic tissue fragments of normal and diabetic rats. Pancreatic tissue fragments of normal and streptozotocin-induced diabetic rats were incubated for 1 h with different concentrations of Leu-Enk (10(-12)-10(-6)M) alone or in combination with either atropine or yohimbine or naloxone. After the incubation period the supernatant was assayed for insulin and glucagon using radioimmunoassay techniques. Leu-Enk (10(-12 )-10(-6)M) evoked large and significant increases in insulin secretion from the pancreas of normal rats. This Leu-Enk-evoked insulin release was significantly (p < 0.05) blocked by atropine, naloxone and yohimbine (all at 10(-6)M). In the same way, Leu-Enk at concentrations of 10(-12)M and 10(-9)M induced significant (p < 0.05) increases in glucagon release from the pancreas of normal rats. Atropine, yohimbine but not naloxone significantly (p < 0.05) inhibited Leu-Enk-evoked glucagon release from normal rat pancreas. In contrast, Leu-Enk failed to significantly stimulate insulin and glucagon secretion from the pancreas of diabetic rats. In conclusion, Leu-Enk stimulates insulin and glucagon secretion from the pancreas of normal rat through the cholinergic, alpha-2 adrenergic and opioid receptor pathways.

Antagonistic effect of orphanin FQ on opioid analgesia in rat.

AIM: To study the effect of orphanin FQ (OFQ), a newly discovered heptadecapeptide, on nociception and opioid analgesia. METHODS: The intracerebroventricular (i.c.v.) and intrathecal (i.t.h.) injections were used to give the drugs. The tail-flick model of rats were used to test the pain threshold. RESULTS: OFQ (i.c.v. or i.t.h.) 0.1 microgram had no effect on nociception but 0.5-10 micrograms induces hyper-reaction of rat to noxious electric stimulus; the decapeptide (OFQ1-10 i.c.v.), a fragment of the OFQ, did not affect the pain reaction of rats. Fentanyl (1 microgram, i.c.v. or i.t.h.), a selective mu-receptor agonist, DSLET (5 micrograms, i.c.v. or i.t.h.), a selective delta-receptor agonist, or U50488H (1 microgram, i.t.h.), a kappa-receptor agonist, induced an increase in pain threshold, when OFQ (0.1 or 1 microgram) was added together with one of them (except for the ith injection of DSLET), the increase of pain threshold was reduced obviously. CONCLUSION: OFQ induces hyperalgesia and antagonizes opioid analgesia mediated by mu- and delta-receptors in the brain and by mu- and kappa- but not delta-receptors in the spinal cord of rats.

Enkephalins modulate differentiation of normal human keratinocytes in vitro.

Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine-enkephalin, leucine-enkephalin and the synthetic DADL) inhibited cell differentiation dose-dependently, while beta-endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine-enkephalin and leucine-enkephalin, but not that of DADL. Furthermore, methionine-enkephalin had a slight inhibitory effect on the proliferation of keratinocytes. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.

Cardiovascular effects of Leu-enkephalin in the nucleus tractus solitarius of the rat.

Microinjections of Leu-enkephalin into the dorsal vagal complex induced hypotension and bradycardia. Both naloxone, given at a dose conferring selectivity for mu receptors, and the delta antagonist ICI 154,129 prevented the cardiovascular effects of Leu-enkephalin. Naloxone was also found to decrease the gain of the baroreflex. These results suggest that Leu-enkephalin is involved in cardiovascular regulation through activation of delta-, and possibly mu-, opioid receptors in the dorsal vagal complex.

Antisense oligodeoxynucleotide to the Gi2 protein alpha subunit sequence inhibits an opioid-induced increase in the intracellular free calcium concentration in ND8-47 neuroblastoma x dorsal root ganglion hybrid cells.

In ND8-47 cells, a neuroblastoma x dorsal root ganglion hybrid cell line, activation of delta-opioid receptors induced an increase in the intracellular free calcium concentration ([Ca2+]i) through dihydropyridine-sensitive calcium channels. This effect was mediated by pertussis toxin-sensitive G proteins. The G protein alpha subunits alpha i2, alpha i3, alpha q, and alpha s were detected using Western blots, whereas alpha o and alpha i1 were not found in ND8-47 cell membranes. To identify the specific G protein alpha subunit(s) responsible for the increase in [Ca2+]i, we treated ND8-47 cells with antisense oligodeoxynucleotides (AS) complementary to the mRNA for each G protein alpha subunit (alpha i2, alpha i3, or alpha s), at a concentration of 10 microM, for up to 6 days and examined their effects on opioid-induced increases in [Ca2+]i and on the levels of G protein alpha subunits. [Ca2+]i was measured in adherent cells using the fluorescent dye fura-2. Treatment of cells with alpha i2-AS (10 microM, for 6 days) resulted in a 73% inhibition of the [D-Ser2,Leu5]-enkephalin-Thr-induced increase in [Ca2+]i. In contrast, pretreatment of cells with alpha i3-AS (10 microM, for 6 days) or alpha s-AS (10 microM, for 6 days) had no effect on the [D-Ser2,Leu5]-enkephalin-Thr-induced responses. Western blots indicated that the levels of alpha i2 were decreased when cells were exposed to alpha i2-AS (10 microM) for 6 days, whereas the levels of alpha i3, alpha s, and alpha q were not affected by this treatment. Treatment of the cells with alpha i3-AS or alpha s-AS for 6 days significantly reduced alpha i3 or alpha s levels, respectively. These results indicate that the opioid-induced increase in [Ca2+]i in ND8-47 cells is mediated by G alpha i2.

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis.

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

Responses of rat nucleus submedius neurons to enkephalins applied with micropressure.

The purpose of this study was to determine what effects leucine-enkephalin and D-Ala2-D-Leu5-enkephalin have on both the background and naturally evoked activity of thalamic nucleus submedius neurons responsive to mechanical cutaneous stimulation. Thirty-five neurons in the nucleus submedius were fully characterized during single-unit extracellular recordings as nociceptive, low-threshold mechanoreceptive (LTM) or unresponsive. Micropressure was used to apply the opioids. Eighteen neurons were inhibited; 13 of these were nociceptive and one was LTM. Six units were activated; two of these were nociceptive and three were LTM. The remaining 11 units were unaffected. Opioid responses were tested for antagonism by naloxone in 12 neurons; eight of these responses were antagonized by naloxone. Statistical analyses indicated that the effects of enkephalins on nociceptive neurons were selective for neuronal modality. The opioids also altered the response of some nociceptive neurons to receptive field stimulation. The presence of nociceptive neurons in the nucleus submedius that are selectively inhibited by opioids provides additional support for the involvement of submedius neurons in nociception. The results of this study suggest that this involvement is more than merely transmission of nociceptive input, since the opioids may be selectively modulating the type of information that is transmitted to the cortex.

[Leu5]enkephalin-like immunoreactive amacrine cells are under nicotinic excitatory control during darkness in chicken retina.

Based on the principle that retinal levels of [Leu5]enkephalin-like immunoreactivity (LELI) are set by the rate of release and thus reflect neural activity, we partially defined the dark-associated increase in excitatory control of LELI amacrine cells in chicken. Retinal levels of LELI were measured by radioimmunoassay (RIA). Intravitreal injection of cholinergic antagonists decreased the rate of depletion of LELI during the dark phase, suggesting the presence of cholinergic excitatory control of the LELI neurons. This cholinergic control involves nicotinic rather than muscarinic receptors, as tubocurarine appeared over 100 times more effective than atropine in inhibiting the decrease in retinal levels of LELI in the dark. (The ED50s were estimated at 3.2 and 450 nmol, respectively.) The lack of effect of the antagonists when applied during the light phase, suggest that there is little cholinergic input to the LELI amacrine cells in the light. Superfusing isolated retinas with buffer containing tubocurarine (10 microM) decreased the efflux of LELI by 35%, compared to the spontaneous release during the dark. Atropine (10 microM) had no effect on the release of LELI, and pilocarpine (100 microM) increased the release of LELI from retinas superfused in the light by 20%. We conclude that, in addition to previously reported glycinergic and dopaminergic inhibition, the LELI amacrine cells receive cholinergic excitatory input. A shift in balance between glycinergic and dopaminergic inhibitory, and cholinergic excitatory control may underly the light-driven variation in activity of the LELI neurons in chicken retina.

Involvement of delta 2 opioid receptors in acute dependence on morphine in mice.

The effects that naltriben (NTB) and naltrindole 5'-isothiocyanate (5'-NTII) have on acute morphine dependence were investigated in mice. The degree of acute dependence was estimated by the ED50 value of s.c.-administered naloxone that was required to precipitate withdrawal jumping 4 hr after s.c. injection of 100 mg/kg of morphine sulfate (MS-100). Pretreatments with NTB (10 mumol/kg, s.c., 30 min before MS-100) and 5'-NTII (10 mumol/kg, s.c., 24 hr before MS-100) increased the ED50 values of naloxone compared to those of the respective saline-treated control mice. Although NTB itself (up to 10 mumol/kg s.c.) did not precipitate jumping 4 hr after MS-100, small doses of NTB (1 and 2 mumol/kg, s.c.) administered 30 min before the challenge doses of naloxone increased substantially the ED50 value of naloxone. These results suggest that delta 2 opioid receptors are involved in acute dependence on morphine in mice.

Electrophysiological actions of delta opioids in CA1 of the rat hippocampal slice are mediated by one delta receptor subtype.

Various opioid agonists and antagonists were examined for their ability to alter extracellularly and intracellularly recorded CA1 pyramidal cell activity. All opioid agonists tested, with the exception of [D-ala2]deltorphin II, increased primary population spike amplitude. Of these active agonists, all except DPDPE and p-Cl-DPDPE produced secondary population spikes. DSLET and DAMGO, but not DPDPE, reduced the amplitude of the orthodromically stimulated IPSP. Naltrexone antagonized the actions of all agonists tested. The actions of DPDPE and p-Cl-DPDPE, but not those of DSLET, DAMGO or morphine, were antagonized by the delta antagonist naltrindole. Similarly, the delta antagonist ICI-174,864 blocked the actions of DPDPE, but not DSLET or DAMGO. Based on the inactivity of [D-ala2]deltorphin II and the lack of delta antagonist-sensitive actions of DSLET, the data suggest that the delta 1 subtype is the predominant delta subtype in the CA1 region of the hippocampus.

Inhibition of spinal opioid analgesia by supraspinal administration of selective opioid antagonists.

The effect of intracerebroventricular administration of a selective mu- (CTOP) or delta- (ICI 174,864) opioid receptor antagonist on the antinociceptive effects produced by intrathecal administration of selective mu- (DAMGO), delta- (DPDPE) and kappa- (U50-488H) opioid receptor agonists was evaluated using the Randall-Selitto paw-withdrawal test, in the rat. While the intracerebroventricular administration of CTOP or ICI 174,864, alone, had no effect on nociceptive thresholds, intracerebroventricular administration of CTOP and ICI 174,864 produced marked antagonism of the antinociceptive effects of intrathecal DAMGO. The antinociceptive effects of intrathecal administration of DPDPE or U50,488H were not antagonized by intracerebroventricular administration of CTOP or ICI 174,864. These data suggest that, in the rat, along with the established descending antinociceptive pathways, there is an ascending antinociceptive control mechanism projecting from the spinal cord to the brainstem. The ascending antinociceptive control involves mu- and delta-opioid agonism at supraspinal sites and appears to be mediated selectively by mu-, but not by delta- or kappa-opioid agonism at the spinal level.

Effects of the delta-opioid receptor antagonist naltrindole on antinociceptive responses to selective delta-agonists in post-weanling rats.

1. Antagonism, by the selective delta-opioid receptor antagonist naltrindole, of the antinociceptive effects of [D-Pen2, D-Pen5] enkephalin (DPDPE), [D-Ser2, Leu5, Thr6] enkephalin (DSLET) and D-Ala2 deltorphin I (DELT I) has been studied in 25 day old rats. 2. Antinociception was measured by the 50 degrees C tail immersion test following i.p. administration of agonists and/or antagonists. 3. Dose-related antinociception was observed with DPDPE, DSLET and DELT I and ED75 doses were computed (0.66 mg kg-1, 0.65 mg kg-1, 0.032 mg kg-1 respectively) and used for antagonism studies. 4. Naltrindole (0.01 mg kg-1) significantly attenuated the antinociceptive effects of DPDPE and DSLET with 0.1 mg kg-1 producing complete reversal of the effects of the ED75 dose. In contrast, naltrindole at 0.01 and 0.1 mg kg-1 did not alter antinociceptive responses to DELT I. Naltrindole at 1 mg kg-1 significantly attenuated DELT I antinociception. 5. Naloxone (1 mg kg-1) produced equivalent degrees of antagonism of the antinociceptive effects of DPDPE, DSLET and DELT I. ICI 174,864 (1 mg kg-1) also antagonized antinociception with a differential degree of attenuation (DSLET > DPDPE > DELT I). 6. Naltrindole (1 mg kg-1) had no effect on the antinociception induced by the selective mu-agonist alfentanil (60 micrograms kg-1). Naltrindole, naloxone or ICI 174,864 had no effect on nociceptive latencies. 7. The differential antagonism by naltrindole of the effects of three selective delta-agonists suggests delta-receptor heterogeneity.Further, the lower sensitivity of response to DELT I suggests that this agent may exert its antinociceptive effects at a different 6 receptor subtype from DPDPE or DSLET.

Effect of centrally administered enkephalins on carrageenin-induced paw oedema in rats.

Intracerebroventricularly (icv) administered met-enkephalin, leu-enkephalin, and morphine induced dose-related attenuation of carrageenin-induced acute paw oedema in rats. Naloxone (10 micrograms, icv) antagonized the anti-inflammatory effects of the enkephalins (20 micrograms) and morphine (20 micrograms), but itself induced an anti-inflammatory effect at a higher dose (50 micrograms, icv). The anti-inflammatory effects of the enkephalins, morphine, and the higher dose of naloxone were significantly inhibited by metyrapone, an inhibitor of endogenous corticoid synthesis. The icv-administered doses of the enkephalins and morphine induced insignificant inflammation-attenuating effects when administered i.p. Results suggest that the anti-inflammatory effects of the enkephalins and morphine are exerted through central opiate receptors. Furthermore, the inflammation-attenuating effects of these drugs and the higher dose of naloxone appear to be dependent upon endogenous corticoids, suggesting that activation of the hypothalamo-pituitary-adrenocortical axis may be involved.

Differential antagonism of opioid delta antinociception by [D-Ala2,Leu5,Cys6]enkephalin and naltrindole 5'-isothiocyanate: evidence for delta receptor subtypes.

The present study has investigated the direct opioid delta receptor-mediated antinociception produced by i.c.v. administration of the highly selective delta agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, as well as that of the less delta-selective [D-Ser2,Leu5,Thr6]enkephalin (DSLET), by using two novel nonequilibrium opioid antagonists, [D-Ala2,Leu5,Cys6] enkephalin (DALCE) and naltrindole 5'-isothiocyanate (5'-NTII). At times ranging from 8 to 48 hr after a single i.c.v. pretreatment of mice with 5'-NTII, the antinociceptive effects of [D-Ala2] deltorphin II were significantly antagonized. In contrast, 5'-NTII pretreatment at times between 10 min and 24 hr failed to antagonize the antinociceptive effects of DPDPE. Previous studies have shown that pretreatment with i.c.v. DALCE produces a dose- and time-related antagonism of DPDPE, but not morphine, antinociception. However, pretreatment with i.c.v. DALCE failed to antagonize the antinociceptive effects of [D-Ala2]deltorphin II. Similarly, i.c.v. administration of DSLET produced time- and dose-related antinociception which was partially antagonized by either beta-funaltrexamine (beta-FNA) or by ICI 174,864 (N,N-dialyl-Tyr-Aib-Aib-Phe-Leu-OH), suggesting mixed activity at mu and delta receptors. ICI 174,864 produced essentially complete antagonism of DSLET antinociception in beta-FNA-pretreated mice. Pretreatment with 5'-NTII (at -8 to -48 hr), blocked the antinociception produced by DSLET in control or in beta-FNA-pretreated mice. In contrast, pretreatment with DALCE failed to antagonize the antinociception produced by i.c.v. DSLET in either control or in beta-FNA-pretreated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

[The influence of piracetam on the effects of narcotic analgesics]. / Vliianie piratsetama na éffekty narkoticheskikh anal'getikov.

In experiments on white male mice there was studied the influence of piracetam (250-300 mg/kg) on the analgesic effect of ligands of different types of opioid receptors (morphine, 7.5 mg/kg, DADLE, 7.5 mg/kg, pentazocine, 15 mg/kg) and also on the action of morphine concerning the cardiovascular system and respiration. Piracetam was shown to possess the antagonistic properties with respect to some effects of morphine, however they are not of the universal character and do not depend on the interaction with a certain type of opioid receptors.

Interaction of newly synthesized N-cyclopropylmethyl derivatives of (-)-6 beta-acetylthionormorphine with opioid receptors.

1. Analgesic activities of N-cyclopropylmethyl derivatives of (-)-6 beta-acetylthionormorphine, KT-89 and KT-90 and their interactions with opioid receptors were studied. 2. KT-89 and KT-90, as well as morphine inhibited the twitch response of the guinea-pig ileal preparation to electrical stimulation. Their pD2 values indicated that KT-89 and KT-90 are about 6.5 and 10 times as potent as morphine, respectively. In guinea-pig ileal preparation KT-89 and KT-90 also behaved as a mu-antagonist. 3. In rabbit vas deferens which contains kappa-receptors, these substances inhibited the twitch response to electrical stimulation and were about 6 times as potent as dynorphin. 4. Their effects on specific binding of [3H]naloxone (mu-selective ligand), [3H]ethylketocyclazocine (kappa-selective ligand) and [3H]D-Ala2-D-Leu5-enkephalin (delta-selective ligand) to the synaptosomal fractions from rat brain were tested. Though both drugs had a nonselectively high affinity to mu-, kappa- and delta-receptors, affinities of KT-89 and KT-90 to kappa-receptors were about 6 and 13 times higher than that of morphine, respectively. 5. Analgesic activities of KT-89 and KT-90 were 6 and 10 times as potent as morphine in an acetic acid-induced writhing test, and 4 and 5 times as potent in a pressure test. 6. The present results suggest that KT-89 and KT-90 induced analgesic actions are mediated through an activation of kappa-receptors. Both the drugs acted as delta-receptor antagonists. Further experiments are needed to study effects of their property as a delta-antagonist on analgesic action.