D-Pen2,D-Pen5]enkephalin Research Articles

Pharmacological characterization of KT-90 using cloned μ-, δ- and κ-opioid receptors

We analyzed the pharmacological characteristics of (−)-3-acetyl-6β-acetylthio- N-cyclopropylmethyl-normorphine (KT-90) using Chinese hamster ovary (CHO) cells expressing cloned rat μ-, δ- and κ-opioid receptors. KT-90 displaced the specific binding of the following radiolabeled ligands selective to the μ-, δ- and κ-opioid receptors, [ 3H][ d-Ala 2,MePhe 4,Gly(ol) 5]enkephalin (DAMGO), [ 3H][ d-Pen 2, d-Pen 5]enkephalin (DPDPE), [ 3H] (+)-(5α,7α,8β)- N-methyl- N-[7-(1-pyrrolidinyl)-1-oxaspiro-4,5)dec-8-yl]benzeneacetamide (U69,593), with K i values of 3.3 ± 0.7, 22.8 ± 1.5 and 1.9 ± 0.3 nM, respectively. In CHO cells expressing the μ-, δ- and κ-opioid receptors, KT-90 inhibited forskolin (10 μM)-induced cyclic AMP accumulation in a concentration-dependent manner with IC 50 values of 2337 ± 750, 17.3 ± 4.6 and 2.0 ± 0.1 nM, respectively. The maximal inhibitory effects of KT-90 in the cells expressing μ-, δ- and κ-opioid receptors were significantly lower than those of the type-selective agonists DAMGO, DPDPE and U69,593, respectively. These results indicated that KT-90 acts as a partial agonist on μ-, δ and κ-opioid receptors. KT-90 (10 and 100 nM), when added with morphine, produced a rightward shift of the concentration-response curve of morphine to inhibit the cyclic AMP accumulation in CHO cells expressing μ-, but not δ- or κ-, opioid receptors. This finding is consistent with the findings that lower doses of KT-90 antagonize morphine analgesia in vivo.

Gastric effects of μ-, δ- and κ-opioid receptor agonists on brainstem unitary responses in the neonatal rat

Single units in the medial subnucleus of the nucleus tractus solitarii, responding to electrical stimulation of subdiaphragmatic vagal fibers, were recorded extracellularly in an in vitro neonatal rat brainstem-gastric preparation. Selective opioid receptor agonists were applied only to the gastric compartment of the bath chamber and therefore, the brainstem functions of the preparation were not affected. The peripheral gastric effects of the μ-opioid receptor agonist, [ d-Ala 2, N-MePhe 4,Gly 5-ol]enkephalin (DAMGO) and κ-opioid receptor agonist, { trans-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate hydrate} (U-50,488H), were evaluated on 69 tonic units that received the subdiaphragmatic vagal input. For approximately 75% of the units observed, DAMGO (1.0 μM; IC 70: 80 nM) and U-50,488H (1.0 μM; IC 70: 200 nM) induced a concentration-dependent inhibition of 62.7 ± 8.9% (mean ± S.D.) and 50.6 ± 6.2% of the control level of the brainstem neuronal activity, respectively. The μ-opioid selective receptor antagonist, naltrexone and non-selective opioid receptor antagonist, naloxone, respectively, blocked the inhibitory effects by DAMGO and U-50,488H. The δ-opioid receptor agonist, [ d-Pen 2, d-Pen 5]enkephalin (DPDPE) (10 μM; IC 70: 400 nM) produced a lesser extent of inhibition of 21.9 ± 8.0% in only 10 out of 51 (20%) neurons tested, and this effect was blocked by naloxone. The area of the stomach where gastric opioid receptors contributed most to brainstem unitary activity was also examined. This was achieved by comparing the opioid effects on a whole-stomach preparation to its effects on a partial-stomach preparation. Our data indicated that the distal stomach containing the pylorus played a key role in the gastric effects of μ- and κ-opioid receptors on brainstem neuronal activity. These results suggest that the μ- and κ-opioid receptors of the distal stomach are important in modulation of brainstem neuronal activity and may play a role in regulating the digestive process.

Effect of nitric oxide synthase inhibition on tolerance to the analgesic action of D-Pen 2, D-Pen 5 enkephalin and morphine in the mouse

The effects of N G-nitro-L-arginine (NNA), an inhibitor of nitric oxide synthase (NOS) on the development of tolerance to the analgesic action of D-Pen 2, D-Pen 5 enkephalin (DPDPE), a δ 1 opioid receptor agonist, and morphine were determined in the mouse. Tolerance to DPDPE was induced in male Swiss-Webster mice by twice daily intracerebroventricular (i.c.v.) injections of the drug (20 μg/mouse) for 4 days. NNA was injected intraperitoneally (i.p.) 10 min before each injection of DPDPE. Chronic injections of DPDPE resulted in development of tolerance to its analgesic action. Multiple injections of NNA by itself did not modify the analgesic response to DPDPE. Concurrent injections of NNA did not affect the development of tolerance to the analgesic action of DPDPE. Twice daily injections of morphine (15 mg/kg s.c.) for 4 days resulted in the tolerance to its analgesic action. Concurrent administration of NNA with morphine attenuated the development of tolerance to morphine. It is concluded that NOS inhibition attenuates morphine, but not δ 1 opioid agonist-induced tolerance in the mouse.

Alkaloid delta Agonist BW373U86 Increases Hypoxic Tolerance

Activation of delta opioid receptors increases survival time during acute, lethal hypoxia in mice. delta Agonists therefore present a promising avenue for therapeutic application to reduce the morbidity and mortality associated with clinical hypoxia in settings such as drowning, head injury apnea, and complicated childbirths. However, most delta agonists now available are peptides, and may have limited clinical utility. In the present study, we evaluate the neuroprotective ability of an alkaloid delta agonist, BW373U86. Alkaloid compounds, due to increased stability and increased systemic distribution, may be more favorable for clinical use. We found that BW373U86, like the peptide delta agonist, DPDPE ([D-Pen2, D-Pen5]-enkephalin), increases survival time of mice during lethal hypoxia. The mechanism of neuroprotection induced by delta receptor activation appears to involve decreasing body temperature. Further, using selective opioid receptor antagonists, it appears that BW373U86 exerts these neuroprotective effects by acting at delta-opioid receptors.

Variations of motor activity following intraventricular (D-Pen 2, D-Pen 5)-enkephalin administration: Interaction with acute uncontrollable foot-shock in mice

The effects of intraventricular administration of (D-Pen 2, D-Pen 5)-enkephalin (DPDPE) (0.005, 1.0, and 2.5 μg/μl in a 1-μl vol.) on horizontal activity, rearing, and exploratory head dipping were assessed among CD-1 mice exposed to acute uncontrollable foot-shock 15 min following stressor termination. Foot-shock reduced horizontal activity, rearing, and exploratory head dipping during the immediate 15-min poststressor interval. Mice challenged with 0.005 μg DPDPE were behaviorally indistinguishable from vehicle-treated subjects. The 1.0-μg dose of DPDPE increased horizontal activity in stressed and nonstressed subjects. Intraventricular infusion of 2.5 μg DPDPE potentiated horizontal activity in previously stressed mice but had no effect in nonstressed animals. The suppression of rearing and exploratory head dipping following uncontrollable foot-shock was not ameliorated by the δ-receptor agonist, and DPDPE was without effect on rearing and exploratory head dipping in nonstressed animals. Potential neurochemical mechanisms associated with the expression of these Stressor associated behaviors are discussed.

Synergy between [formula omitted] receptors mediates adenosine release from spinal cord synaptosomes

Morphine releases adenosine from the spinal cord and this contributes to spinal antinociception. The present study examined possible interactions between μ- and subclasses of δ-opioid receptors in the release of adenosine. Nanomolar (10 −8, 10 −9 M) concentrations of morphine release adenosine from spinal cord synaptosomes under conditions of partial depolarization with elevated K +, and this component of release is mediated by activation of μ-opioid receptors. Subnanomolar (10 −10, 10 −11 M) concentrations of the μ-opioid receptor agonists morphine, [ N-MePhe 3, d-Pro 4]morphiceptin, and [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO) have minimal effects on the release of adenosine from the spinal cord. However, [ d-Pen 2, d-Pen 5]enkephalin (DPDPE), a δ 1-opioid receptor agonist, and [ d-Ala 2,Cys 4]deltorphin, a δ 2-opioid receptor agonist, at doses which exhibit no intrinsic effects (10 −8 and 10 −7 M), shifted the dose-response curve for μ-opioid receptor-evoked adenosine release to the left in a dose-dependent manner. DPDPE was more potent than [ d-Ala 2,Cys 4]deltorphin when combined with the highly selective μ-opioid receptor agonist [ N-MePhe 3, d-Pro 4]morphiceptin, but these agents showed similar activity with the less selective agonists DAMGO and morphine. Simultaneous activation of μ- and δ-opioid receptors generates a synergistic release of adenosine from spinal cord synaptosomes. Although agonists for both δ 1- and δ 2-opioid receptor subtypes produce this response, the δ 1-opioid receptor agonist is more potent at eliciting this effect when the most selective μ-opioid receptor ligand is used.

Continuous cocaine administration enhances μ- but not δ-opioid receptor-mediated inhibition of adenylyl cyclase activity in nucleus accumbens

Cocaine alters opioid receptor densities in rat brain. To investigate the functional consequences of such opioid receptor changes, adenylyl cyclase activity was measured in rat nucleus accumbens and caudate putamen following continuous cocaine administration (50 mg/kg/day, 7 days). In the nucleus accumbens chronic cocaine led to an increase in both the number of μ-opioid receptors and the maximal inhibition of adenylyl cyclase activity by DAMGO ([ d-Ala 2, N-methyl-Phe 4,Glyol]enkephalin). There was no effect on inhibition of adenylyl cyclase activity by DPDPE ([ d-Pen 2, d-Pen 5]enkephalin). There were no changes in the caudate putamen. Thus, continuous cocaine administration for 7 days results in a selective increase in μ-opioid receptor-mediated effector function in the nucleus accumbens.

Effect of antagonism of the NMDA receptor on tolerance to [D-Pen2,D-Pen5]enkephalin, a delta 1-opioid receptor agonist.

The effects of MK-801, a noncompetitive antagonist of the N-methy1-D-aspartate (NMDA) receptor, and of LY 235959, a competitive antagonist of the NMDA receptor, on the development of tolerance to the analgesic action of [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta 1-opioid receptor agonist were determined in mice. Tolerance to DPDPE was induced in male Swiss Webster mice by twice daily ICV injections of the drug (20 micrograms/mouse) for 4 days. To determine the effects of NMDA receptor antagonists, the drugs were injected IP in appropriate doses 10 min before each injection of DPDPE. The doses of MK-801 were 0.01, 0.03, and 0.10 mg/kg whereas those of LY 235959 were 1, 2, and 4 mg/kg. Chronic administration of NMDA receptor antagonists by themselves did not alter the tail flick response or the analgesic effect of DPDPE (10 micrograms/mouse, ICV). MK-801 given concurrently with DPDPE dose-dependently inhibited the development of tolerance to DPDPE, the significant effect being observed at 0.10 mg/kg dose. Similar inhibitory effects on the development of tolerance to DPDPE were observed with LY 235959 at 1, 2, and 4 mg/kg doses. It is concluded that antagonism of NMDA receptors blocks tolerance to the analgesic action of delta 1-opioid receptor agonist in the mouse.

The effect of intrathecal opioid-receptor agonists on visceral noxious stimulation in rabbits

BACKGROUND & AIMS: Conflicting results have been published concerning the effects of different opioid-receptor agonists against visceral noxious stimulation. The introduction of colorectal distention facilitates research in this field. The aim of this study was to examine intrathecally administered opioid agonists against colorectal distention in conscious rabbits. METHODS: Rabbits were equipped with a subcutaneous intrathecal injection system. Colorectal distention was induced by inflation of a balloon inserted into the descending colon. The test parameter was the pressure eliciting a characteristic visceromotor response. Examinations were performed before and after administration of the following drugs: morphine, U50488H, [D-Pen2, D- Pen5]enkephalin (DPDPE), naloxone, MR2266, naltrindole, saline, and acidified saline. RESULTS: The visceromotor response to colorectal distention was inhibited in a dose-dependent fashion by intrathecal opioids acting as agonists at all three types of opioid receptors. Morphine was antagonized more effectively by intrathecal than intramuscular naloxone. U50488H and DPDPE were equally antagonized by the specific antagonists MR2266 and naltrindole. Electrical thresholds in the lumbar region were increased, although they remained unaltered in the cervical region after administration of all three agonists. CONCLUSIONS: Intrathecal administration of different opioid agonists produces a dose-dependent spinal effect. The rank order of potencies in this model is DPDPE > U50488H > morphine > saline = 0. (Gastroenterology 1996 Jan;110(1):139-46)

Systemic and supraspinal, but not spinal, opiates suppress allodynia in a rat neuropathic pain model

The effects of intraperitoneal (IP), intracerebroventricular (ICV) and intrathecal (IT) opiates were studied in the rat neuropathic pain model of Kim and Chung. Dose dependent reduction of allodynia was observed after IP and ICV morphine, but not after IT morphine, IT or ICV c[ d-pen2 d-pen5]enkephalin (DPDPE) (δ agonist), or IT or ICV U50488H (κ agonist). The effects of ICV morphine were blocked by IP naloxone, but not by IT methysergide, phentolamine or 8-sulfophenyltheophylline. Catalepsy (immobility) was observed after IT, ICV and IT morphine but this was not reliably associated with a reduction of allodynia. IP and ICV morphine may thus reduce tactile allodynia via supraspinal, but not spinal, μ opioid receptors.

Delta opioid receptors: reflexive, defensive and vocal effective responses in female rats

Ultrasonic vocalizations may be an expression of the affective pain response in laboratory animals. The present experiment compares the effects of morphine to the delta agonist, DPDPE (D-Pen2,D-Pen5 enkephalin) on a range of reflexive, behavioral and affective responses during an aggressive interaction. In experiment 1, naive female Long-Evans rats received morphine (0, 1, 3, 6, 10 micrograms ICV), or DPDPE (0, 30, 60, 100 micrograms ICV). In experiment 2, female rats were treated with naltrindole (1.0 mg/kg IP) 20 min before DPDPE (0, 60, 100 micrograms ICV). The following endpoints were measured: (1) latency to tail flick in response to heat stimuli; (2) high (33-65 kHz) and low (20-32 kHz) frequency ultrasonic and audible vocalizations; (3) defensive behavior; and (4) motoric activity. Following a brief exposure to attack, rats were threatened by the aggressor but protected from further attack by a large, wire mesh cage, thereby allowing for continued behavioral and vocal measurement without the risk of physical injury; video and audio recordings were made during the attack and then during a portion of the protected encounter (2 min). Morphine suppressed pain reactions varying in complexity from a spinal reflex, to an organized escape reaction, to an affective vocal response. The delta agonist, DPDPE, attenuated high frequency ultrasonic calling and tail flick responding. Defensive behaviors were also modulated by DPDPE at doses that had no effect on walking or rearing, indicating behavioral specificity. By contrast, doses of morphine that decreased defensive upright and escape also decreased motor activity. In female rats, morphine and DPDPE share a common profile of effects on a range of functional end-points, but DPDPE appears to modulate more selectively the reactions related to aversiveness without exerting sedative effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of calcitonin gene related peptide and its receptor binding sites during the development of tolerance to mu and delta opioids.

Calcitonin gene related peptide (CGRP), one of the most abundant peptides in the spinal cord, is localized in primary afferents and released following nociceptive stimuli. Its colocalization and corelease with substance P, a well-known nociceptive neuropeptide, support the importance of CGRP in pain mechanisms. However, its distinctive function in that regard remains to be fully established. Recently, we reported that increases in CGRP-like immunostaining and decrements in specific 125I-labelled human CGRP alpha ([125I]hCGRP alpha) binding sites in the spinal cord were correlated with the development of tolerance to the spinal antinociceptive action of a mu opioid agonist, morphine. The goal of the present study was to investigate whether the development of tolerance to other classes of opioids, namely, delta and kappa agonists, can also alter CGRP-like immunostaining and receptors in the rat spinal cord. The antinociceptive effects of all opioids were monitored by the tail-immersion test. Tolerance to their antinociceptive properties was induced by the infusion for 7 days of mu (morphine sulfate, 7.5 micrograms/h), delta D([D-Pen2,D-Pen5]enkephalin (DPDPE), 2.0 micrograms/h), and kappa (U-50488H, 10.0 micrograms/h) related agonists at the spinal level (L4), using osmotic minipumps. We confirmed that rats chronically treated with morphine showed significant decreases in [125I]CGRP alpha binding in laminae I, II, and III of the L4 spinal cord, while CGRP-like immunostaining was increased in these same laminae. Similar effects were observed following a treatment with the delta agonist, DPDPE, while the kappa agonist, U-50488H, apparently only slightly decreased [125I]CGRP alpha] binding in lamina II. Binding in other laminae and CGRP-like immunostaining were not affected. These results suggest a specific interaction between spinal CGRP systems and the development of tolerance to the spinal antinociceptive effects of mu- and delta-related agonists.

ACTH-(1–24) blocks the decompensatory phase of the haemodynamic response to acute hypovolaemia in conscious rabbits

Graded caval occlusion in conscious rabbits caused a biphasic cardiovascular response. Phase I was characterized by a fall in systemic vascular conductance so that arterial pressure was maintained. When cardiac output had fallen to 64 ± 3% of its baseline level, phase II supervened. During phase II, conductance rose abruptly and arterial pressure fell to a life-threatening level (< 40 mm Hg). Intravenous (i.v.) or central (fourth ventricular) administration of the adrenocorticotrophin (ACTH) fragment ACTH-(1–24) prevented the occurrence of phase II. The central dose of ACTH-(1–24) needed to block the occurrence of phase II was ∼ 39 times less than the i.v. dose. Central administration of the δ 1-opioid receptor agonist [ d-Pen 2, d-Pen 5]enkephalin (DPDPE) reversed this effect of both central and i.v. ACTH-(1–24). I.v. ACTH-(1–24) also lowered arterial pressure while raising cardiac output and vascular conductance. These effects were insensitive to propranolol and hyoscine methyl bromide, and were not mimicked by cortisol or adrenaline. It is concluded that ACTH-(1–24) has an acute, adrenal-independent, peripheral vasodilator effect as well as a central, anti-shock, effect.

Effects of Intrathecal mu, delta, and kappa Agonists on Thermally Evoked Cardiovascular and Nociceptive Reflexes in Halothane-Anesthetized Rats

Despite significant opioid binding in the intermediolateral cell column, the effects of intrathecal injections of mu, delta, and kappa opioid agonists on the cardiovascular response to noxious stimulation have not been examined systematically. The pharmacology of intrathecally administered opioid agonists (mu, morphine, [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAGO); delta, metkephamid, [D-Ala2-D-Leu5]enkephalin (DADL), [D-Pen2,D-Pen5]enkephalin (DPDPE); kappa, U50488H and PD117,302) or agonist-antagonist (nalbuphine) on somatomotor (tail-flick) and cardiovascular changes (blood pressure and heart rate) evoked by immersing the tail in 53 degrees C water were examined in rats anesthetized with halothane (0.75%) and in which intrathecal catheters had been chronically implanted. Intrathecal administration of mu and delta, but not kappa agonists or agonist-antagonist produced a dose-dependent block of tail-flick and evoked cardiovascular responses with the order of activity being as follows: DAGO > metkephamid DADL > morphine > DPDPE >> nalbuphine = PD117,302 = U50488H = 0. These effects were reversed readily by the opioid antagonist naloxone. In addition, intrathecal administration of mu and delta but not kappa or agonist-antagonist had little effect on resting heart rate and blood pressure. These data indicate that the agonist occupancy of spinal mu and delta, but not kappa agonists can profoundly modulate the autonomic and somatomotor response evoked by high threshold thermal stimuli.

Evidence for lack of modulation of μ‐opioid agonist action by δ‐opioid agonists in the mouse vas deferens and guinea‐pig ileum

1. There is evidence from in vivo studies for an interaction of mu- and delta-opioid ligands. In the present work this concept has been investigated using the mouse vas deferens and guinea-pig ileum myenteric plexus-longitudinal preparations. 2. In field stimulated vasa deferentia of the mouse, co-administration of sub-effective concentrations of the delta-opioid agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) and [Met5]- or [Leu5]enkephalin had no effect on the dose-response curves of the mu-agonists [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAMGO) and morphine. Similarly, the delta-opioid agonists did not alter the potency of morphine and DAMGO when added at different times prior to the mu-opioid agonists, or when EC50 concentrations of delta-opioid ligands were co-administered. Compounds with preferred activity for the putative delta 1-(DPDPE) or delta 2-([D-Ala2,Glu4]deltorphin II (Delt II)) opioid receptors were ineffective in this respect. 3. The guinea-pig ileum contains delta-opioid receptors. No function of these receptors in mediating blockage of field-stimulated contractions was observed with ligands having affinity for the putative delta 1 or delta 2 subtypes nor were the agonists able to modulate responses to mu-opioid ligands in this tissue. 4. The results demonstrate the modulation of mu-opioid agonists by delta-opioid agonists does not occur in the isolated peripheral tissues examined. Thus the findings do not support the concept of a functional coupling of opioid receptors, though the results may be explained by differences between opioid systems in the brain and peripheral tissues examined.

Non-NMDA receptor antagonist attenuates antinociception induced by morphine but not β-endorphin, D-Pen 2-D-Pen 5-enkephalin, and U50, 488H administered intracerebroventricularly in mice

The antinociception induced by morphine but not β-endorphin, D-Pen 2-D-Pen 5-enkephalin (DPDPE), or U50, 488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide) administered intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the N-Methyl-D-Aspartic Acid (NMDA) receptor. The present study was designed to determine if non-NMDA receptors are involved in opioid-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. Various doses of CNQX (6-Cyano-7-nitroquinoxaline-2,3-dione), a competitive non-NMDA receptor antagonist, (0.001 to 0.5 μg) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. CNQX (0.01 to 1 μg, i.c.v.) dose-dependently attenuated the inhibition of the tail-flick response induced by morphine (1 μg). However, inhibition of the tail-flick response induced by i.c.v. administered β-endorphin (1 μg), DPDPE (10 μg), or U50, 488H was not affected by i.c.v. administered CNQX. It is concluded that non-NMDA receptors are involved in i.c.v. morphine-induced antinociception. However, non-NMDA receptors are not involved in i.c.v. administered β-endorphin-, DPDPE-, and U50, 488H-induced antinociception at the supraspinal level.

Opioid effects on spinal [ 3H]5-hydroxytryptamine release are not related to their antinociceptive action

Several opioid compounds were evaluated for an ability to modulate the K +-stimulated release of [ 3H]serotonin ([ 3H]5-hydroxytryptamine, [ 3H]5-HT) from rat spinal cord synaptosomal and tissue slice preparations. Selective κ-opioid receptor agonists depressed K +-stimulated release of the radiolabelled transmitter from both tissue preparations, an effect which was reversed by norbinaltorphimine. Conversely, the selective μ- and δ-opioid receptor agonists [ d-Ala 2,NMePhe 4,Gly-ol 5]enkephalin (DAMGO) and [ d-Pen 2, d-Pen 5]enkephalin (DPDPE), respectively, enhanced the K +-stimulated release of [ 3H]5-HT. This effect was only seen using the tissue slice preparation. When used at concentrations near its reported K d for μ-opioid receptors, the selective μ-opioid receptor antagonist d-Phe-Cys-Tyr- d-Trp-Orn-Thr-Pen-Thr-NH 2 (CTOP) blocked the action of DAMGO, but had no effect on the action of DPDPE. However, higher concentrations of CTOP, as well as all effective concentrations of selective δ-opioid receptor antagonists, blocked the action of both DAMGO and DPDPE. All agonist effects on spinal 5-HT release, regardless of the tissue preparation, were only seen at high (μM) concentrations. Moreover, effects of the opioid agonists were not consistent with the reported involvement of spinal 5-HT neurotransmission in the mediation of their antinociceptive action. Thus, the ability of opioids to modulate spinal 5-HT release appears to be of minimal physiological significance.

Treatment with antisense oligodeoxynucleotide to a conserved sequence of opioid receptors inhibits antinociceptive effects of delta subtype selective ligands.

Previous work has suggested the existence of subtypes of the delta opioid receptor (DOR) which have been termed delta1 and delta2. [D-Ala2, Glu4]deltorphin has been suggested to selectively elicit antinociception via the delta2 receptor while [D-Pen2, D-Pen5]enkephalin (DPDPE) is thought to act via the delta1 receptor. Treatment with an antisense oligodeoxynucleotide (oligo) directed towards the N-terminal portion of the cloned DOR has been demonstrated to selectively inhibit the antinociceptive actions of [D-Ala2, Glu4]deltorphin, but not of DPDPE, suggesting that the cloned DOR corresponds to that pharmacologically defined as delta2. Here, an antisense oligo (or a mismatch sequence) was designed to target a conserved region of the cloned mu, delta and kappa opioid receptor. These oligos were employed in order to determine whether the antinociceptive effects of [D-Ala2, Glu4]deltorphin, as well as DPDPE, could be inhibited. The data indicate that the antinociceptive actions of both ligands were inhibited by treatment with this antisense, but not with the mismatch oligo. Taken together, the results of the treatments with oligos directed towards the N-terminal portion of the cloned DOR and with that directed to the conserved region of the opioid receptors suggest that (a) DPDPE effects are mediated by a subtype of the DOR which shares a domain common to the cloned opioid receptors, and (b) the N-terminal region differs between these putative DOR subtypes.

Characterization of functional opioid delta receptors in a luteinizing hormone-releasing hormone-producing neuronal cell line.

Endogenous opioids participate in the regulation of gonadotropin secretion through an influence on the release of the hypothalamic LHRH. However, it is not clear whether opioids exert a direct effect on LHRH-producing neurons or interfere with other systems able to influence LHRH release. A neuronal LHRH-producing cell line (GT1) developed recently provides a good model to study the mechanisms controlling LHRH release. In the present study, the presence of opioid receptors on a subclone of GT1 cells (GT1-1) has been investigated. A specific and saturable binding of the 3H-labeled nonselective opioid ligand diprenorphine ([3H]DIP) was detected by a receptor binding assay on both intact GT1-1 cells and crude membrane preparations obtained from these cells. Analysis of saturation curves revealed that [3H]DIP apparently binds to a single class of sites with a Kd of 0.2 nM and a binding capacity of 125 fmol/mg protein, corresponding to approximately 20,000 sites/cell. Selective displacement of the binding of [3H]DIP to GT1-1 cells by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, [D-Pen2,D-Pen5]enkephalin (DPDPE), and U50488H, which are selective ligands, respectively, for mu-, delta-, and kappa-receptors, was also evaluated. Only the specific delta-ligand DPDPE produced a significant inhibition of the binding of [3H]DIP. [D-Ala2,N-Me-Phe4,Gly5-ol]Enkephalin and U50488H were totally ineffective. The inhibitory effect of the agonist DPDPE on the binding of [3H]DIP was decreased by the presence of sodium ions, a typical characteristic of the binding of agonists to opioid receptors. Finally, it has been observed that treatment with prostaglandins E1 and E2 produces a dramatic increase in cAMP accumulation in GT1-1 cells, and DPDPE is highly effective in suppressing this effect. On the basis of these results, it is possible to postulate the presence of functional delta-opioid receptors on GT1-1 cells. By extrapolation, one might suggest that endogenous opioids may affect LHRH neurons by two mechanisms: a direct one, acting via delta-receptors, and an indirect one, through the activation of neurons impinging on the LHRH system, which uses mu-receptors.

New developments of the μ-selective opioid 3,8-diazabicyclo[3.2.1]octanes

BW 373U86 is a novel, non-peptidic δ-opioid receptor ligand with agonist properties in mouse brain and in the mouse isolated vas deferens. The sensitivity of BW 373U86, and of the peptidic δ-opioid agonists [d-Pen2, D-Pen5]enkephalin (DPDPE) and [d-Ala2,Glu4]deltorphin, to regulation by guanine nucleotides and sodium was evaluated in competition studies against the 5 selective radioligand [3H]naltrindole. The IC50 values for DPDPE and [d-Ala2,Glu4]deltorphin were significantly increased in brain and mouse vas deferens in the presence of Gpp(NH)p and NaCl. In contrast, the IC50 values for BW 373U86 were not altered in the presence of Gpp(NH)p and NaCl in either tissue. The data indicate that the agonist properties of BW 373U86 may not be affected by the supposed uncoupling of the α-subunit of the G-protein from a receptor thought to be G-protein linked.