Endogenous Enkephalins Research Articles

Vulnerability to stressor-induced disturbances in self-stimulation from the dorsal and ventral A10 area: Differential effects of intraventricular D-Ala 2-Met 5-enkephalinamide, D-Ala 2, N-Me-Phe 4, Gly-Ol 5-enkephalin, and D-Pen 2, D-Pen 5- enkephalin administration

D-Ala 2-Met 5-enkephalinamide (DALA) (1.0 μg/μl) was administered intraventricularly to mice responding for electrical stimulation from the dorsal or ventral aspects of the VTA immediately prior to footshock (Experiment 1). Predictably, footshock reduced self-stimulation from the dorsal but not the ventral VTA immediately, 24, and 168 h following the stressor. Intraventricular DALA administration effected a partial attenuation of stressor-induced self-stimulation reductions from the dorsal VTA immediately and 24 h poststressor. Deficits appeared among DALA-Shocked mice responding for brain stimulation from the ventral VTA during comparable test intervals. The long-term depressant influence of footshock on self-stimulation from the dorsal VTA was abolished among DALA-treated mice and DALA-associated reductions in self-stimulation from the ventral A10 region among stressed mice were not evident 1 week later. Administration of D-Ala 2, N-Me-Phe 4, Gly-Ol 5-enkephalin (DAGO) (0.01 μg/μl) or D-Pen 2, D-Pen 5-enkephalin (DPDPE) (1.0 μg/μl) intraventricularly prior to footshock effected an immediate and a delayed antagonism, respectively, of the stressor on self-stimulation from the dorsal VTA, which persisted for 1 week. Prophylactic administration of 0.001 μg/μl DAGO or 0.01 μg/μl DPDPE prior to the stressor failed to influence self-stimulation from the ventral VTA (Experiment 2). Administration of 0.01 μg/μl DAGO or 1.0 μg/μl DPDPE among mice responding for brain stimulation from the dorsal VTA following footshock produced a weak therapeutic effect immediately poststressor, but effected protracted amelioration of footshock-induced reductions of self-stimulation from the dorsal VTA (Experiment 3). Taken together, μ, δ, and μ-δ activation influenced self-stimulation differentially from the dorsal and ventral VTA according to the temporal order of opioid peptide challenge relative to stressor imposition. These data are discussed with respect to stressors, motivational alterations, and the putative modulating influence of endogenous enkephalin activity in subareas of the VTA.

Involvement of δ-opioid receptors in the effects induced by endogenous enkephalins on learned helplessness model

Pharmacological, neurochemical and behavioural findings support a possible role of endogenous opioids in clinical depression. There is evidence from animal studies that δ-opioid receptors are involved in several behavioural responses to opioids, including motivational activities. In the present study, the mixed enkephalin catabolism inhibitor, RB 101 ( N( R, S)-2-benzyl-3[( S)-(2-amino-4-methylthiobutyldithio]-1-oxopropyl)- l-phenylalanine benzyl ester) (1.25, 2.5 and 5 mg/kg), induced a dose-dependent antidepressant-like effect in a learned helplessness model. Thus, RB 101 reversed escape deficits in rats previously subjected to inescapable shocks, suggesting the involvement of endogenous enkephalins in depression. Similar effects were observed after administration of the selective δ-opioid receptor agonist, BUBU (Tyr-D.Ser-( O- tert-butyl)-Gly-Phe-Leu-Thr(O-Tet-butyl-OH)) (1 and 2 mg/kg). Moreover, RB 101 effects were antagonized by administration of naltrindole (NTI) (0.1 mg/kg), which points to a preferential involvement of δ-opioid receptors in this enkephalin-controlled behaviour. As RB 101 has been reported to be almost devoid of opiate-related side-effects, it could represent a promising alternative in the treatment of depressive patients who are unresponsive to, or intolerant of, classical antidepressants.

Submillimolar levels of calcium regulates DNA structure at the dinucleotide repeat (TG/AC)n.

Submillimolar levels of calcium, similar to the physiological total (bound + free) intranuclear concentration (0.01-1 mM), induced a conformational change within d(TG/AC)n, one of the frequent dinucleotide repeats of the mammalian genome. This change is calcium-specific, because no other tested cation induced it and it was detected as a concentration-dependent transition from B- to a non-B-DNA conformation expanding from 3' end toward the 5' of the repeat. Genomic footprinting of various rat brain regions revealed the existence of similar non-B-DNA conformation within a d(TG/AC)28 repeat of the endogenous enkephalin gene only in enkephalin-expressing caudate nucleus and not in the nonexpressing thalamus. Binding assays demonstrated that DNA could bind calcium and can compete with calmodulin for calcium.

Role of endogenous pro-enkephalin A-derived peptides in human T cell proliferation and monocyte IL-6 production

In this paper, we describe that met-enkephalin and/or enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes. The peptides are produced after stimulation with the mitogenic monoclonal antibodies anti-CD2.1/2.2 and anti-CD28. In monocytes, enkephalin synthesis was induced by stimulation with lipopolysaccharide. We demonstrate here that these immune cell-derived enkephalins play an important regulatory role in the immune response. By using an anti-sense oligonucleotide strategy we could block the production of enkephalins. Blockade of the production of met-enkephalin and enkephalin-containing intermediary peptides resulted in enhancement of the proliferative T cell response and inhibition of monocyte IL-6 secretion. In vitro reconstitution of the anti-sense treated cultures with synthetic met-enkephalin or the delta-type specific opioid receptor agonist deltorphin could reverse inhibition of monocyte IL-6 production, suggesting that endogenous enkephalins act via membrane opioid receptors. In contrast, addition of met-enkephalin or deltorphin to the anti-sense treated T cell cultures did not have any effect on T cell proliferation.

A new example of a morphine‐sensitive neuro‐effector junction: adrenergic transmission in the mouse vas deferens

A new example of a morphine‐sensitive neuro‐effector junction: adrenergic transmission in the mouse vas deferens

RB 101, a purported pro drug inhibitor of enkephalin metabolism, is antinociceptive in pregnant mice.

In an earlier study, we demonstrated the enhancement of pregnancy-induced analgesia with an inhibitor of endogenous enkephalin metabolism. The purpose of the present study was to evaluate the antinociceptive effect of another inhibitor of enkephalin metabolism, RB 101, on pregnant mice. Further, since other studies have shown RB 101 to be free of opioid side effects, we examined its effect on respiratory rate. Analgesia was assessed using the hot plate test, and respiratory rate was measured by recording the output from an end-tidal carbon dioxide detector. In pregnant mice, experiments were conducted on Day 17 or Day 18 of pregnancy; mice usually deliver on Day 19. For the hot plate test, animals were tested in the following groups: Group 1, RB 101 150 mg/kg (n = 15); Group 2, RB 101 50 mg/kg (n = 15); Group 3, RB 101 vehicle (n = 15); Group 4, morphine 5 mg/kg (n = 14); and Group 5, RB 101 150 mg/kg + naloxone 5 mg/kg (n = 10). The test was repeated on the second day after delivery in animals in Groups 1 and 3 (given RB 101 150 mg/kg and RB 101 vehicle, respectively). RB 101 150 mg/kg and morphine 5 mg/kg were significantly different (mean percentage of maximum possible effect 30.0 and 37.7, respectively, at 30 min and 41.6 and 32.6, respectively, at 60 min) in their antinociceptive effect in pregnant animals from all other groups. Naloxone, when coadministered with RB 101, prevented the development of antinociception. RB 101 150 mg/kg was not antinociceptive after delivery. Depression of respiratory rate was tested in a separate set of animals in the following groups: Group 1, RB 101 150 mg/kg (n = 16); Group 2, morphine 5 mg/kg (n = 16); Group 3, RB 101 vehicle (n = 15). Morphine 5 mg/kg produced significant depression of respiratory rate at 30 min postinjection when compared with RB 101 150 mg/kg and RB 101 vehicle (mean percent change in respiratory rate was 78.5% compared with 87.7% and 92.4%, respectively, where 100% = no change). These results suggest that drugs such as RB 101 may produce antinociception with minimal effects on respiration.

Opposite role of delta 1- and delta 2-opioid receptors activated by endogenous or exogenous opioid agonists on the endogenous cholecystokinin system: further evidence for delta-opioid receptor heterogeneity.

Using the mouse caudate-putamen, where delta-opioid receptor subtypes have been shown to regulate adenylyl cyclase activity, we show in this study that endogenous enkephalins inhibit enzyme activity through activation of delta 1- and delta 2-opioid receptors. Thus, naltriben or 7-benzylidenenaltrexone as well as the delta-selective antagonist naltrindole (mixed delta 1 and delta 2 antagonist) antagonized inhibition of adenylyl cyclase activity induced by methionine- or leucine-enkephalin, while the micro-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) was without effect. Furthermore, we have previously shown that activation of delta-opioid receptors increases cholecystokinin release in the central nervous system, resulting in a potentiation of micro-opioid antinociceptive responses, and the respective role of delta 1- and delta 2-opioid receptors in this facilitatory effect has now been evaluated. Activation of delta 2-opioid receptors, either by endogenous enkephalins protected from catabolism by the complete enkephalin-degrading enzyme inhibitor N-((R,S)-2-benzyl-3((S)(2-amino-4-methyl-thio) butyldithio)-1-oxopropyl)-L-phenyl-alanine benzyl ester (RB 101), or by the delta 2-selective agonist Tyr-D-Ser(O-tert-butyl)-Gly-Phe-Leu-Thr(O-tert-butyl) (BUBU), potentiated micro-opioid antinociceptive responses in the hot-plate test in mice. This effect was antagonized by a selective cholecystokinin-A antagonist. Activation of delta 1-opioid receptors by endogenous opioid peptides decreased the micro-opioid responses. These results suggest that stimulation of delta 2-opioid receptors potentiates micro-opioid analgesia in the hot-plate test in mice through an increase in endogenous cholecystokinin release, while activation of delta 1-opioid receptors could decrease it. Thus, the pre-existing physiological balance between opioid and cholecystokinin systems seems to be modulated in opposite directions depending on whether delta 1- or delta 2-opioid receptors are selectively activated. This is the first demonstration that endogenous enkephalins, methionine- and leucine-enkephalin, are the natural ligands of delta-opioid receptor subtypes, and that delta 2-opioid receptor activation may facilitate the endogenous cholecystokinin-related modulation of micro-opioid analgesia, while the delta 1-opioid receptors may have an inhibitory role. These results could have important applications for the characterization of opioid delta 1 and delta 2 as subtypes or subsites and in pain alleviation.

Opioid receptor-mediated control of acetylcholine release in human neocortex tissue.

The effects of various opioid receptor agonists and antagonists on evoked acetylcholine release were studied in slices of human neocortex prelabelled with [3H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K+ (20 mM). The delta-opioid receptor agonist DPDPE and the kappa-opioid receptor agonist U50488 reduced the evoked [3H]-overflow (acetylcholine release) in a concentration-dependent fashion; the delta-opioid receptor antagonist naltrindole and the kappa-opioid receptor antagonist norbinaltorphimine, respectively, antagonized these effects. Application of the mu-opioid receptor agonist DAGO also resulted in an inhibition of acetylcholine release; however, both delta- and kappa-opioid receptor antagonists were able to block this effect. The mu-opioid receptor agonists morphine and (+)-nortilidine had no effect. These results indicate that acetylcholine release in human neocortex is inhibited through delta- and kappa-opioid receptors, but not through mu-opioid receptors. Acetylcholine release was significantly increased by the delta-opioid receptor antagonist naltrindole in the presence of a mixture of peptidase inhibitors providing evidence for a delta-opioid receptor-mediated inhibition of acetylcholine release by endogenous enkephalin. K(+)-evoked acetylcholine release in the presence of TTX was inhibited by U50488, but not by DPDPE, suggesting the presence of kappa-opioid receptors on cholinergic terminals and the localization of delta-receptors on cortical interneurons. Therefore, the potent effect of DPDPE on acetylcholine release is likely to be indirect, by modulation of intrinsic cortical neurons. These interneurons probably do not use GABA as neurotransmitter since both GABAA and GABAB receptor agonists (muscimol and baclofen, respectively) were without effect on acetylcholine release.

Antiallodynic effects of a CCKB antagonist in rats with nerve ligation injury: role of endogenous enkephalins

Cholecystokinin (CCK) may act as an endogenous anti-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid delta (δ) receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish the release and/or availability of endogenous enkephalins. The present studies have further evaluated this possibility by studying the antiallodynic actions of a CCK B antagonist (L365,260) alone, or in the presence of thiorphan (a neutral endopeptidase inhibitor) in a model of peripheral neuropathy. Animals subjected to nerve injury, but not sham controls, exhibited long lasting, stable mechanical allodynia. Intrathecal (i.t.) administration of L365,260 or thiorphan alone did not alter allodynia. However, co-administration of these compounds produced a significant antiallodynic action which was antagonized by receptor selective doses of naltrindole, an opioid δ receptor antagonist. In addition, antisera to [Leu 5 ]enkephalin, but not to [Met 5 ]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCK B receptors may enhance the actions or availability of endogenous [Leu 5 ]enkephalin or a like substance which can elicit a significant antiallodynic action via opioid δ receptors when its degradation is by inhibited by thiorphan. The data suggest that δ opioids are involved in regulation of some aspects of nerve-injury induced pain.

Similar involvement of several brain areas in the antinociception of endogenous and exogenous opioids

The complete inhibitor of the enkephalin degrading enzymes, RB 101, N-{( R,S)-2-benzyl-3[( S)-(2-amino-4-methylthio)butyldithio]-1-oxopropyl}- l-phenylalanine benzyl ester, which crosses the blood-brain barrier, induced antinociceptive effects similar to those of exogenous opiates. The almost complete absence of tolerance and dependence after chronic administration of RB 101 is therefore due to limited stimulation of opioid receptors by ‘protected’ endogenous enkephalins. In order to clarify the mechanisms involved in these response, we have investigated the participation of several brain structures in the antinociceptive effects induced by systemic administration of morphine or RB 101. Rats were implanted with bilateral cannulae into the ventro-basal thalamus, central amygdala and periaqueductal gray matter, or with a cannula into the raphe magnus nucleus. The antinociceptive responses induced by systemic morphine or RB 101 were measured by using the tail-electrical stimulation test, where three different thresholds were determined: motor response, vocalization and vocalization post-discharge. The ability of the opioid receptor antagonist methylnaloxonium to block these antinociceptive responses was evaluated after local injection into the different brain structures. The blockade of morphine- and RB 101-induced antinociception was similar, and was stronger when methylnaloxonium was injected into the periaqueductal gray matter and raphe magnus nucleus than when it was injected into the ventro-basal thalamus and amygdala. These results suggest that brain structures related to the control of pain seem to be the same for the antinociception induced by exogenous opiates and endogenous opioids.

Antiallodynic effects of a CCK B antagonist in rats with nerve ligation injury: role of endogenous enkephalins

Cholecystokinin (CCK) may act as an endogenous anti-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid delta (δ) receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish the release and/or availability of endogenous enkephalins. The present studies have further evaluated this possibility by studying the antiallodynic actions of a CCK B antagonist (L365,260) alone, or in the presence of thiorphan (a neutral endopeptidase inhibitor) in a model of peripheral neuropathy. Animals subjected to nerve injury, but not sham controls, exhibited long lasting, stable mechanical allodynia. Intrathecal (i.t.) administration of L365,260 or thiorphan alone did not alter allodynia. However, co-administration of these compounds produced a significant antiallodynic action which was antagonized by receptor selective doses of naltrindole, an opioid δ receptor antagonist. In addition, antisera to [Leu 5]enkephalin, but not to [Met 5]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCK B receptors may enhance the actions or availability of endogenous [Leu 5]enkephalin or a like substance which can elicit a significant antiallodynic action via opioid δ receptors when its degradation is by inhibited by thiorphan. The data suggest that δ opioids are involved in regulation of some aspects of nerve-injury induced pain.

Similar decrease in spontaneous morphine abstinence by methadone and RB 101, an inhibitor of enkephalin catabolism.

1. The dual inhibitor of enkephalin degrading enzymes, RB 101, is able to block endogenous enkephalin metabolism completely, leading to potent antinociceptive responses potentiated by blockade of CCKB receptors. In this study we have investigated the effects induced by RB 101 given alone, or with the CCKB antagonist, PD-134,308, on a model of spontaneous morphine withdrawal and substitutive maintenance in rats. 2. Animals were chronically treated with morphine for 7 days followed, 36 h after the interruption of drug administration, by a maintenance treatment for 5 days with methadone (2 mg kg-1, i.p.), clonidine (0.025 mg kg-1, i.p.), RB 101 (40 mg kg-1, i.p.), PD-134,308 (3 mg kg-1, i.p.) or a combination of RB 101 plus PD-134,308. Several behavioural observations were made during this period in order to evaluate the acute effects as well as the consequence of chronic maintenance induced on spontaneous withdrawal by the different treatments. 3. Methadone was the most effective compound in decreasing the spontaneous withdrawal syndrome after acute administration. Both, methadone and RB 101 had similar effectiveness in reducing opiate abstinence during the period of substitutive treatment. PD-134,308 did not show any effect when administered alone and did not modify the effect of RB 101. 4. Naloxone (1 mg kg-1, s.c.) failed to precipitate any sign of withdrawal when injected at the end of the chronic maintenance treatment suggesting that, under the present conditions, methadone and RB 101 did not induce significant physical opiate-dependence. 5. The mildness of the side effects induced by chronic RB 101, suggests that systemically active inhibitors of enkephalin catabolism could represent a promising treatment in the maintenance of opiate addicts.

Expression and function of proenkephalin A messenger ribonucleic acid in murine fetal thymocytes.

In recent years we examined the function of the endogenous enkephalins encoded by PEA messenger RNA (mRNA) expressed in murine thymocytes, the precursor cells to T lymphocytes, which are the primary effector cells in cell- mediated immune responses. In the present study, we examined the expression and function of PEA mRNA and enkephalins in fetal thymocytes. By Northern gel and in situ hybridization techniques, we show that PEA mRNA is constitutively expressed in fetal thymocytes early in gestation, with maximal expression occurring on day 15. By birth, PEA mRNA is no longer constitutively expressed, but can be induced by culturing newborn thymocytes with the T cell-specific mitogen, Concanavalin-A. Both a delta-opioid receptor antagonist, naltrindole, and a PEA mRNA-specific antisense complementary DNA enhance the spontaneous proliferation of day 15, but not day 14, fetal thymocytes, consistent with the observation that PEA mRNA is expressed in thymocytes on day 15, but not day 14, of gestation. The enhanced proliferation of day 15 fetal thymocytes is reversed by a delta-opioid receptor agonist, deltorphin. The data suggest that endogenous enkephalins encoded by PEA mRNA expressed in day 15 fetal thymocytes act to inhibit the spontaneous proliferation of these cells, perhaps so that their differentiation into mature T lymphocytes can occur.

The CCKB antagonist PD-134,308 facilitates rewarding effects of endogenous enkephalins but does not induce place preference in rats.

The interaction between cholecystokinin and endogenous opioid systems on rewarding responses was examined. Motivational effects induced by peripheral administration of a complete inhibitor of enkephalin catabolism, RB 101 or the CCKB antagonist PD-134,308, and by both compounds in combination were evaluated in the conditioned place preference test in rats. RB 101 (5, 10, 20, 40 and 80 mg/kg, IP, and 20 mg/kg, IV) given alone produced a bell-shaped dose-effect function. A significant increase of the preference for the drug-associated compartment was only observed at doses of 10 and 20 mg/kg (IP). The effect observed with morphine was stronger, and all the doses used of this compound (1.25, 2.5 and 5 mg/kg, SC) were found to be active. These results suggest that the inhibitor of enkephalin catabolism has weak rewarding properties. Pretreatment with the CCKB antagonist PD-134,308 (0.1, 0.3, 1 and 3 mg/kg, IP) alone failed to produce a reliable aversion or preference on the paradigm studied. When PD-134,308 (0.3 mg/kg, IP) was coadministered with a subthreshold dose of morphine (0.6 mg/kg, SC) or RB 101 (5 mg/kg, IP), a conditioned place preference was observed, indicating that the CCKB antagonist facilitated the motivational responses induced by endogenous enkephalins as compared to morphine. This suggests that endogenous cholecystokinin, acting through CCKB receptors, modulates the rewarding effects of endogenous enkephalins.

Opioid receptor types for endogenous enkephalin in the thoracic ganglion of the crab, Carcinus maenas

In crustaceans, the endogenous opioid peptides, enkephalins, are known to be concentrated in the thoracic ganglion, although they have been demonstrated in all parts of the nervous system. Bmax and Kd measurements have been obtained for the binding of ligands used to characterize delta- and kappa-type opioid receptors in vertebrates. High- and low affinity binding of [3H] [2-D-Pen5-D Pen] enkephalin ([3H]DPDPE) has been measured with a Kd = 9.2 +/- 2.4 nM, Bmax = 153 fmol/mg, and Kd = 243 +/- 27 nM, Bmax = 1.785 pmol/mg, respectively. In addition a kappa-type receptor with Kd 85.5 +/- 12.6 nM and Bmax = 21.138 pmol/mg protein has been recorded. Binding characteristics of several ligands were monitored. Electrophoretic studies of affinity chromatographically purified receptor fractions revealed a molecular mass of 60 kDa. Isoelectric focusing showed a specific binding of [3H]DPDPE to thoracic ganglion membranes at a pl of 5.5.

Weak tolerance to the antinociceptive effect induced by the association of a peptidase inhibitor and a CCK B receptor antagonist

We have recently shown that CCK B receptor antagonists such as PD-134,308, are able to strongly potentiate antinociception induced by endogenous enkephalins, protected from degrading enzymes by the mixed inhibitor RB 101, ester, at both spinal and supraspinal levels. In this study, the duration of this facilitatory response and the possible development of tolerance to this synergistic effect were investigated in the rat tail-flick test after acute and chronic treatment with PD-134,308 and RB 101. PD-134,308 facilitated and prolonged the antinociceptive responses induced by RB 101 (20 mg/kg, i.v.). The duration of the effect induced by PD-134,308 was also investigated by injecting this compound at different times before RB 101 administration. In the case of the tail-flick test, the improvement of RB 101 antinociceptive response was still significant 6 h after PD-134,308 (3 mg/kg, i.p.), whereas in the hot-plate test, this enhancement was only effective for 3 h after CCK B receptor antagonist administration. In the case of a repeated administration of RB 101, the potentiation induced by PD-134,308 on the antinociceptive effect produced by the first injection of RB 101 (20 mg/kg, i.v.), was found almost identical after a second administration of RB 101 performed 190 min later. Chronic administration of RB 101 (20 mg/kg, i.v.) plus PD-134,308 (3 mg/kg, i.p.) administered for 5 days both once or twice per day, did not induce the development of tolerance to antinociception at the peak effect time. However, a decrease in the duration of the antinociceptive response was observed. These results indicate that the potent and long-lasting antinociceptive response induced by the coadministration of the peptidase inhibitor and the CCK B receptor antagonist could have interesting perspectives in the clinical treatment of pain.

The effect of prolonged treatment with imipramine and electroconvulsive shock on the levels of endogenous enkephalins in the nucleus accumbens and the ventral tegmentum of the rat.

The present study was designed to find out whether the prolonged administration of imipramine (IMI) or electroconvulsive shock (ECS) influences levels of endogenous enkephalins in the nucleus accumbens (NAS) and the ventral tegmentum (VTA) of the rat. Ressults indicate that treatment with IMI as well as with ECS has a profound effect on the levels of enkephalins in both structures. In the NAS both treatments lead to an increase in the levels of endogenous enkephalins and this effect is accompanied by an increase in mRNA coding for proenkephalin (measured by in situ hybridization) in this structure, indicating the enhancement of biosynthesis of endogenous enkephalinergic peptides following antidepressant treatment. The results are discussed in the light of the hypothesis concerning the influence of endogenous enkephalins on mesolimbic dopamine neurons, the activity of which plays a crucial role in the etiology of depression.

The peristaltic reflex in the rat ileum: evidence for functional mu- and delta-opiate receptors.

The potency order of opiate agonists at decreasing the rate of peristalsis in the rat isolated ileum was: difenoxin > loperamide > DADLE (D-Ala2-D-Leu5-enkephalin) > morphine > DSLET (D-Ser2,Leu5-Thr6-enkephalin). U-50488 (trans 3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl) cyclohexyl) benzeneacetamide methane sulphonate) was inactive at 300 nM. Naloxone (400 nM) caused a significant 1.52-fold increase in the rate of peristaltic contractions and inhibited the effects of the active opiate agonists. The apparent pA2 values of naloxone were similar using difenoxin, loperamide and morphine as agonists, but the value was slightly, though significantly lower when DADLE was the agonist. It is suggested that the previously identified delta-opiate receptors of the rat small intestine have a functional role in suppressing the peristaltic reflex. The same response is subserved by mu-opiate receptors and either of these opiate-receptor subtypes could be activated by endogenous enkephalins.

Opposite role of CCKA and CCKB receptors in the modulation of endogenous enkephalin antidepressant-like effects.

Systemic administration of RB 101, a complete inhibitor of the enkephalin degrading enzymes, has been reported to induce naltrindole-reversed antidepressant-like effects in the conditioned suppression of motility (CSM) test in mice. The selective CCKB antagonist L-365,260 also elicits the same naltrindole-blocked responses on CSM. The aim of this study was therefore to investigate the possible modulation of RB 101 induced behavioral responses by activation or blockade of CCK receptors. Thus, the effects induced by RB 101 administered alone or associated with an ineffective dose of a selective CCKB agonist (BC 264), a CCKB antagonist (L-365,260) or a CCKA antagonist (L-364,718), were evaluated on the CSM in mice. RB 101 alone decreased the stress-induced loss of motility, as previously reported. The antidepressant-like effect of RB 101 was potentiated by L-365,260, and suppressed by BC 264 and to a lesser extent by L-364,718. The facilitatory effect induced by L-365,260 on RB 101 responses was blocked by the delta selective antagonist naltrindole. All these effects occurred only in shocked animals. The present results suggest that the activation of CCKA and CCKB receptors by endogenous CCK, could play an opposite role in the control of behavioral responses induced by endogenous enkephalins. Delta opioid receptors seem to be selectively involved in this interaction.

Met-enkephalin-containing peptides encoded by proenkephalin A mRNA expressed in activated murine thymocytes inhibit thymocyte proliferation.

Murine thymocytes activated with the mitogen Con A express proenkephalin A mRNA (PEA mRNA) and met-enkephalin and/or met-enkephalin-containing peptides ("enkephalins"). This Con A-induced expression of PEA mRNA is modulated by the delta opioid receptor agonist, deltorphin I, in a biphasic, dose-dependent manner. That is, 10(-13) M to 10(-11) M deltorphin enhanced PEA mRNA expression 3- to 3.5-fold over the level induced by Con A alone, and 10(-9) M to 10(-7) M deltorphin inhibited it 40 to 70%. delta opioid receptor antagonists recognizing the delta-2 (naltrindole (NTI) and naltriben (NTB)), but not the delta-1 (7-benzylidenenaltrexone (BNTX)), subtype of opioid receptor described in brain, reversed both the enhancing and inhibiting effects of deltorphin on Con A-induced PEA mRNA expression. In addition, the delta-2 receptor-specific antagonists, NTI and NTB, directly inhibited Con A-induced PEA mRNA expression. The function of the enkephalins expressed by thymocytes was examined by using 1) delta opioid receptor antagonists, 2) PEA mRNA-specific antisense cDNA, and 3) Ab to met-enkephalin, and measuring cell proliferation. All three reagents caused enhancement of Con A-induced proliferation, with effects ranging from two- to fourfold over the response to Con A alone. Again, the delta-2 subtype-specific antagonists, NTI and NTB, were functional and the delta-1 subtype-specific antagonist, BNTX, was not. The PEA mRNA-specific antisense cDNA blocked translation but not transcription of PEA mRNA. The data suggest that 1) endogenous enkephalins induced in thymocytes modulate their own expression through delta-2-like opioid receptors, and 2) these endogenous enkephalins function to inhibit the proliferation of activated thymocytes.