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.

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.

Hyperphagia induced by 2-deoxy-D-glucose in the presence of the delta-opioid antagonist ICI 174,864.

The effect of the selective delta-opioid antagonist ICI 174,864 (N,N-bisallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib=alpha-aminoisobutyric acid) on the hyperphagia induced by 2-deoxy-D-glucose (2-DG) was investigated in non-deprived rats. The increase in food intake produced by 2-DG (500 mg/kg i.p.) was not reduced by ICI 174,864 at a dose (3 micrograms/rat i.c.v.) which totally abolished the feeding response to the delta-agonist D-Ala2-D-Leu5-enkephalin (10 micrograms/rat i.c.v.). These findings suggest that the appetitive effects of 2-DG are not mediated by an enkephalinergic/delta-receptor system. They do not, however, preclude the possible involvement of endogenous opioids acting at other sub-types of opioid receptor in this glucoprivic ingestional response, which is suppressed by less specific opioid antagonists such as naloxone.

Beta-endorphin-(1-27) antagonizes beta-endorphin- but not morphine-, D-Pen2-D-Pen5-enkephalin- and U50, 488H-induced analgesia in mice.

beta-Endorphin-(1-27), administered intraventricularly has been previously reported to block the analgesia induced by beta-endorphin injected intraventricularly. The present study was to determine if the blocking effect of beta-endorphin-(1-27) was specific to beta-endorphin which stimulates epsilon receptors, but not to other opioids with activity at different opioid receptors. The antagonistic effects of beta-endorphin-(1-27) on the analgesia induced by beta-endorphin (epsilon-opioid receptor agonist), D-Ala2-NMePhe4-Gly-ol-enkephalin(DAGO) and morphine, (mu-opioid receptor agonists), D-Pen2-D-Pen5-enkephalin(DPDPE) and D-Ala2-D-Leu5-enkephalin(DADLE) (delta-opioid receptor agonists) and U-50, 488H (kappa-opioid receptor agonist) were studied. beta-Endorphin-(1-27) injected intraventricularly, at doses which, when injected alone did not produce analgesia, antagonized the analgesia induced by beta-endorphin given intraventricularly. However, the analgesia induced by DAGO, morphine, DPDPE, DADLE and U-50, 488H given intraventricularly was not antagonized by beta-endorphin-(1-27). The data suggest that beta-endorphin-(1-27) selectively blocks the analgesia induced by the stimulation of epsilon receptors but not by the stimulation of mu, delta, and kappa receptors. The results support the previously proposed hypothesis that beta-endorphin produces its analgesia by stimulating specific epsilon receptors.

Multi-dimensional analysis of behavior in mice treated with the delta opioid agonists DADL (D-Ala2-D-Leu5-enkephalin) and DPLPE (D-Pen2-L-Pen5-enkephalin).

The effects of intracerebroventricular injection of the delta-selective opioid peptides, DADL (D-Ala2-D-Leu5-enkephalin) and DPLPE (D-Pen2-L-Pen5-enkephalin), on spontaneous locomotor activity were investigated in mice using multi-dimensional behavioral analysis, based upon a capacitance system. The analysers classified the movements into 9 sizes (1/1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 and 1/256). Specific patterns of behavior were each registered on these sizes of movement. At 1.0 and 3.0 micrograms, DADL produced a significant increase in circling (1/4 size of movements) within 15 min after the start of measurements, while it produced a marked increase in linear locomotion (1/2 size), circling (1/4 size), rearing (1/16 size) and grooming (1/32, 1/64 and 1/128 sizes) within 15-30 min after the start. At 10.0 micrograms, DPLPE decreased linear locomotion (1/1 size) and conversely increased circling behavior (1/4 size) within 15 min after the start, whilst this peptide at 3.0 or 10.0 micrograms, produced a marked increase in linear locomotion (1/2 size), circling (1/4 size) and grooming (1/128 size) within 15-30 min after the start. The behavioral effects induced by DADL (3.0 micrograms) and DPLPE (10.0 micrograms) were completely reversed by naloxone (1.0 and 2.0 mg/kg). These results obtained with DPLPE, a delta-selective peptide and DADL, a less delta-selective peptide, indicate a common pattern of activity which was presumably delta receptor-mediated. However, one component (linear locomotion, at times immediately after administration of the peptide) did clearly differ between these two peptide analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Only one pharmacophore is required for the kappa opioid antagonist selectivity of norbinaltorphimine.

We have investigated whether one or two pharmacophores are required for the kappa opioid receptor selectivity of the bivalent opioid antagonist norbinaltorphimine, (-)-1 (nor-BNI), by the synthesis and testing of its meso isomer 2. In smooth muscle preparations 2 was more potent than 1 and about half as selective as a kappa antagonist. Since 2 contains only one antagonist pharmacophore but yet retains substantial kappa selectivity, it is concluded that kappa selectivity is not dependent on the presence of two (-)-naltrexone-derived pharmacophores of 1. It is suggested that the kappa selectivity of (-)-1 and 2 is derived from the portions of the second halves of these molecules in that they mimic key "address" components of dynorphin at kappa opioid receptors.

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.

Guanine nucleotide-mediated inhibition of opioid agonist binding. Modulatory effects of ions and of receptor occupancy.

We have analysed the potency of GTP, GDP and their analogues in reducing [3H]DADLE binding to opioid receptors in NG 108-15 cell membranes. Under conditions where non-specific hydrolysis and transphosphorylation is inhibited, the following rank order of potency was found: GDP greater than or equal to GTP gamma S greater than GTP greater than GDP beta S greater than or equal to GDPNH2 greater than GppNHp much greater than GMP. Remarkably, the slopes for the inhibition curves of GTP, GDP and their thiosubstituted analogues, but not of GDPNH2 and GppNHp, were extremely shallow, indicating either negative cooperativity or the existence of two states for the guanine nucleotide binding proteins, that both can mediate the effect of nucleotides on agonist receptor binding. The potencies of the different guanine nucleotide analogues, except that of GppNHp, were increased by the presence of sodium or chloride ions in the assay medium. Magnesium also affected GTP-mediated inhibition of opioid agonist binding since it decreased the IC50 of the nucleotide and steepened the slope of the inhibition curve. The IC50s of nucleotides and the slopes of their inhibition curves were also dependent on the extent of receptor occupancy by the agonist. From these data we conclude that (1) either diphospho- or triphosphonucleotides can regulate agonist binding. (2) Magnesium, sodium and chloride, by acting at different components of the receptor/G protein complex produce similar effects on nucleotide mediated regulation of agonist binding. (3) A mutual influence exists between receptor occupancy by agonists and G protein-mediated guanine nucleotide effect on the receptor.

Interactions of leucine-enkephalin with alpha-adrenoceptors in the conscious dog.

Leucine-enkephalin (Leu-ENK) administered systemically to the conscious dog increases heart rate and blood pressure, a response prevented by naloxone and hence mediated by opiate receptors. Clonidine attenuates the heart rate increase without affecting the pressor response. Propranolol also reduces the magnitude of the heart rate increase but to a lesser degree than does clonidine. Clonidine appears to inhibit the Leu-ENK-induced heart rate increase by both a centrally mediated increase in vagal tone and a peripheral presynaptic alpha 2-agonist effect. The presence of a pressor response to Leu-ENK after prazosin pretreatment suggests that Leu-ENK may also increase blood pressure independently of the alpha 1-adrenoceptor. Thus, Leu-ENK appears to act via a naloxone-sensitive receptor (perhaps a vagal afferent chemoreceptor), the response involving both sympathetic and parasympathetic mechanisms. The pressor response may also involve a direct effect on peripheral vessels or a non-alpha 1-adrenergic efferent pathway.

ICI 174,864, a selective delta opioid antagonist, reverses the learning impairment produced by [leu]enkephalin.

The role of opioid delta receptors in the learning impairment produced by [leu]enkephalin (LE) in one-way active avoidance conditioning was investigated in mice. LE (30 and 100 micrograms/kg) impaired acquisition of the avoidance response, whereas ICI 174,864 (3.0 mg/kg), a selective delta opioid receptor antagonist, enhanced acquisition. The impairment produced by 100 micrograms/kg LE was completely reversed by 1.0 mg/kg ICI 174,864, a dose of the antagonist that by itself had no effect. Control studies provided evidence that the effects of ICI 174,864 and LE on conditioning cannot be explained by performance variables such as alterations in activity levels or footshock sensitivity. The results suggest that opioid delta receptors play an important role in the modulation of learning, and that the effects of LE on avoidance conditioning are mediated by delta receptors.

Modification of place preference conditioning in mice by systemically administered [Leu]enkephalin.

[Leu]enkephalin (300 micrograms/kg, i.p.) induced either a positive or negative conditioned place preference response in mice depending on whether the animals were trained against or towards their initial preference. Induction of a positive preference (300 micrograms/kg) was partially blocked by simultaneous addition of methylnaloxonium (10 mg/kg, i.p.), an opioid antagonist that does not readily cross the blood-brain barrier; methylnaloxonium alone (3 or 10 mg/kg) had no effect on the place preference response. The results indicate that [Leu]enkephalin treatment reverses the initial preference of the animal regardless of training, and that some aspect of the [Leu]enkephalin effect on place preference conditioning is mediated by peripheral opioid receptors. These findings challenge the notion that place preference conditioning is a simple measure of opioid reward.

[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.

Cyclic AMP or forskolin rapidly attenuates the depressant effects of opioids on sensory-evoked dorsal-horn responses in mouse spinal cord-ganglion explants.

Exposure of fetal mouse spinal cord-ganglion explants to morphine (greater than 0.1 microM) results in naloxone-reversible, dose-dependent depression of sensory-evoked dorsal-horn synaptic-network responses within a few minutes. After chronic opiate exposure (1 microM) for 2-3 days, these dorsal cord responses recover and can then occur even in greater than 10 microM morphine. In the present study, when naive explants were treated with forskolin (10-50 microM)--a selective activate activator of cyclase (AC)--for 10-30 min prior to and during exposure to morphine (0.1-0.3 microM) or D-Ala2-D-Leu5-enkephalin (0.03-0.1 microM), the usual opioid depressant effects on dorsal-horn responses generally failed to occur (10-30 min tests). Dibutyryl cyclic AMP (10 microM) or the more lipid-soluble analog, dioctanoyl cyclic AMP (0.1 mM), produced a similar degree of subsensitivity to opiates as 10 microM forskolin. With high levels of forskolin (50 microM), even concentrations of morphine up to 1-10 microM were far less effective in depressing cord responses. These effects of exogenous cAMP analogs and forskolin on cord-ganglion explants are probably both mediated by increases in intracellular cAMP. The marked decrease in opioid sensitivity of cAMP or forskolin-treated cord-ganglion explants provides significant electrophysiologic data compatible with the hypothesis that neurons may develop tolerance and/or dependence during chronic opioid exposure by a compensatory enhancement of their AC/cAMP system following initial opioid depression of AC activity. Previous evidence relied primarily on behavioral tests and biochemical analyses of cell cultures. It will be of interest to determine if dorsal-horn tissues of cord-ganglion explants do, in fact, develop increased AC/cAMP levels as they express physiologic signs of tolerance during chronic exposure to opioids.

Intracerebral opiates block the epileptic effect of intracerebroventricular (ICV) leucine-enkephalin.

Intracerebroventricular (ICV) injection of both enkephalin (100 micrograms) and morphine (200 micrograms produces characteristic electrographic seizures. Injection of low doses of either morphine or levorphanol into the lateral ventricle of the brain prior to the administration of epileptogenic doses of enkephalin can block the induction of such seizures. A similar trend was observed when either opiate preceded ICV morphine. Microinjections of both morphine (30 micrograms) or levorphanol (40 micrograms) into the periaqueductal gray area (PAG) or into the nucleus accumbens resulted in potent analgesia. However, only morphine injected into the nucleus accumbens was effective in blocking electrographic seizures induced by ICV enkephalin. On the basis of this and other previous findings we propose that the excitatory-epileptic and the inhibitory-antiepileptic action of opiates and opioids are mediated by two different systems. Furthermore, we propose that such systems may differ both in their anatomical distribution and in the classes of opiate receptors underlying their action.

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.

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.

Parasympathetic ganglia: naloxone antagonizes inhibition by leucine-enkephalin and GABA.

Synaptic transmission in parasympathetic ganglia of the cat urinary bladder was depressed by low doses (0.1-10 micrograms i.a.) of Leu- or Met-enkephalin but only by larger doses (10 micrograms-1 mg i.a.) of morphine. Naloxone blocked the depressant effects of the opiates as well as the depression produced by GABA, but did not block the depressant effects of norepinephrine. Intracellular recording revealed that Leu-enkephalin reduced EPSP-amplitude and lowered the probability of synaptically evoked firing without altering postsynaptic membrane potential or resistance. These findings suggest that enkephalinergic inhibition in bladder ganglia is mediated at least in part by a presynaptic site of action on delta opiate receptors.

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.