Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex.

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express ß-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Morphine-like opiates selectively antagonize receptor-arrestin interactions.

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of mu and delta receptors with G protein or beta-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

A2A receptor knockout worsens survival and motor behaviour in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative genetic disorder that leads to motor, cognitive, and psychiatric disturbances. The primary neuropathological hallmark is atrophy of the striatum. HD preferentially affects efferent striato-pallidal neurons that express enkephalin as well as dopamine D2 and A(2A) adenosine receptors (A(2A)Rs). Expression and function of A(2A)Rs are altered in HD but, despite being an important modulator of the striato-pallidal function, the subsequent pathophysiological consequence of such changes remains unclear. Whether blockade of A(2A)Rs is of therapeutic interest in HD remains ill-defined. In the present work, we aimed to determine the pathophysiological consequences of genetic deletion of A(2A)Rs in HD by crossing A(2A)R knockout mice with the N171-82Q HD transgenic model. Our data demonstrate that knockout of A(2A)Rs moderately but significantly worsens motor performances and survival of N171-82Q mice and leads to a decrease in striatal enkephalin expression. These results support that early and chronic blockade of A(2A)Rs might not be beneficial in HD.

Opiate peptide modulation of amino acid responses suggests novel form of neuronal communication.

Mouse spinal neurons grown in tissue culture were used to study the electrophysiological pharmacology of the opiate peptide leucine-enkephalin. Enkephalin depressed glutamate-evoked responses in a noncompetitive manner independent of any other effects on membrane properties. The results demonstrate a neuromodulatory action of opiate peptide functionally distinct from the conventional neurotransmitter class of operation.

Selective protection of methionine enkephalin released from brain slices by enkephalinase inhibition.

Methionine enkephalin release was evoked by depolarization of slices from rat striatum with potassium. In the presence of 0.1 microM thiorphan [(N(R,S)-3-mercapto-2-benzylpropionyl)glycine], a potent inhibitor of enkephalin dipeptidyl carboxypeptidase (enkephalinase), the recovery of the pentapeptide in the incubation medium was increased by about 100 percent. A similar effect was observed with the dipeptide phenylalanylalanine, a selective although less potent enkephalinase inhibitor. Inhibition of other known enkephalin-hydrolyzing enzymes--aminopeptidase by 0.1 mM puromycin or angiotensin-converting enzyme by 1 microM captopril--did not significantly enhance the recovery of released methionine enkephalin. These data indicate that enkephalinase is critically involved in the inactivation of the endogenous opioid peptide released from striatal neurons.

Stimulation of dopamine synthesis in caudate nucleus by intrastriatal enkephalins and antagonism by naloxone.

The intraventricular injection of methionine-enkephalin (50 to 100 micrograms) or [d-Ala2]-methionine-enkephalinamide (1.5 to 12 micrograms), a synthetic enkephalin analog resistant to enzyme degradation, caused a marked dose-dependent increase in dihydroxyphenylacetic acid and homovanillic acid concentrations in the rat striatum. The [d-Ala2] analog increased the accumulation of dopa in the striatum after aromatic amino acid decarboxylase inhibition, indicating that it increased dopamine synthesis. At the highest doses used both enkephalins failed to modify brain serotonin metabolism. The monolateral microinjection of the [d-Ala2]] analog (3 to 6 micrograms) into the caudate nucleus increased the concentration of dihydroxyphenylacetic acid in the injected side, whereas bilateral injection increased the concentration of this compound in both caudate nuclei and caused catalepsy. The stimulant effect of the [d-Ala2] analog on dopamine synthesis in the striatum persisted after destruction of striatal postsynaptic dopamine receptors with kainic acid. The biochemical and behavioral effects of enkephalins were prevented by naloxone, a specific narcotic antagonist. The results indicate that enkephalins stimulate dopamine synthesis by an action on opioid receptors localized on dopaminergic nerve terminals.

Dual actions of substance P on nociception: possible role of endogenous opioids.

Substance P produces analgesia when administered to mice in very small doses by the intraventricular route (1.25 to 5 nanograms per mouse). The analgesic effect can be blocked by naloxone. At higher doses (greater than 50 nanograms per mouse), this activity is lost. At these higher doses, however, substance P produced hyperalgesia when combined with naloxone and analgesia when combined with baclofen [beta-(4-chlorophenyl)-gamma-aminobutyric acid]. Substance P may have dual actions in brain, releasing endorphins at very low doses and directly exciting neuronal activity in nociceptive pathways at higher doses.

Anticonvulsants specific for petit mal antagonize epileptogenic effect of leucine enkephalin.

The anticonvulsants ethosuximide, sodium valproate, and trimethadione that are specific for petit mal epilepsy abolished in rats the electrical seizure activity and behavioral abnormalities produced by leucine enkephalin, whereas phenobarbital and phenytoin had no effect. The dose-response curve for naloxone against seizure activity induced by leucine enkephalin was the same as that in gamma-hydroxybutyrate-induced petit mal. These data indicate that the epileptic properties of leucine enkephalin are petit mal-like and raise the possibility of involvement of enkephalinergic systems in. The dose-response curve for naloxone against seizure activity induced by leucine enkephalin was the same as that in gamma-hydroxybutyrate-induced petit mal. These data indicate that the epileptic properties of leucine enkephalin are petit mal-like and raise the possibility of involvement of enkephalinergic systems in petit mal epilepsy.

Disparate effects of enkephalin and morphine upon insulin and glucagon secretion by islet cell cultures.

Insulin and glucagon release from monolayer pancreatic islet cell cultures were inhibited in a dose-response fashion by various enkephalins. Morphine, however, stimulated insulin and glucagon release. Both effects were blocked by naloxone, while naloxone alone had no effect. In isolated and denervated islet cells, opiates directly influence secretion from islet cells.

Effects of [D-Ala2]-methionine-enkephalin on blood pressure, heart rate, and baroreceptor reflex sensitivity in conscious cats.

Effects of intracerebroventricular (i.c.v) injection of [D-Ala2]-methionine-enkephalinamide (DAME) on blood pressure (BP), heart rate, and baroreceptor reflex sensitivity were studied in conscious cats. DAME was administered at doses between 5 and 100 nmoles. Blood pressure and heart rate increased dose dependently. The sensitivity of the baroreceptor reflex was attenuated for 15 to 60 minutes after DAME administration; this was independent of the BP changes. The effects of enkephalin on BP and baroreceptor reflex were abolished by i.c.v. naloxone. DAME caused pathological changes in the electroencephalogram (EEG) characterized by sharp waves in the hippocampus recordings and a loss of theta activity in the electrocorticogram. Behavioral changes were characterized by decreased physical mobility and anxiousness. These behavioral and EEG changes lasted for a longer period of time than the cardiovascular changes; they were also counteracted by naloxone. It is concluded that DAME produces a centrally mediated vasopressor response and a baroreceptor reflex attenuation and that, with respect to the time course, the effects on the baroreceptor reflex are separated from those on BP behavior and EEG, but not on heart rate. The fact that all effects of enkephalin on the parameters tested in the present experiment were completely antagonized by naloxone suggests that they are mediated by naloxone-sensitive enkephalin brain receptors.

Effect of mixed (RB 38A) and selective (RB 38B) inhibitors of enkephalin degrading enzymes on a model of depression in the rat.

This is a study of the effects of the endogenous opioid peptides, enkephalins, on learned helplessness, an experimental model of depression in rats. For this purpose, the responses induced by RB 38A, a mixed inhibitor of enkephalin catabolism, and RB 38B, a selective inhibitor of neutral endopeptidase EC 3.4.24.11, were compared with the antidepressive effect induced by imipramine. RB 38A and RB 38B induced an imipramine-like effect in reducing helpless behavior, as illustrated by the decrease in the number of escape failures. According to the different pharmacological potential of both inhibitors to reduce enkephalin metabolism, complete inhibition of enkephalins (RB 38A) produced a higher response than that obtained with a partial inhibitor (RB 38B). On the other hand, naloxone (NLX) was found to facilitate the induction of learned helplessness, and to antagonize the effect of both enkephalin-degrading enzyme inhibitors. These results suggest that modifications in the activity of the endogenous opioid system could take place in this model of depression. The antidepressant-like effects induced by RB 38B, and especially by RB 38A, in the learned helplessness paradigm suggest that new mixed enkephalinase inhibitors, able to cross the blood-brain barrier, could provide a new strategy in the treatment of affective disorders.

In vivo release of CCK-8 from the dorsal horn of the rat: inhibition by DAGOL.

There is evidence that CCK-8 may interact with opioids and that both systems are probably implicated in pain modulation. In order to elucidate this relationship we sought to examine factors governing the movement of CCK-8 from the spinal cord into the extracellular space. We report that CCK-8 like immunoreactivity, as measured by RIA, is released from the spinal cord of the rat in vivo, following potassium stimulation and by direct activation of high threshold peripheral afferents by stimulation of the sciatic nerve. Also, we show that CCK-8 release is inhibited by the mu-selective opioid receptor agonist DAGOL. Naloxone totally reversed the effect produced by DAGOL, implying an opiate mediated mechanism.

Treatment of chronic allodynia in spinally injured rats: effects of intrathecal selective opioid receptor agonists.

We examined the effects of intrathecal (i.t.) selective opioid receptor agonists in alleviating mechanical and cold allodynia in spinally injured rats. Both DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-opioid receptor agonist) and DPDPE ([D-Phe2,D-Phe5]-enkephalin, a delta-opioid receptor agonist) dose-dependently relieved the chronic allodynia-like behavior at doses selective for their respective receptors. The anti-allodynic effect of DAMGO and DPDPE was reversed by the selective mu- and delta-opioid receptor antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) and naltrindole, respectively. In contrast, the selective kappa-opioid receptor agonist U50488H did not alleviate the allodynia-like behavior, but rather enhanced it. The anti-nociceptive and anti-allodynic effect of i.t. DAMGO was blocked by U50488H. Thus, activation of spinal mu- and delta-, but not kappa-opioid receptors produced anti-allodynic effect in this model of central pain. Drugs which act selectively on opioid receptor subtypes may be useful in managing chronic central pain of spinal cord origin.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation.

RB101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl)-L-phenylalanine benzyl ester) is a recently developed full inhibitor of the enkephalin-catabolizing enzymes able to cross the blood-brain barrier, whereas RB38A ((R)-3-(N-hydroxycarboxamido-2-benzylpropanoyl)-L-phenylalanine) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freund's complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg, i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non-inflamed paws. RB101 (10 and 20 mg/kg, i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg, i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 micrograms) blocked the effect of morphine only in non-inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues.

Differences in central effects of beta-endorphin and enkephalins: beta-endorphin. A potent psychomotor stimulant.

The endogenous opiate-like peptides, beta-endorphin, methionine- and leucine-enkephalin have been investigated in unanaesthetized cats after intracerebroventricular injection. beta-Endorphin produced marked and prolonged psychomotor stimulation (restlessness, apprehension, looking around, vacant stare and impelling locomotion), accompanied by pupillary dilation and tremor which was prevented by nalorphine. In contrast to beta-endorphin, the enkephalins did not cause affective behavioural phenomena. However, the enkephalins evoked transient and inconsistent vomiting which was also prevented by nalorphine. It is apparent, therefore, that morphinomimetic brain peptides are involved in at least two functions in the central nervous system: beta-endorphin subserves the mediation of a long-lasting psychomotor stimulation, while the enkephalins mediate vomiting of a transient character.

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)

New kelatorphan-related inhibitors of enkephalin metabolism: improved antinociceptive properties.

In order to improve the in vivo protection of enkephalins from enzymatic degradation, a new series of inhibitors derived from kelatorphan [HONHCOCH2CH(CH2Ph)CONHCH(CH3)COOH], the first-described complete inhibitor of enkephalin metabolism, were designed by modification of the C-terminal amino acid. The progressive lengthening of the chain of this residue shows that a beta-alanine seems to be the best basic model for the conception of such types of compounds. On the other hand, the methylation of the amide bond, which is well accepted by aminopeptidase N (EC 3.4.11.2) and dipeptidylaminopeptidase, induced a significant loss of affinity for neutral endopeptidase -24.11. Starting from these data, compounds containing a variously substituted beta-alanine residue and corresponding to the general formula HONHCOCH2CH(CH2Ph)CONHCH(R1)CH(R2)COOH were synthesized. All these molecules inhibit neutral endopeptidase -24.11 and dipeptidylaminopeptidase in the nanomolar range, and those containing an aromatic chain (compound 7A, R1 = CH2Ph,R2 = H, and compound 8A, R1 = Ph, R2 = H) inhibit the biologically relevant aminopeptidase N, with IC50's around 10(-8) M. Intracerebroventricular injection in mice of these multienzyme inhibitors produced an efficient and naloxone-reversible analgesic response (hot plate test): compounds 7A and 8A were shown to be more potent than kelatorphan in increasing the jump latency time, in agreement with their in vitro properties, and these new compounds were found to increase the forepaw lick latency, a reflex considered as a typical morphine response.

[L-Ala3]DPDPE: a new enkephalin analog with a unique opioid receptor activity profile. Further evidence of delta-opioid receptor multiplicity.

To investigate delta-opioid receptor topography near the 3-position of [D-Pen2,D-Pen5]enkephalin (DPDPE), a series of small-group 3-position analogs of DPDPE have been synthesized and assayed for binding potencies and in vitro biological activities. L-Amino acid substitutions at this position are highly favored over D-amino acid substitutions, with the smallest, [L-Ala3]DPDPE (DPADPE), being the most favored in the series investigated. [L-Ala3]DPDPE is nearly as delta-potent and more delta-selective in both rat brain binding (18 nM vs [3H] [p-ClPhe4]DPDPE and mu/delta = 610) and peripheral bioassays (12 nM in the MVD and GPI/MVD = 4500) when compared to DPDPE (8.5 nM, mu/delta = 73 and 4.1 nM, GPI/MVD = 1800, respectively). Whereas DPDPE is a potent analgesic when given icv, [L-Ala3]DPDPE is only a weak analgesic. However, [L-Ala3]DPDPE has been found to antagonize DPDPE, but not Deltorphin II, in a moderately potent (pA2 = 5.7) and selective fashion in vivo. Thus, [L-Ala3]DPDPE is a fairly potent agonist at peripheral delta receptors and is a moderately potent (mixed) antagonist of delta 1 receptors in the brain. It appears that [L-Ala3]DPDPE does not interact in any significant manner with delta 2 or mu receptors in the brain.

7-Spiroindanyl derivatives of naltrexone and oxymorphone as selective ligands for delta opioid receptors.

A series consisting of spiroindanyl (5-7), benzospiroindanyl (8-10), and spiroperinaphthyl (11) derivatives of naltrexone and oxymorphone were synthesized in order to investigate the role of an orthogonal-oriented "address" for delta opioid receptors. All of the ligands exhibited a preference for delta receptors in vitro. The 7-benzospiroindanyl derivative 8 (BSINTX) was the most selective delta opioid receptor antagonist in vitro. In mice BSINTX antagonized the delta 1-selective agonist, [D-Pen2,D-Pen5]enkephalin without significantly affecting the antinociceptive potency of delta 2, mu, and kappa agonists. The results of this study are consistent with an orthogonally-oriented address favoring delta 1 activity.