D-Ala2,D-Leu5]enkephalin Research Articles

Chronic opioid treatment attenuates carbachol-mediated polyphosphoinositide hydrolysis in chick embryo neuronal cultures

Opiate binding sites on cultured neurons derived from 6-day-old (E6) chick embryo cerebral hemispheres (CH), shown to be cholinergic by choline acetyltransferase immunostaining, were labeledd with [ 3H]etorphine (μ and δ opiate receptors expression) and [ 3H]morphine (mostly μ). When examined by light microscope autoradiography, opiate receptors were found to be expressed by most neurons and were distributed predominantly on neuronal perikarya. Muscarnic and opiate receptors in E6CH cultured neurons were found to be functionally coupled when the effects of opiate receptor occupancy on the inositol phosphate-linked muscarinic receptors was studied. Carbachol stimulated the release of [ 3H]inositol phosphates (InsP) from cultures preincubated with [ 3H]inositol and LiCl, in a dose-dependent manner, and the functional expression of muscarinic receptors peaked in number at day 7 in culture, declining thereafter. Short-term (<1h) treatment of E6 neuronal cultures with 1 μM opioid peptides such as morphiceptin or d-Ala 2- d-Leu 5-enkephalin (DADLE) did not not inhibit the release of inositol phosphates in response to 1 mM carbachol whereas forskolin, which also activates adenylate cyclase and raises cAMP levels, inhibited InsP release by about 25%. In contrast, long-term (48 h) opioid treatment with either morphiceptin or DADLE (1–10 μM) inhibited the carbachol-stimulated inositol phosphate release by ⩾50%. Prolonged treatment with morphiceptin also inhibited the bradykinin-mediated release of InsP from E6CH cells. In both cases, the inhibition was blocked by the continuous presence of naloxone, suggesting that the inhibition was mediated through opiate receptors. When E6CH cells were depolarized by 30 mM K +, allowing [Ca 2+]i to increase, the inhibition of inositol phosphate formation caused by long-term morphiceptin exposure was partially relieved. This 48 h (chronic) exposure to opioids may involve some depletion of available [Ca 2+]i in cholinergic neurons. We propose that these interactions between the opiate and muscarinic receptor transducing systems may represent the molecular basis of the neuromodulating activity of opioids upon the cholinergic system early in development.

Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus

The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture. Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations. β-Endorphin 1–31 caused a 65% maximal inhibitory effect (IC 50 = 6.1 × 10 −9 M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin A 1–8 and DADLE ([ d-Ala 2, d-Leu 5] enkephalin). The effects of β-endorphin 1–31 and Met-enkephalin were completely reversed by the δ opioid antagonist ICI 174,864 while the κ-receptor specific antagonist binaltorphimine or the σ-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A 1–8 and of DADLE. The μ-receptor specific agonist DAGO ( [ d-Ala 2,N- Me-Phe 4, Gly 5- OL]enkephalin ) did not affect POMC mRNA levels. The failure of the dopaminergic D 2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the δ-, κ- and σ- (but not μ-) receptors in vivo.

Increased dopamine receptor sensitivity in the rat following acute administration of sufentanil, U50,488H and d-Ala2-d-Leu5-enkephalin

The effects of acutely administered opioid receptor agonists sufentanil, U50,488H and [D-Ala2,D-Leu5]-enkephalin (DADL) were observed upon dopamine D1 and D2 binding site density in the striatum of the rat. In addition, the functional implications of opioid-induced changes in dopamine receptor sensitivity were studied using the behavioural profile elicited by apomorphine in the rat. The mu-agonist sufentanil (1 or 20 micrograms/kg, i.p.), the kappa-agonist U50,488H (10 mg/kg, i.p.) and DADL (1 microgram/animal, i.c.v.) all significantly elevated D2 but not D1 binding site density in rat striatum. Pretreatment with sufentanil (1 microgram/kg, i.p.) induced an elevation in apomorphine-induced stereotyped behaviour, but attenuated locomotor activity. Following administration of U50,488H (10 mg/kg, i.p.), both the degree of stereotypy and the intensity of the locomotor activity were enhanced. Contralateral rotation was observed in animals pretreated with DADL (1 microgram/animal, i.c.v.) following challenge with apomorphine. It is concluded that the opioid agonists studied induce a significant elevation in functional D2 sites to the exclusion of D1 sites. However, the precise mechanism by which this effect is elicited remains to be established.

Ontogeny and distribution of opioid receptors in the rat brainstem

The distribution and postnatal ontogeny of opioid receptors have been investigated using in vitro quantitative receptor autoradiography. Rats were studied at postnatal day 1 (P1), P5, P10, P21 and P120 (adult). Opioid receptor sites for ( d-Ala 2,N-MePhe 4,Gly-ol 5)-enkephalin (DAMGO) binding were labelled with 4 nM of 3H-DAMGO; ( d-Ala 2, d-Leu 5)-enkephalin (DADLE) binding sites were labelled with 4 nM of 3H-DADLE in the presence of 1 μM unlabelled μ-agonist (N-MePhe 3, d-Pro 4)-morphiceptin (PL017). We found that both binding sites have strikingly different distributional patterns. [ 3H]DADLE binding sites were rather homogeneous, whereas the distribution of [ 3H]DAMGO binding was very heterogeneous with the highest density in the nucleus of the solitary tract (NTS), ambiguus nucleus, dorsal motor nucleus of the vagus and the parabrachial areas. [ 3H]DAMGO binding density was 2- to 40-fold higher than [ 3H]DADLE binding sites in most brainstem nuclei. [ 3H]DAMGO binding sites appeared in most brainstem nuclei at birth, with a high density in cardiorespiratory-related nuclei, whereas [ 3H]DADLE binding sites were too scarce to be quantitated at P1. Both binding sites increased with age, but the developing patterns depended on the nucleus and the type of binding site. In most areas, the densities of both binding sites reached a maximum between P10 and P21 and then decreased to an adult level, but in some nuclei (e.g. the caudal part of the NTS and dorsal raphe nucleus), [ 3H]DAMGO binding sites kept increasing until adulthood. In contrast with the brainstem, cortical areas had a lower binding density in the newborn and reached peak levels later than brainstem regions (post P21). We conclude that (1) since [ 3H]DAMGO binding sites mainly reflect μ-receptors and [ 3H]DADLE binding sites δ-receptors (in the presence of PL017), the brainstem is essentially a μ-receptor region though δ-receptors are present; (2) both opioid receptors are present at birth but δ-receptors are very scarce in the newborn; (3) both receptors increase with age, but the time course depended on various nuclei and receptor types; (4) cardiorespiratory-related nuclei have high density of μ-receptors at all ages; and (5) opioid receptors develop earlier in the brainstem than in the cortex.

Evidence for opioid receptor-mediated activation of the G-proteins, Go and Gi2, in membranes of neuroblastoma x glioma (NG108-15) hybrid cells

In membranes of neuroblastoma x glioma (NG108-15) hybrid cells, the photoreactive GTP analog, [alpha-32P] GTP azidoanilide, was incorporated into 39-41-kDa proteins comigrating in urea-containing sodium dodecyl sulfate-polyacrylamide gels with immunologically identified G-protein alpha-subunits, i.e. a 39-kDa Go alpha-subunit, a 40-kDa Gi2 alpha-subunit, and a 41-kDa Gi alpha-subunit of an unknown subtype. The synthetic opioid, D-Ala2,D-Leu5-enkephalin (DADLE), stimulated photolabeling of the 39-41-kDa proteins. In the presence of GDP, which increased the ratio of agonist-stimulated to basal photolabeling, DADLE at a maximally effective concentration stimulated photolabeling of the 39- and the 40-kDa protein 2-3-fold. Somatostatin, adrenaline, and bradykinin were less potent than DADLE and, to varying degrees, stimulated photolabeling of the 40-kDa protein more than that of the 39-kDa protein. Prostaglandin E1 was inactive. The present data represent direct evidence for an activation of endogenous Go and Gi2 via opioid receptors and other receptors in the native membrane milieu.

Pharmacological profile of various kappa-agonists at kappa-, mu- and delta-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the rat brain.

1. The potency, relative efficacy and selectivity of a series of kappa-opioid receptor agonists at the mu-, delta- and kappa-opioid receptors mediating inhibition of electrically-induced (radiolabelled) neurotransmitter release from superfused rat brain slices was determined. 2. With regard to their potencies at kappa-receptors mediating inhibition of striatal [3H]-dopamine release, the highest pD2 value (8.7) was found for bremazocine and the lowest (7.1) for U50488; the pD2 values for ethylketocyclazocine (EKC), tifluadom, U69593 and PD117302 were between 8.0 and 8.3. There were no marked differences between the relative efficacies of the kappa-agonists (maximum inhibition being 60-70%). In contrast to the other kappa-agonists, at a concentration of 1 microM, PD117302 caused a significant (25-40%) increase of the spontaneous efflux of tritium. 3. None of the kappa-agonists significantly affected striatal [14C]-acetylcholine (ACh) release, with the exception of a slight inhibitory effect of EKC. The delta-receptor-mediated inhibitory effect of [D-Ala2, D-Leu5]enkephalin (DADLE) on [14C]-ACh release was antagonized in a concentration-dependent manner by bremazocine (0.1 and 1.0 microM) and also partially by EKC (1 microM), but not by the other kappa-agonists. The pA2 value for bremazocine as an antagonist at the delta-receptors involved was 8.0, compared to 7.6 for naloxone. 4. None of the kappa-agonists significantly affected cortical [3H]-noradrenaline (NA) release, with the notable exception of tifluadom, which strongly inhibited release by activating mu-receptors. The mu-receptor-mediated inhibitory effect of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) on [3H]-NA release was antagonized in a concentration-dependent manner by bremazocine and EKC, but not by the other K-agonists. The pA2 value for bremazocine as an antagonist at the mu-receptors involved was 8.2, compared to 8.6 for naloxone. 5. Thus, whereas U69593 and PD1 17302 display high potency and selectivity towards K-opioid receptors, the potent benzomorphan K-agonists bremazocine and EKC also appear to be strong mu-opioid receptor antagonists.

State-dependent variation in the inhibitory effect of [D-Ala 2, D-Leu 5]- enkephalin on hippocampal serotonin release in ground squirrels

Accumulated evidence has suggested that increased endogenous opioid activities may facilitate the onset of hibernation either directly or possibly through modulation of other neurotransmitter systems. The seasonal change of [D-Ala 2, D-Leu 5]-enkephalin (DADLE), a σ receptor agonist, in modulating K + (35 mM)- induced [ 3H]-5-hydroxytryptamine (5-HT) release from the hippocampal and hypothalamic slices of euthermic and hibernating Richardsons' ground squirrels was therefore investigated. DADLE (0.1 – 10 μM) had no effect on 5-HT release in the hypothalamic slices but elicited a dose-related inhibition on [ 3H]-5-HT release from the hippocampal slices of the euthermic ground squirrel. The inhibitory effect of DADLE was completely reversed by naloxone (10 μM), but not by tetrodotoxin (1 μM). In contrast, DADLE failed to alter the K +-induced 5-HT release from the hippocampal slices of the hibernating ground squirrel. This state-dependent reduction in responsiveness to an opioid is consistent with the hypothesis that enhanced endogenous opioid activity in the hibernating phase could lead to down regulation of the opioid receptors and minimize its inhibition on hippocampal serotonergic activity. A high 5-HT activity would inhibit midbrain reticular activating system indirectly through non-serotonergic fibers, which in turn facilitate the onset or maintenance of hibernation.

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro

Several peptides of diverse structure, reported to possess high affinity and selectivity for the δ opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu 5]enkephalin, the cyclic enkephalin analogs [D-Pen 2, D-Pen 5] enkephalin (DPDPE) and [D-Pen 2, p-F-Phe 4, D-Pen 5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala 2, D-Leu 5]enkephalin (DADLE) and [D-Ser 2(O-tBu), Leu 5, Thr 6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH 2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH 2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH 2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC 50 = 1.13 X 10 −10 molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.

Enkephalins modulate inhibitory neuromuscular transmission in circular muscle of human colon via delta‐opioid receptors.

1. A sucrose-gap technique was used to investigate the neuromodulatory actions of enkephalins on non-adrenergic, non-cholinergic inhibitory junction potentials (IJPs) in the circular muscle of the human large intestine. 2. The native enkephalins, [Leu5]enkephalin (LENK) and [Met5]enkephalin (MENK) caused a concentration-dependent reduction in amplitude of IJPs without a significant effect on the smooth muscle membrane. 3. The actions of LENK and MENK were mimicked by the delta-selective opioid receptor agonists [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Ala2, D-Leu5]enkephalin (DADLE). 4. The actions of LENK, MENK and DPDPE were antagonized to similar extents by the delta-selective opioid receptor antagonist ICI 174,864. 5. The mu-selective opioid receptor agonist [D-Ala2, Me Phe, Gly-ol5]enkephalin was approximately 100-fold less potent than any of the native or synthetic enkephalins at reducing the amplitude of the IJP. Dynorphin A and beta-endorphin both had very weak activity. 6. Responses to all of the agonists were inhibited by naloxone. The degree of antagonism of DPDPE or DADLE by naloxone (1 microM) was the same as that of LENK or MENK. 7. Neither MENK nor LENK affected hyperpolarization of the smooth muscle membrane induced by ATP or 5-hydroxytryptamine. Vasoactive intestinal polypeptide (1 pM-1 microM) did not produce any observable responses and this lack of reactivity was not affected by the enkephalins. 8. It is concluded that in the circular muscle of the human colon, LENK and MENK can act on prejunctional delta-opioid receptors to produce inhibition of non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. Possible physiological significance of this prejunctional receptor is discussed.

Peptide opioids and morphine effects on inflammatory process

Morphine was found to inhibit human granulocyte aggregation and ATP, thromboxane B2 (TxB2), and leukotriene B4 (LTB4) secretion during cell aggregation. None of the opioid peptides tested [(D-Ala2, D-Leu5)-enkephalin (DADL), (D-Ala2, N-Me-Phe4, Gly-ol5)-enkephalin (DAGO) or dynorphin 1-9 (Dyn 1-9)] was capable of mimicking morphine effects, while Dyn 1-9 per se induced TxB2 and LTB4 secretion from granulocytes. Morphine inhibition of both cell aggregation and ATP, but not of arachidonic acid metabolism product secretion, was prevented by naloxone. The naloxone-sensitive impairment by morphine of CD11b-CD18 complex surface expression observed could play a role in opioid inhibition of granulocyte activation.

Opioid receptors in the accessory optic system of the rat: Effects of monocular enucleation

The presence and concentrations of each of the three subtypes of opioid receptors (mu, kappa, and delta) has been studied in the accessory optic nuclei (dorsal, lateral, and medial terminal nuclei and the interstitial nucleus of the superior fasciculus, posterior fibers: DTN, LTN, MTN, and inSFp) in normal young rats with radioligands directed towards each opioid receptor subtype. The changes in mu opioid receptors have also been investigated in monocularly enucleated rats in which one eye was removed and the rats sacrificed at postoperative day (PO) 2, 3, 5, 7, 14, and 30. As the MTN is the only accessory optic nucleus of the rat large enough for semiquantitative evaluation, the mu receptor population of the MTN has been subjected to optical microdensitometric analysis. All four of the accessory optic nuclei (AOS nuclei) are found to contain exceedingly high levels of mu opioid receptor binding with the selective radioligand [3H]-[D-Ala,MePhe4, Gly-ol5] (DAGO), low levels of kappa opioid receptor binding using the radioligand [3H]-[ethylketocyclazocine] (EKC) together with the competing agents [D-Pro4]-morphiceptin and [D-Ser2,Thr6]-Leu-enkephalin, and an absence of delta opioid receptor binding with the radioligand [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) combined with the competing agent [D-Pro4]-morphiceptin. Monocular enucleation, as studied on the mu opioid receptor population with this experimental approach, results in virtually a complete loss of mu opioid receptors throughout all four of the contralaterally located AOS nuclei, including both dorsal and ventral subdivisions of the medial terminal nucleus (MTNd,v). Kappa and delta receptors are very few (kappa receptors) or are lacking (delta receptors) in the AOS nuclei, and for this reason, the effects of monocular enucleation on these two opioid receptor subtypes have not been investigated. Monocular enucleation also produces a significant lowering in mu receptor binding in other primary optic nuclei (the lateral geniculate nuclei, nucleus of the optic tract, and superficial layers of the superior colliculus) and in the pars principalis of the medial geniculate nucleus (description of changes in mu receptors in non-accessory optic primary optic nuclei will be considered elsewhere). Microdensitometric study of the MTNd,v shows that the decreased binding of mu receptors in this nucleus is barely detectable (about 6%) at PO2 and rises to 6-15% at PO3. At PO5 receptor loss reaches approximately 62%, whereas at PO7 it is about 81% complete. At PO14 and PO30, the mu receptor loss is nearly complete at around 93%.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperpolarizing action of enkephalin on neurons in the dorsal motor nucleus of the vagus, in vitro

Intracellular recordings were made from neurons of the dorsomotor vagal nucleus (DMV) in slices of rat medulla oblongata. [D-Ala 2, D-Leu 5]-enkephalin (DADLE), applied by perfusion (0.01–3 μM) or droplets, dose-dependently hyperpolarized 85% of the DMV neurons tested. The hyperpolarization, associated with a decrease in membrane resistance, persisted after elimination of synaptic activity by perfusion with Ca 2+-free/high-Mg 2+ solution or with 1 μM TTX solution. The opioid antagonist, naloxone, reversibly inhibited DADLE-induced hyperpolarization. The hyperpolarization depended on extracellular K + concentration and reversed at about −90 mV. DADLE also decreased Ca 2+-dependent spike duration and after-hyperpolarization (AHP). DAGO (a selective μ-receptor agonist), but not DPLPE (a selective δ-receptor agonist), mimicked DADLE's effects on membrane potential, Ca 2+-dependent spike duration, and AHP. It is concluded that DADLE, through postsynaptic μ-type opioid receptors, hyperpolarized DMV neurons by increasing K + conductance, which may have an inhibitory effect on DMV output. DADLE-induced decrease of spike duration and AHP was also mediated by μ-receptors and could have additional effects on functions of the DMV neuron by virtue of reduction in Ca 2+ entry.

Simultaneous development of opioid tolerance and opioid antagonist-induced receptor upregulation

Mice treated chronically with opioid antagonists have increased receptor density in brain and are supersensitive to the pharmacodynamic action of morphine. In the present study mice were implanted subcutaneously with naltrexone or placebo pellets for 8 days. During implantation mice received daily injections of morphine (100 or 250 mg/kg) or saline. Morphine analgesia was completely blocked in mice that were implanted with naltrexone at the low dose of morphine; while some analgesic action was observed at the higher dose. Mice implanted with placebo were analgesic following the daily morphine treatment. At the end of 8 days the pellets were removed and 24 h later some mice were tested for morphine analgesia while others were examined in binding studies. Naltrexone treatment increased [ 3H]naloxone, 3H[ d-Ala 2- d-Leu 5]enkephalin (DADLE) and 3H[ d-Ala 2,NMePhe 4,Gly-ol 5]enkephalin (DAGO) binding compared to controls and increased the analgesic potency of morphine. Daily treatment with morphine did not alter brain opioid binding or naltrexone-induced receptor upregulation. Mice injected daily with morphine were significantly less sensitive to morphine (tolerant) than their respective saline control group for both the placebo and the naltrexone-treated groups. However, naltrexone-treated mice were more sensitive to morphine than placebo controls regardless of whether they were injected daily with morphine or not. These results indicate that if naltrexone-induced opioid receptor upregulation occurs in the presence of repeated agonist administration, the new binding sites mediate tolerance via desensitization to morphine.

Opiate receptors in neuronal primary cultures

The occurrence and characteristics of μ-, δ- and k-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of cAMP in neuronal enriched primary cultures from the cerebral cortex of foetal rats. Morphine or [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAGO) were used as μ-receptor agonists. [ d-Ala 2, d-Leu 5-Enkephalin (DADLE) or [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE) were used as δ -receptor agonists and dynorphin 1–13 (Dyn) or U-50, 488H were used as k-receptor agonists. In the presence of 10 −8–10 −5 M morphine or 10 −8–10 −5 M DAGO, there was a dose-related inhibition of the 10 −5 M forskolin-stimulated accumulation of cAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10 −9–10 −6 M DADLE or 10 −9–10 −6 M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of cAMP and a similar result was obtained in the presence of 10 −9–10 −5 M Dyn or 10 −9–10 −5 M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express μ-, δ- and k-receptors, that inhibit the forskolin-stimulated accumulation of cAMP. Administration of 10 −5 M morphine and 10 −6 M DADLE or 10 −6 M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of cAMP, indicating the presence of both μ- and δ -receptors on the same population of cells.

Effects of chronic clomipramine on central DADLE antinociception

Equipotent antinociceptive doses, as determined by a tail-flick response, for centrally administered (periaqueductal gray) morphine (M) and d-Ala 2, d-Leu 5 enkephalin (DADLE) were established as 5 μg and 19 μg, respectively. Chronic (28 day) subcutaneous infusion of clomipramine (CMI) via an Alzet minipump attenuated both central M-and DADLE-induced analgesia by day 15; attenuation persisted for the duration of the infusion (day 29). Within 7 days following removal of the pump, antinociceptive responses to M and DADLE returned to near pre-CMI Ievels. Our results indicate a similarity between M and DADLE with regard to attenuation of their antinociceptive action by chronic CMI. This attenuation may be due to decreased mu opioid receptor sensitivity or density resulting from chronic tricyclic antidepressant administration.

Decrease in μ-opioid receptor binding capacity in rat brain after chronic PL017 treatment

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or d-Ala 2, d-Leu 5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, μ-opioid receptor binding capacity was attenuated together with the δ-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both μ and δ receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the μ-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5–20 μg/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD 50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED 50 of the peptide to elicit the catatonic effect. When μ- and δ-binding was determined using [ 3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20–30%) in the μ-opioid receptor binding but not in δ-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [ 3H]DAMGO saturation binding data revealed a decrease inB max values and no change in theK d values. Hence, μ-opioid receptors can be specifically regulated by ligand in the brain as δ-receptors are in neuroblastoma × glioma NG 108-15 cells. Chronic activation of the μ-opioid receptor in the brain would result in the down-regulation of the binding sites.

Computer modeling of subtypes of κ opioid receptors in adrenal medulla

The existence of multiple subtypes of κ opioid receptors in brain and adrenal medulla, has been controversial. We have characterized opioid receptors in frozen membranes from bovine adrenal medulla by use of objective mathematical modeling. [ 3H]Etorphine, [ 3H]ethylketocyclazocine (EKC) and [ 3H][D-Ala 2, D-Leu 5]enkephalin (DADL) were utilized as labeled ligands. Self- and cross-displacement curves were constructed using the three corresponding unlabeled ligands in the presence or absence of increasing concentrations of DADL. Results indicated: (1) each of the three ligands studied individually showed the presence of heterogeneity of binding sites; (2) sites labeled by etorphine were heterogeneous: 84% of etorphine binding was displaceable by 10 −4 M DADL, while the remaining 16% was DADL non-suppressible; (3) 75% of the binding of EKC was displaceable by DADL while 25% was non-suppressible; (4) mathematical modeling showed the presence of three subtypes of κ binding sites (a) κ 1, showing slight selectivity for EKC relative to etorphine; (b) κ 2, with K d ≈ 1 nM for etorphine, and sufficiently high affinity for DADL (K d ≈ 150 nM) so that it is suppressible by 100 μM DADL; and (c) K 3, with no measurable affinity for DADL and a 27-fold selectivity for etorphine relative to EKC. The three subtypes of κ sites were present at concentrations of 7.4, 75, and 55 fmol/mg protein, respectively. The relative affinities of a series of κ agonists for the etorphine-binding sites were characterized. The present studies confirm the existence of three subtypes of κ opioid receptors in bovine adrenal medulla, and indicate the utility of mathematical modeling for characterization of complex receptor systems.

Effect of opioids on acetylcholine release evoked by K + or glutamic acid from rat neostriatal slices

Endogenous acetylcholine (ACh) release from rat striatal slices was measured by a chemiluminescent method. Several opiate agents were tested for their ability to modulate ACh release evoked by potassium ions (K +) or glutamic acid (GLU). Morphine, [ d-Ala 2, Gly(ol) 5]-enkephalin (DAGO), [ d-Ala 2, d-Leu 5]-enkephalin (DADLE) and [ d-Pen 2- d-Pen 5]-enkephalin (DPDPE) were found to have an inhibitory effect on K +- or GLU-evoked ACh release. This effect was completely blocked by naloxone, but this antagonist by itself had no effect on ACh release. The action of μ-opiate agonists (morphine and DAGO) on ACh release evoked by K + was sensitive to tetrodotoxin (TTX), but that of δ-opiate agonists (DADLE and DPDPE) was insensitive. The release evoked by GLU was abolished in the presence of TTX. The activation of κ-opiate receptor by dynorphin-(1–13) had no effect on K +- or GLU-evoked ACh release. It is concluded that μ- and δ-opiate agonists, but not κ, exert an inhibitory control on striatal cholinergic interneurons, but with a different mechanism of action or localization of the receptors. Corticostriatal glutamatergic neurons have an important role in the interaction of the ACh-opioid systems.

Modulation of Histamine Release in the Rat Brain by K‐Opioid Receptors

The opioid modulation of histamine release was studied in rat brain slices labeled with L-[3H]histidine. The K(+)-induced [3H]histamine release from cortical slices was progressively inhibited by the preferential kappa-agonists ketocyclazocine, dynorphin A (1-13), Cambridge 20, spiradoline, U50,488H, and U69,593 in increasing concentrations. In contrast, the mu-agonists morphine, morphiceptin, and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) were ineffective as were the preferential delta-agonists [D-Ala2,D-Leu5]enkephalin (DA-DLE) and [D-Pen2,D-Pen5]enkephalin (DPDPE). Nor-binaltorphimine (nor-BNI) and MR 2266, two preferential kappa-antagonists, reversed the inhibitory effect of the various kappa-agonists more potently than did naloxone, with mean Ki values of 4 nM and 25 nM, respectively. The effects of ketocyclazocine and naloxone also were seen in slices of rat striatum, another brain region known to contain histaminergic nerve endings. We conclude that kappa-opioid receptors, presumably located on histaminergic axons, control histamine release in the brain. However, nor-BNI and naloxone failed, when added alone, to enhance significantly [3H]histamine release from cerebral cortex or striatum, and bestatin, an aminopeptidase inhibitor, failed to decrease K(+)-evoked [3H]histamine release. These two findings suggest that under basal conditions these kappa-opioid receptors are not tonically activated by endogenous dynorphin peptides. The inhibition of cerebral histamine release by kappa-agonists may mediate the sedative actions of these agents in vivo.

Kappa‐Selective Opioid Receptor Agonists Leumorphin and Dynorphin Inhibit Supraoptic Neurons in Rat Hypothalamic Slice Preparations

Abstract To clarify the effects of opioid peptides, and in particular the effects of kappa-receptor agonists on the activity of supraoptic neurons, extracellular recordings were made from 71 spontaneously firing neurons in the rat hypothalamic slice preparation. Of 71 neurons, 28 showed a phasic firing pattern (phasic neurons: putative vasopressin neurons). The mean firing rate of phasic neurons was 2.6 spikes/s (intraburst firing rate 5.4 +/- 2.2 spikes/s). The mean firing rate of neurons classified as non-phasic neurons (putative oxytocin neurons) was 4.5 spikes/s. Following bath application of leumorphin (LM) at 10(-7) M, which has potent opioid activity at kappa-receptors, 17 (61%) of 28 phasic neurons were inhibited and 22 (51%) of 43 non-phasic neurons were inhibited. Excitation was observed in only one non-phasic neuron. The dose-dependence of the response to LM was tested in five supraoptic neurons. There was an inverse relationship between LM concentration and percent change in firing rate. The threshold concentration of LM was approximately 10(-8) M. The relatively selective kappa-receptor antagonist, MR-2266, completely blocked the LM-induced responses. Its effects were long-lasting and only partial recovery was observed 2 h after the application of MR-2266. Dynorphin had similar inhibitory effects on supraoptic neurons to those obtained with LM when tested on the same neurons. In another series of experiments the mu-receptor agonist morphine and the delta-receptor agonist [D-Ala, D-leu]-enkephalin (DADLE) were applied to 28 supraoptic neurons (12 phasic and 16 non-phasic neurons) at 10(-7) M and their actions compared directly with that of LM. Only two of 12 phasic neurons tested were inhibited by DADLE and none of five phasic neurons tested was inhibited by morphine, while eight of the 12 neurons tested were inhibited by LM. By contrast the non-phasic neurons tested were inhibited by application of each of the peptides; seven of 16 neurons tested were not only inhibited by LM, but also five of 11 neurons by DADLE and seven of 15 by morphine. The magnitude of the responses varied from cell to cell. These results suggest that LM acts at the same receptors as dynorphin, and that opioids acting preferentially at kappa-receptors inhibit both vasopressin and oxytocin neurons while delta- and mu-receptor agonists inhibit primarily oxytocin neurons.