Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance.

We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.     

Brief treatment of sensory ganglion neurons with GM1 ganglioside enhances the efficacy of opioid excitatory effects on the action potential

In previous studies, we showed that low (nM) concentrations of opioid prolong the action potential duration (APD) of many mouse dorsal root ganglion (DRG) neurons via Gs-linked excitatory opioid receptors, whereas micromolar opioid levels shorten the APD via Gi/Go-linked inhibitory receptors. In addition, cholera toxin-B subunit (CTX-B) selectively blocks opioid- but not forskolin-induced prolongation of the APD in DRG neurons. Since CTX-B binds with selective high affinity to GM1 ganglioside located on the cell surface, the results suggest that GM1 plays an essential role in regulating excitatory opioid receptor functions. This hypothesis was tested by treating DRG neurons in mouse DRG-cord explants with exogenous gangliosides and determining whether the efficacy of opioid agonists in prolonging the APD is enhanced. The threshold concentration of the opioids, dynorphin(1-13) and morphine required to prolong the APD in many DRG neurons was markedly decreased from nM to fM levels after bath exposure to 10 nM to 1 microM GM1 ganglioside for less than 5 min. In contrast, GM2 and GM3 gangliosides and asialo-GM1 ganglioside were ineffective, even when DRG neurons were exposed to high concentrations (1-10 microM) for periods greater than 1 h. Although GD1a, GD1b and GQ1b gangliosides appeared to be as effective as GM1 when tested at microM concentrations for 15 min, tests at lower concentrations, shorter periods, and/or at lower temperature (24 degrees vs 34 degrees C), showed that they were significantly less effective than GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic opioid treatment of neuroblastoma X dorsal root ganglion neuron hybrid F11 cells results in elevated GM1 ganglioside and cyclic adenosine monophosphate levels and onset of naloxone-evoked decreases in membrane K+ currents

Prolongation of the action potential duration of dorsal root ganglion (DRG) neurons by low (nM) concentrations of opioids occurs through activation of excitatory opioid receptors that are positively coupled via G_s regulatory protein to adenylate cyclase. Previous results suggested GM1 ganglioside to have an essential role in regulating this excitatory response, but not the inhibitory (APD-shortening) response to higher (μM) opioid concentrations. Furthermore, it was proposed that synthesis of GM1 is upregulated by prolonged activation of excitatory opioid receptor functions. To explore this possibility we have utilized cultures of hybrid F11 cells to carry out closely correlated electrophysiological and biochemical analyses of the effects of chronic opioid treatment on a homogeneous population of clonal cells which express many functions characteristic of DRG neurons. We show that chronic opioid exposure of F11 cells does, in fact, result in elevated levels of GM1 as well as cyclic adenosine monophosphate (AMP), concomitant with the onset of opioid excitatory supersensitivity as manifested by naloxone-evoked decreases in voltage-dependent membrane K⁺ currents. Such elevation of GM1 would be expected to enhance the efficacy of excitatory opioid receptor activation of the G_s/adenylate cyclase/cyclic AMP system, thereby providing a positive feedback mechanism that may account for the remarkable supersensitivity of chronic opioid-treated neurons to the excitatory effects of opioid agonists as well as antagonists. These in vitro findings may provide novel insights into the mechanisms underlying naloxone-precipitated withdrawal syndromes and opioid-induced hyperalgesia after chronic opiatf addiction in vivo. © 1995 Wiley-Liss, Inc.     

Cholera toxin-A subunit blocks opioid excitatory effects of sensory neuron action potentials indicating mediation by Gs-linked opioid receptors

Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances. In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system. In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses. After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected. Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD. The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium. The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioids excite rather than inhibit sensory neurons after chronic opioid exposure of spinal cord-ganglion cultures

Tests were carried out to determine if the tolerance that develops in dorsal-horn network responses of mouse dorsal root ganglion (DRG)-spinal cord explants after chronic exposure to opioids could be accounted for by alterations in the excitability and pharmacologic properties of the afferent DRG cells. Intracellular recordings were made from DRG neurons in organotypic DRG-cord explants after chronic treatment with 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 4 days in vitro. Acute application of 10 microM DADLE shortened the duration of the Ca2+ component of the somatic action potential (APD) in only 5% of the treated neurons (4 out of 79 cells), in contrast to about 50% of the cells in naive explants (36 out of 74). Thus many DRG neuron perikarya became tolerant to the APD-shortening effects of DADLE. Furthermore, 77% of the treated DRG cells (61 out of 79) showed prolongation of the APD in response to an acute increase in DADLE concentration vs 34% in naive explants (25 out of 74). However, when the DADLE responsivity tests were carried out in the presence of multiple K+ channel blockers, only 20% of the treated DRG neurons showed APD prolongation (3 out of 15 cells), whereas 73% showed APD-shortening responses (11 out of 15 cells). The results suggest that: (1) DADLE-induced APD prolongation of the treated DRG neurons is mediated by opioid receptor subtypes that decrease a voltage-sensitive K+ conductance; (2) the DADLE-induced APD-shortening effects which are unmasked during more complete K+ channel blockade are mediated by opioid-receptor subtypes in the same neuron that reduce a voltage-sensitive Ca2+ conductance (resembling kappa receptors). DRG neurons did not become tolerant to either of these two opioid effects after chronic exposure to DADLE. Opioid shortening of the APD of DRG neuron perikarya has been generally accepted to be a model of opioid inhibition of calcium influx and transmitter release at presynaptic DRG terminals6,52,53,65,75,76. It is postulated that the opioid-induced APD prolongation observed in the present study provides evidence that opioids can also evoke direct excitatory effects on neurons. The enhancement of DADLE-induced excitatory responses and attenuation of DADLE-induced inhibitory responses of DRG neurons after chronic exposure to this opioid show striking similarities to the effects of forskolin or pertussis toxin treatment. These in vitro studies may provide clues to compensatory mechanisms underlying physiologic expression of tolerance to opioid analgesic effects in primary afferent synaptic networks.     

Cholera toxin-B subunit blocks excitatory opioid receptor-mediated hyperalgesic effects in mice, thereby unmasking potent opioid analgesia and attenuating opioid tolerance/dependence

In a previous study we demonstrated that injection (i.p.) of low doses of GM1 ganglioside in mice rapidly attenuates morphine’s analgesic effects. This result is consonant with our electrophysiologic studies in nociceptive types of dorsal root ganglion (DRG) neurons in culture, which showed that exogenous GM1 rapidly increased the efficacy of excitatory (Gs-coupled) opioid receptor functions. By contrast, treatment of DRG neurons with the non-toxic B-subunit of cholera toxin (CTX-B) which binds selectively to GM1, blocked the excitatory, but not inhibitory, effects of morphine and other bimodally-acting opioid agonists, thereby resulting in a net increase in inhibitory opioid potency. The present study provides more direct evidence that endogenous GM1 plays a physiologic role in regulating excitatory opioid receptor functions in vivo by demonstrating that cotreatment with remarkably low doses of CTX-B (10 ng/kg, s.c.) selectively blocks hyperalgesic effects elicited by morphine or by a kappa opioid agonist, thereby unmasking potent opioid analgesia. These results are comparable to the effects of cotreatment of mice with morphine plus an ultra-low dose of the opioid antagonist, naltrexone (NTX) which blocks opioid-induced hyperalgesic effects, unmasking potent opioid analgesia. Low-dose NTX selectively blocks excitatory opioid receptors at their recognition site, whereas CTX-B binds to, and interferes with, a putative allosteric GM1 regulatory site on excitatory opioid receptors. Furthermore, chronic cotreatment of mice with morphine plus CTX-B attenuates development of opioid tolerance and physical dependence, as previously shown to occur during cotreatment with low-dose NTX.     

Inhibitor of cyclic AMP-dependent protein kinase blocks opioid-induced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures

The duration of the calcium component of the action potential (APD) of dorsal root ganglion (DRG) neurons in mouse spinal cord-ganglion explants has been shown to be dually modulated via excitatory and inhibitory opioid receptors. In order to determine if opioid-induced APD prolongation is modulated by receptors that are positively coupled to the adenylate cyclase (AC)/cyclic AMP second messenger system, whole-cell recordings were made from mouse DRG neurons grown in dissociated cell cultures. Tests for opioid responsivity were carried out after intracellular dialysis of an inhibitor of cAMP-dependent protein kinase (PKI). In control recordings, both DADLE-induced APD prolongation as well as shortening were prevented by co-perfusion with the opioid antagonist, diprenorphine (10 nM). Intracellular dialysis of PKI in these neurons completely blocked opioid-induced APD prolongation but did not attenuate APD shortening generally elicited by higher opioid concentrations. Bath perfusion of 10 nM DADLE elicited APD prolongation in 59% of the DRG neurons (n = 34) tested with control solution in the recording pipette, whereas none showed APD prolongation when the pipette contained PKI (n = 18). In control tests with 1 microM DADLE, the APD was prolonged in 37% of the cells and shortened in 26% (n = 19); in contrast, a matched group of PKI-treated cells showed no APD prolongation, whereas 42% showed APD shortening (n = 26). The results support the hypothesis that opioid-induced APD prolongation in DRG neurons is mediated by opioid receptor subtypes that are positively coupled via Gs to AC/cAMP-dependent voltage-sensitive ionic conductances.     

Etorphine elicits anomalous excitatory opioid effects on sensory neurons treated with GM1 ganglioside or pertussis toxin in contrast to its potent inhibitory effects on naive or chronic morphine-treated cells

The ultra-potent opioid analgesic, etorphine, elicits naloxone-reversible, dose-dependent inhibitory effects, i.e. shortening of the action potential duration (APD) of naive and chronic morphine-treated sensory dorsal root ganglion (DRG) neurons, even at low (pM-nM) concentrations. In contrast, morphine and most other opioid agonists elicit excitatory effects, i.e. APD prolongation, at these low opioid concentrations, require much higher (ca. 0.1–1 μM) concentrations to shorten the APD of naive neurons, and evoke only excitatory effects on chronic morphine-treated cells even at high > 1–10 wM concentrations. In addition to the potent agonist action of etorphine at μ-, δ- and κ-inhibitory opioid receptors in vivo and on DRG neurons in culture, this opioid has also been shown to be a potentantagonist of excitatory μ-, δ- and κ-receptor functions in naive and chronic morphine-treated DRG neurons. The present study demonstrates that the potent inhibitory APD-shortening effects of etorphine still occur in DRG neurons tested in the presence of a mixture of selective antagonists that blocks all μ-, δ- and κ-opioid receptor-mediated functions, whereas addition of the epsilon (ε)-opioid-receptor antagonist, β-endorphin(1–27) prevents these effects of etorphine. Furthermore, after markedly enhancing excitatory opioid receptor functions in DRG neurons by treatment with GM1 ganglioside or pertussis toxin, etorphine showsexcitatory agonist action onnon-μ-/δ-/κ-opioid receptor functions in these sensory neurons, in contrast to its usual potent antagonist action on μ-, δ- and κ-excitatory receptor functions in naive and even in chronic morphine-treated cells which become supersensitive to the excitatory effects of μ-, δ- and -opioid agonists. This weak excitatory agonist action of etorphine on non-μ-/δ-/κ-opioid receptor functions may account for the tolerance and dependence observed after chronic treatment with extremely high doses of etorphine in vivo.     

Opioids at low concentration decrease openings of K+ channels in sensory ganglion neurons.

Previous studies showed that low concentrations of opioids prolong the calcium-dependent component of the action potential duration (APD) of dorsal root ganglion (DRG) neurons, whereas higher concentrations shorten the APD. In the present study whole-cell voltage-clamp, as well as cell-attached membrane-patch voltage-clamp, recordings demonstrate that application of picomolar to nanomolar concentrations of mu, delta or kappa opioid agonists (DAGO, DPDPE or dynorphin) to DRG neurons in dissociated cell cultures reversibly decreased the activities of voltage-sensitive K+ channels. Pretreatment of DRG neurons with the opioid receptor antagonists, naloxone (30 nM) or diprenorphine (1 nM) prevented mu/delta or kappa opioid-induced decreases in K+ channel activities, respectively. Since opioids added to the bath solution decreased the activities of K+ channels in the membrane patch sealed off by the pipette tip, our results provide strong evidence that some modes of excitatory modulation of the action potential of DRG neurons are mediated by diffusible second messengers. The data are consonant with our previous studies indicating that opioids can elicit excitatory effects on sensory neurons via cholera toxin-sensitive Gs-linked excitatory opioid receptors coupled to cyclic AMP-dependent ionic channels.     

Neuraminidase inhibitor, oseltamivir blocks GM1 ganglioside-regulated excitatory opioid receptor-mediated hyperalgesia, enhances opioid analgesia and attenuates tolerance in mice

The endogenous glycolipid GM1 ganglioside plays a critical role in nociceptive neurons in regulating opioid receptor excitatory signaling demonstrated to mediate "paradoxical" morphine hyperalgesia and to contribute to opioid tolerance/dependence. Neuraminidase (sialidase) increases levels of GM1, a monosialoganglioside, in these neurons by enzymatic removal of sialic acid from abundant polysialylated gangliosides. In this study, acute treatment of mice with the neuraminidase inhibitor, oseltamivir enhanced morphine analgesia. Acute oseltamivir also reversed "paradoxical" hyperalgesia induced by an extremely low dose of morphine, unmasking potent analgesia. In chronic studies, co-administration of oseltamivir with morphine prevented and reversed the hyperalgesia associated with morphine tolerance. These results provide the first evidence indicating that treatment with a neuraminidase inhibitor, oseltamivir, blocks morphine's hyperalgesic effects by decreasing neuronal levels of GM1. The present study further implicates GM1 in modulating morphine analgesia and tolerance, via its effects on the underlying excitatory signaling of Gs-coupled opioid receptors. Finally, this work suggests a remarkable, previously unrecognized effect of oseltamivir-which is widely used clinically as an antiviral agent against influenza-on glycolipid regulation of opioid excitability functions in nociceptive neurons.     

Acute thermal hyperalgesia elicited by low-dose morphine in normal mice is blocked by ultra-low-dose naltrexone, unmasking potent opioid analgesia

Our previous electrophysiologic studies on nociceptive types of dorsal root ganglion (DRG) neurons in culture demonstrated that extremely low fM-nM concentrations of morphine and many other bimodally-acting mu, delta and kappa opioid agonists can elicit direct excitatory opioid receptor-mediated effects, whereas higher (microM) opioid concentrations evoked inhibitory effects. Cotreatment with pM naloxone or naltrexone (NTX) plus fM-nM morphine blocked the excitatory effects and unmasked potent inhibitory effects of these low opioid concentrations. In the present study, hot-water-immersion tail-flick antinociception assays at 52 degrees C on mice showed that extremely low doses of morphine (ca. 0.1 microg/kg) can, in fact, elicit acute hyperalgesic effects, manifested by rapid onset of decreases in tail-flick latency for periods >3 h after drug administration. Cotreatment with ultra-low-dose NTX (ca. 1-100 pg/kg) blocks this opioid-induced hyperalgesia and unmasks potent opioid analgesia. The consonance of our in vitro and in vivo evidence indicates that doses of morphine far below those currently required for clinical treatment of pain may become effective when opioid hyperalgesic effects are blocked by coadministration of appropriately low doses of opioid antagonists. This low-dose-morphine cotreatment procedure should markedly attenuate morphine tolerance, dependence and other aversive side-effects.     

Ultra-low doses of naltrexone or etorphine increase morphine's antinociceptive potency and attenuate tolerance/dependence in mice

In previous studies we showed that low (pM) concentrations of naloxone (NLX), naltrexone (NTX) or etorphine selectively antagonize excitatory, but not inhibitory, opioid receptor-mediated functions in nociceptive types of sensory neurons in culture. Cotreatment of these neurons with pM NTX or etorphine not only results in marked enhancement of the inhibitory potency of acutely applied nM morphine [or other bimodally-acting (inhibitory/excitatory) opioid agonists], but also prevents development of cellular manifestations of tolerance and dependence during chronic exposure to μM morphine. These in vitro studies were confirmed in vivo by demonstrating that acute cotreatment of mice with morphine plus a remarkably low dose of NTX (ca. 10 ng/kg) does, in fact, enhance the antinociceptive potency of morphine, as measured by hot-water tail-flick assays. Furthermore, chronic cotreatment of mice with morphine plus low doses of NTX markedly attenuates development of naloxone-precipitated withdrawal-jumping in physical dependence assays. The present study provides systematic dose-response analyses indicating that NTX elicited optimal enhancement of morphine's antinociceptive potency in mice when co-administered (i.p.) at about 100 ng/kg together with morphine (3 mg/kg). Doses of NTX as low as 1 ng/kg or as high as 1 μg/kg were still effective, but to a lesser degree. Oral administration of NTX in the drinking water of mice was equally effective as i.p. injections in enhancing the antinociceptive potency of acute morphine injections and even more effective in attenuating development of tolerance and NLX-precipitated withdrawal-jumping during chronic cotreatment. Cotreatment with a subanalgesic dose of etorphine (10 ng/kg) was equally effective as NTX in enhancing morphine's antinociceptive potency and attenuating withdrawal-jumping after chronic exposure. These studies provide a rationale for the clinical use of ultra-low-dose NTX or etorphine so as to increase the antinociceptive potency while attenuating the tolerance/dependence liability of morphine or other conventional bimodally-acting opioid analgesics.     

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.     

Delta opioid peptide DADLE and naltrexone cause cell cycle arrest and differentiation in a CNS neural progenitor cell line

Opioids have been demonstrated to play an important role in CNS development by affecting proliferation and differentiation in various types of neural cells. This study examined the effect of a stable delta opioid peptide [D ‐Ala(2), D ‐Leu(5)]‐enkephalin (DADLE) on proliferation and differentiation in an AF5 CNS neural progenitor cell line derived from rat mesencephalic cells. DADLE (1 pM, 0.1 nM, or 10 nM) caused a significant growth inhibition on AF5 cells. The opioid antagonist naltrexone at 0.1 nM also caused growth inhibition in the same cells. When DADLE and naltrexone were both added to the AF5 cells, the resultant growth inhibition was apparently additive. DADLE alone or DADLE in combination with naltrexone did not cause apoptosis as evidenced by negative TUNEL staining. The cell‐cycle progression analysis indicated that both DADLE (0.1 nM) and naltrexone (0.1 nM) caused an arrest of AF5 cell cycle progression at the G1 checkpoint. Neuronal marker indicated that DADLE‐ or naltrexone‐treated AF5 cells tend to differentiate more when compared to controls. Results demonstrate the nonopioid action of both DADLE and naltrexone on cell cycle arrest and differentiation in a CNS neural progenitor cell line. Results also suggest some potential utilization of DADLE and/or naltrexone in stem cell research. Synapse 64:267–273, 2010. © 2009 Wiley‐Liss, Inc.     

Regional cerebral opioid receptor studies with [11C]diprenorphine in normal volunteers

The results are described of the cerebral uptake and heterogeneous retention of [11C]diprenorphine after intravenous injection in 4 normal volunteers. This potent opioid antagonist (Kd = 0.2 nM) was chosen because of its safety, lack of side-effects at trace doses in human pilot studies, rapid cerebral uptake and high percentage (80-90%) specific binding in animal in vivo studies. High uptake of [11C]diprenorphine was demonstrated in regions such as the thalamus, caudate nucleus, temporal, frontal and parietal cortices, which are known from postmortem studies to have high concentrations of opioid receptors. A stable level of activity or a very slow decline in activity was observed between 20 and 50 min after injection in areas such as the caudate nucleus and thalamus. Conversely, rapid washout of activity was observed in the occipital cortex, which is known to have low opioid receptor concentrations. Some 80-90% of maximum binding was naloxone reversible. These results with a ligand that is safe and without side-effects, suggest that this technique is suitable for studies of opioid physiology in man.     

Antiapoptotic and cytotoxic properties of delta opioid peptide [D-Ala(2),D-Leu(5)]enkephalin in PC12 cells.

The delta opioid peptide [D-Ala(2),D-Leu(5)]enkephalin (DADLE) has been shown to promote organ survival and to protect against methamphetamine-induced neurodegeneration. However, the cellular mechanisms of these actions of DADLE are not totally clear. We examined the action of DADLE in serum-deprived pheochromocytoma cells (PC12) and found that DADLE protected against cell death in those cells. However, the dose-response curves of the protective effects of DADLE are U-shaped as judged by three biochemical or morphological assays: the LDH release, the DNA laddering, and the apoptotic nuclei. It was found that femtomolar to picomolar concentrations of DADLE are antiapoptotic, whereas micormolar concentrations of DADLE are cytotoxic in PC12 cells. The protective effect of DADLE could be attenuated by a selective delta2 opioid antagonist and the cytotoxic action of DADLE was reduced by a selective mu opioid receptor antagonist. The treatment of cells with PD98059, a selective inhibitor of ERK kinase (MEK), or the transfection of cells with a dominant interfering form of MEK (MEK-KA97) blocked both the protective effect of DADLE and the ERK phosphorylation induced by DADLE. Cytotoxic concentrations of DADLE, on the other hand, caused an increase of Fas-ligand (FasL) in PC12 cells that was attenuated by a selective mu antagonist. Our results suggest, therefore, that endogenous opioid peptides may, at low concentrations, promote cell survival via the MEK-ERK pathway perhaps through delta2 opioid receptors, whereas they may kill cells at high concentrations via the activation of FasL through an as-yet unknown mechanism involving mu opioid receptors.     

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala,d-Leu]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [d-Ala²,d-Leu⁵]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [³H]diprenorphine binding to the P₂ membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B_max values and no change in the K_d values of [³H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [³H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.     

Enhanced analgesic potency and reduced tolerance of morphine in 129/SvEv mice: evidence for a deficiency in GM1 ganglioside-regulated excitatory opioid receptor functions

10-fold higher doses in SW mice. Furthermore, cotreatment of 129/SvEv mice with morphine plus a low dose of naltrexone (ca. 0.1 microgram/kg) that markedly enhances and prolongs morphine's antinociceptive effects in SW mice did not enhance, and often attenuated6 h. The marked GM1-induced attenuation of morphine's antinociceptive effects in 129/SvEv mice may be due to conversion of some of the opioid receptors in these mice from an inhibitory Gi/Go-coupled to an excitatory Gs-coupled mode. Exogenous GM1 supplementation can, therefore, reverse the anomalous lack of morphine tolerance displayed by this mouse strain in comparison to SW and other mice. The present study may provide insights into factors that regulate the marked variability in nociceptive sensitivity and opioid tolerance/dependence liability among individual humans.     

Decrease in delta-opioid receptor density in rat brain after chronic [D-Ala2,D-Leu5]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [D-Ala2,D-Leu5]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [3H]diprenorphine binding to the P2 membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in Bmax values and no change in the Kd values of [3H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the delta-opioid receptors in these brain regions. When [3H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the delta-opioid receptor binding was observed in all brain areas tested. mu-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of delta-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced mu-opioid receptor binding, chronic DADLE treatment preferentially reduced delta-opioid receptor binding activity.     

Opioid peptides alleviated while naloxone potentiated methamphetamine-induced striatal dopamine depletion in mice

Summary. Delta opioid peptide [d-Ala², d-Leu⁵] enkephalin (DADLE) can partially reverse long-term loss of striatal dopamine transporters induced by multiple doses of methamphetamine via an unknown mechanism. This study was designed to examine the modulating effects of three opioid ligands, DADLE, Leucine enkephalin (L-enk), and naloxone, on the long-lasting dopamine depletion produced by 4 cumulative doses of methamphetamine. Both DADLE (at a dose of 18 mg/kg) and L-enk (100 μg/kg × 2) effectively attenuated methamphetamine-induced dopamine depletion in the striatum while their protective effects were not blocked by coadministration of naloxone. In contrast, naloxone (10 mg/kg × 2) alone potentiated the long-lasting dopamine depletion produced by methamphetamine. Moreover, none of the treatments with DADLE (18 mg/kg), L-enk (100 μg/kg), or naloxone (10 mg/kg) alone affected body temperature. These results suggest that the opioid ligands may, directly or indirectly, modulate this methamphetamine-induced dopamine neurotoxicity in the nigrostriatal system via a temperature-independent mechanism. Received March 6, 2001; accepted June 10, 2001