After chronic opioid exposure sensory neurons become supersensitive to the excitatory effects of opioid agonists and antagonists as occurs after acute elevation of GM1 ganglioside

Mouse sensory dorsal-root ganglion (DRG) neurons chronically exposed to 1 microM D-Ala2-D-Leu5-enkephalin (DADLE) for greater than 1 week in culture become tolerant to opioid inhibitory effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD). Acute application of higher concentrations of DADLE (ca. 10 microM) to these treated neurons not only fails to shorten the APD but, instead, generally elicits excitatory effects, i.e. prolongation of the APD. The present study shows that chronic DADLE- or morphine-treated DRG neurons also become supersensitive to the excitatory effects of opioids. Whereas nM concentrations of dynorphin(1-13) are generally required to prolong the APD of naive DRG neurons, fM levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine do not alter the APD of naive DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the treated cells. Similar supersensitivity to the excitatory effects of opioid agonists and antagonists was previously observed after acute treatment of naive DRG neurons with GM1 ganglioside. Our results suggest that both chronic opioid and acute GM1 treatments of DRG neurons greatly enhance the efficacy of opioid excitatory receptor functions so that even the extremely weak agonist properties of naloxone and diprenorphine become effective in prolonging the APD of these treated cells when tested at low concentrations, whereas their antagonist properties at inhibitory opioid receptors do not appear to be altered. Furthermore, whereas cholera toxin-B subunit (CTX-B; 1-10 nM) blocks opioid-induced APD prolongation in naive DRG neurons (presumably by interfering with endogenous GM1 modulation of excitatory opioid receptors functions), even much higher concentrations of CTX-B were ineffective in chronic opioid-treated as well as acute GM1-elevated neurons. These and related data suggest that opioid excitatory supersensitivity in chronic opioid-treated DRG neurons may be due to a cyclic AMP-dependent increase in GM1 ganglioside levels. Our results may clarify mechanisms of opioid dependence and the paradoxical supersensitivity to naloxone which triggers withdrawal symptoms after opiate addiction.     

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.     

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.     

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)     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Blockade of D-2 dopamine receptors strongly enhances the potency of enkephalins to inhibit dopamine-sensitive adenylate cyclase in rat neostriatum: involvement of delta- and mu-opioid receptors

The interactions between dopamine receptors and opioid receptors coupled to adenylate cyclase in rat neostriatum were investigated. cAMP efflux from neostriatal slices induced by simultaneous activation of (stimulatory) D-1 and (inhibitory) D-2 dopamine receptors with 30 microM dopamine was inhibited by the preferential delta-opioid receptor agonist [D-Ala2-D-Leu5] enkephalin (DADLE) and the mu-opioid receptor agonist morphine with an EC50 of 100 and 800 nM, respectively. On selective D-1 receptor activation (i.e., with D-2 receptors blocked by 10 microM (-)sulpiride), the EC50 of DADLE was strongly reduced to 3 nM, whereas that of morphine was unaffected. When D-1 and D-2 receptors were activated simultaneously, the inhibitory effects of DADLE (0.3 microM) and morphine (3 microM) on cAMP efflux were antagonized equally well by naloxone, a mu-opioid receptor antagonist. In contrast, on selective D-1 receptor activation, naloxone was about 20 times more potent in antagonizing the inhibitory effect of morphine than DADLE. Moreover, the delta-opioid receptor antagonist ICI 174864 (0.75 microM) did not affect the inhibitory effect of morphine but antagonized that of DADLE, provided that D-2 receptors were blocked. The highly selective delta-opioid receptor agonist [D-Pen2-D-Pen5] enkephalin (DPDPE) inhibited dopamine-stimulated cAMP efflux only when D-2 receptors were blocked. Similar results were obtained when the agonists SKF 38393 and LY 141865 were used to activate D-1 and D-2 receptors, respectively. These data indicate that blockade of D-2 receptors in the neostriatum elicits the coupling of delta-opioid receptors to dopamine-sensitive adenylate cyclase, thereby making it considerably more sensitive to inhibition by the enkephalins.     

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors

Recent evidence indicates that agonist ligands of G protein coupled receptors (GPCR) can activate different signaling systems. Such “agonist-directed” signaling also occurs with opioid receptors. Previous work from our laboratory showed that chronic morphine, but not DAMGO, up-regulates the expression of Gα12 and that both morphine and DAMGO decreased Gαi3 expression in CHO cells expressing the cloned human mu opioid receptor. In this study, we tested the hypothesis that chronic opioid regulation of G protein expression is agonist-directed. Following a 20 h treatment of CHO cells expressing the cloned human mu (hMOR-CHO), delta (hDOR-CHO) or kappa (hKOR-CHO) opioid receptors with various opioid agonists, we determined the expression level of Gα12 and Gαi3 by Western blots. Among five mu agonists (morphine, etorphine, DADLE, DAMGO, herkinorin) tested with hMOR-CHO cells, only chronic morphine and etorphine up-regulated Gα12 expression. All five mu agonists decreased Gαi3 expression. Among six delta agonists (SNC80, DPDPE, deltorphin-1, morphine, DADLE, etorphine) tested with hDOR-CHO cells, all six agonists down-regulated Gαi3 expression or moderately up-regulated Gα12 expression. Among five kappa agonists, ((−)-ethylketocyclazocine, salvinorin A, U69,593, etorphine, (−)-U50,488) tested with hKOR-CHO cells, only chronic (−)-U50,488 and (−)-EKC up-regulated Gα12 expression. All kappa agonists decreased Gαi3 expression. These data demonstrate that chronic opioid agonist regulation of G protein expression depends not only on the agonist tested, but also on the type of opioid receptor expressed in a common cellular host, providing additional evidence for agonist-directed signaling.     

Kappa opioid receptors in human lumbo-sacral spinal cord

[3H]Etorphine and [3H]ethylketocyclazocine bind with high affinity (Kd between 0.25-2.0 nM) to a single class of sites in human lumbo-sacral spinal cord. Other ligands such as [3H]morphine, [3H]dihydromorphine and [3H]D-Ala2, D-Leu5-enkephalin (DADLE) did not bind to significant number of sites under our incubation conditions. Ligand selectivity pattern strongly suggests that [3H]etorphine labels kappa opioid binding sites in the human lumbo-sacral spinal cord since benzomorphans and oripavines are much more potent than mu and delta agonists. Furthermore, [3H]etorphine and [3H]ethylketocyclazocine binding is sensitive to high concentrations of DADLE suggesting that these sites are of the kappa 2 sub-type. Finally, the visualization of these sites by receptor autoradiography demonstrates that they are mainly concentrated in lamina II and III of the dorsal horn. Moderate densities of sites are present around the central canal. Thus, it is possible that kappa opioid binding sites could be involved in the control of sensory and autonomic functions in the human lumbo-sacral spinal cord.     

Opioid peptide receptor studies. 16. Chronic morphine alters G-protein function in cells expressing the cloned mu opioid receptor

Chronic morphine treatment results in functional uncoupling of the mu opioid receptor and its G protein in both cell culture and animal models. In the present study, Chinese hamster ovary (CHO) cells stably expressing the cloned human mu opioid receptor (hMOR-CHO cells) were incubated with 1 microM of morphine (or no drug) for 20 h. Subsequently, we assessed DAMGO- and morphine-stimulated [(35)S]-GTP-gamma-S binding and agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. Using a single concentration of [(35)S]-GTP-gamma-S (0.05 nM), chronic morphine treatment did not significantly change basal [(35)S]-GTP-gamma-S binding, shifted the morphine EC(50) from 59 nM to 146 nM, and decreased the maximal stimulation (E(max)) from 201% to 177%. Similar results were observed with DAMGO. Binding surface analysis resolved two [(35)S]-GTP-gamma-S binding sites (high-affinity and low-affinity sites). In control cells, morphine stimulated [(35)S]-GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In morphine-treated cells, morphine stimulated [(35)S]-GTP-gamma-S binding by decreasing the high-affinity K(d) without changing the B(max). Morphine treatment increased the EC(50) (5-11-fold) for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation. These changes were not observed in cells expressing a mutant mu opioid receptor which does not develop morphine tolerance, suggesting that the changes in [(35)S]-GTP-gamma-S binding observed in hMOR-CHO cells result from the development of morphine tolerance.     

Chronic naltrexone increases opiate binding in brain and produces supersensitivity to morphine in the locus coeruleus of the rat

Rats were implanted subcutaneously for 2-4 weeks with slow-release pellets of naltrexone (10 mg) or placebo and then the pellets were removed. One day after removal of the pellet, animals were either (1) sacrificed and various CNS regions examined for specific binding of [3H]naloxone, [3H]etorphine or [3H]rauwolscine or (2) they were anesthetized and prepared acutely for assessing morphine-induced changes in the spontaneous activity of neurons in the locus coeruleus (LC). Naltrexone treatment significantly increased the number of specific binding sites for opiates, but not for alpha 2-adrenergic antagonists, in spinal cord, hypothalamus, striatum and cortex. Specific binding of [3H]naloxone was also increased in the LC. The spontaneous activity of neurons in the LC was reduced by the chronic naltrexone treatment, suggesting that these neurons became supersensitive to the tonic inhibitory effect of endogenous opioid peptides. Moreover, neurons in the LC of chronic naltrexone-treated rats exhibited an enhanced response to the inhibitory effects of morphine administered systemically. These results demonstrate that chronic opiate receptor blockade increases the number of receptor sites for morphine and that this increase in receptors is accompanied by a neuronal supersensitivity in the LC to morphine which can be assessed electrophysiologically.     

Polyarthritis-associated changes in the opioid control of spinal CGRP release in the rat

As a model of chronic inflammatory pain, Freund's adjuvant-induced polyarthritis has been shown to be associated with marked alterations in the activity of opioid- and calcitonin gene-related peptide (CGRP)-containing neurons in the dorsal horn of the spinal cord in rats. Possible changes in the interactions between these two peptidergic systems in chronic inflammatory pain were investigated by comparing the effects of various opioid receptor ligands on the spinal outflow of CGRP-like material (CGRPLM) in polyarthritic and age-paired control rats. Intrathecal perfusion of an artificial cerebrospinal fluid in halothane-anaesthetized animals allowed the collection of CGRPLM released from the spinal cord and the application of opioid receptor ligands. The blockade of κ-opioid receptors similarly increased CGRPLM release in both groups of rats as expected of a κ-mediated tonic inhibitory control of CGRP-containing fibres in control, as well as in polyarthritic rats. In contrast, the higher increase in CGRPLM outflow due to the preferential blockade of μ opioid receptors by naloxone in polyarthritic rats as compared to non-suffering animals supports the idea of a reinforced μ opioid receptor-mediated tonic inhibitory control of CGRP-containing fibres in rats suffering from chronic pain. Even more strikingly, the differences observed in the effects of ∂-opioid receptor ligands on CGRPLM outflow suggest that ∂ receptors are functionally shifted from a participation in a phasic excitatory control in non-suffering rats to a tonic inhibitory control in polyarthritic rats. These data indicate that agonists acting at the three types of opioid receptors all exert a tonic inhibitory influence on CGRP-containing nociceptive primary afferent fibres within the spinal cord of polyarthritic rats. Such a convergence probably explains why morphine and other opioids are especially potent to reduce pain in subjects suffering from chronic inflammatory diseases.     

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-Ala2, D-Leu5]-enkephalin (DADLE), applied by perfusion (0.01-3 microM) 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 Ca2(+)-free/high-Mg2+ solution or with 1 microM 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 Ca2(+)-dependent spike duration and after-hyperpolarization (AHP). DAGO (a selective mu-receptor agonist), but not DPLPE (a selective delta-receptor agonist), mimicked DADLE's effects on membrane potential, Ca2(+)-dependent spike duration, and AHP. It is concluded that DADLE, through postsynaptic mu-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 mu-receptors and could have additional effects on functions of the DMV neuron by virtue of reduction in Ca2+ entry.     

Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.