Change in functional selectivity of morphine with the development of antinociceptive tolerance.

BACKGROUND AND PURPOSE: Opioids, such as morphine, are the most effective treatment for pain but their efficacy is diminished with the development of tolerance following repeated administration. Recently, we found that morphine activated ERK in opioid-tolerant but not in naïve rats, suggesting that morphine activation of µ-opioid receptors is altered following repeated morphine administration. Here, we have tested the hypothesis that µ-opioid receptor activation of ERK in the ventrolateral periaqueductal gray (vlPAG) is dependent on dynamin, a protein implicated in receptor endocytosis. EXPERIMENTAL APPROACH: Rats were made tolerant to repeated microinjections of morphine into the vlPAG. The effects of dynamin on ERK activation and antinociception were assessed by microinjecting myristoylated dominant-negative dynamin peptide (Dyn-DN) or a scrambled control peptide into the vlPAG. Microinjection of a fluorescent dermorphin analogue (DERM-A594) into the vlPAG was used to monitor µ-opioid receptor internalization. KEY RESULTS: Morphine did not activate ERK and Dyn-DN administration had no effect on morphine-induced antinociception in saline-pretreated rats. In contrast, morphine-induced ERK activation in morphine-pretreated rats that was blocked by Dyn-DN administration. Dyn-DN also inhibited morphine antinociception. Finally, morphine reduced DERM-A594 internalization only in morphine-tolerant rats indicating that µ-opioid receptors were internalized and unavailable to bind DERM-A594. CONCLUSIONS AND IMPLICATIONS: Repeated morphine administration increased µ-opioid receptor activation of ERK signalling via a dynamin-dependent mechanism. These results demonstrate that the balance of agonist signalling to G-protein and dynamin-dependent pathways is altered, effectively changing the functional selectivity of the agonist-receptor complex. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Opioid receptor internalization contributes to dermorphin-mediated antinociception.

Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-chlornaltrexamine (beta-CNA) prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABA(A) receptor antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization.

Cellular actions of opioids and other analgesics: implications for synergism in pain relief.

1. mu-Opioid receptor agonists mediate their central analgesic effects by actions on neurons within brain regions such as the mid-brain periaqueductal grey (PAG). Within the PAG, mu-opioid receptor-mediated analgesia results from inhibition of GABAergic influences on output projection neurons. We have established that mu-opioid receptor activation in the PAG causes a presynaptic inhibition of GABA release that is mediated by activation of a voltage-dependent K+ channel via 12-lipoxygenase (LOX) metabolites of arachidonic acid. 2. At a cellular level, mu-opioid agonists have also been shown to open inwardly rectifying K+ channels, close voltage-gated Ca2+ channels and presynaptically inhibit glutamatergic synaptic transmission in the PAG. 3. The mu-opioid receptor-mediated presynaptic inhibition of GABAergic transmission was abolished by phospholipase A2 inhibitors and non-specific LOX and specific 12-LOX inhibitors. Cyclo-oxygenase (COX) and specific 5-LOX inhibitors did not reduce the inhibitory effects of mu-opioid agonists. 4. The opioid actions on GABAergic transmission were mimicked by arachidonic acid and 12-LOX metabolites, but not 5-LOX metabolites. The efficacy of mu-opioids was enhanced synergistically by treatment of PAG neurons with inhibitors of the other major enzymes responsible for arachidonic acid metabolism, COX and 5-LOX. 5. These results explain a previously described analgesic action of COX inhibitors in the central nervous system that was both independent of prostanoid release and inhibited by opioid receptor antagonists and they also explain the synergistic interaction of opioids with COX inhibitors. These findings also suggest new avenues for the development of centrally active analgesic agents involving combinations of lowered doses of opioids and specific 5-LOX inhibitors.

Arachidonic acid stimulates a novel cocaine-sensitive cation conductance associated with the human dopamine transporter.

The dopamine transporter (DAT) exhibits several ionic currents that are either coupled to or uncoupled from the transport of substrate. Second messenger systems have been shown to modulate dopamine (DA) transport, however, the modulation of DAT-associated currents has not been studied in depth. Using the two-electrode voltage-clamp method to record from Xenopus oocytes expressing the human DAT, we examined the effects of arachidonic acid (AA) on membrane currents. AA (10-100 microM) stimulates a novel nonselective cation conductance seen only in oocytes expressing human DA transporter (hDAT). The AA-stimulated conductance is up to 50-fold greater than the current normally elicited by DA, but does not appear to arise from the modulation of previously described hDAT conductances, including the leak current and the current associated with electrogenic transport. In addition, DA dramatically potentiates and cocaine blocks the AA-stimulated DAT current. DA potentiates the AA-induced currents in the absence of sodium and chloride, indicating that these currents arise from processes distinct from those associated with substrate transport. The effects of AA were mimicked by other fatty acids with a rank order of potency correlated with their degree of unsaturation, suggesting that AA directly stimulates the novel cation current. Therefore, AA stimulation of this DAT-associated conductance may provide a novel mechanism for modulation of neuronal signaling.

Opioids, NSAIDs and 5-lipoxygenase inhibitors act synergistically in brain via arachidonic acid metabolism.

We have established that mu-opioid receptor activation causes a presynaptic inhibition of neurotransmitter release that is mediated by 12-lipoxygenase metabolites of arachidonic acid in midbrain neurons [1]. We further demonstrated that the efficacy of opioids was enhanced synergistically by treatment of brain neurons with inhibitors of the other major enzymes responsible for arachidonic acid metabolism; cyclooxygenase (COX-1) and 5-lipoxygenase. These findings explain a mechanism of analgesic action of NSAIDs in the central nervous system that is both independent of prostanoid release and inhibited by opioid antagonists, as well as the synergistic interaction of opioids with NSAIDs. These findings also suggest new avenues for development of centrally active medications involving combinations of lowered doses of opioids and specific 5-lipoxygenase inhibitors.

Enhanced opioid efficacy in opioid dependence is caused by an altered signal transduction pathway.

Chronic morphine administration induces adaptations in neurons resulting in opioid tolerance and dependence. Functional studies have implicated a role for the periaqueductal gray area (PAG) in the expression of many signs of opioid withdrawal, but the cellular mechanisms are not fully understood. This study describes an increased efficacy, rather than tolerance, of opioid agonists at mu-receptors on GABAergic (but not glutamatergic) nerve terminals in PAG after chronic morphine treatment. Opioid withdrawal enhanced the amplitudes of electrically evoked inhibitory synaptic currents mediated by GABAA receptors and increased the frequency of spontaneous miniature GABAergic synaptic currents. These effects were not blocked by 4-aminopyridine or dendrotoxin, although both Kv channel blockers abolish acute opioid presynaptic inhibition of GABA release in PAG. Instead, the withdrawal-induced increases were blocked by protein kinase A inhibitors and occluded by metabolically stable cAMP analogs, which do not prevent acute opioid actions. These findings indicate that opioid dependence induces efficacious coupling of mu-receptors to presynaptic inhibition in GABAergic nerve terminals via adenylyl cyclase- and protein kinase A-dependent processes in PAG. The potential role of these adaptations in expression of withdrawal behavior was supported by inhibition of enhanced GABAergic synaptic transmission by the alpha2 adrenoceptor agonist clonidine. These findings provide a cellular mechanism that is consistent with other studies demonstrating attenuated opioid withdrawal behavior after injections of protein kinase A inhibitors into PAG and suggest a general mechanism whereby opioid withdrawal may enhance synaptic neurotransmission.

Sigma-binding site ligands inhibit K+ currents in rat locus coeruleus neurons in vitro.

Biological actions of novel sigma1- and sigma2-selective binding site ligands (trishomocubanes: 4-azahexacyclo [5.4.1.0.(2,6).0(3,10).0(5,).0(8,11)]dodecanes), and the reference ligands, 1,3-di(2-tolyl)-guanidine (DTG), haloperidol, (+)-pentazocine and dextromethorphan, were studied in rat locus coeruleus neurons using intracellular and whole-cell patch clamp recordings. High concentrations of trishomocubanes produced small inward currents and affected some parameters of action potential waveforms suggesting modest potency to inhibit ionic conductances underlying action potentials. Sigma-ligands produced large inward currents in the presence of mu-opioid, alpha2-adrenoceptor and ORL1 receptor agonists. These reversed polarity near the K+ equilibrium potential, suggesting that sigma-ligands act as ligand activated K+-channel blockers or interfere with the coupling between these receptors and K+-channels. However, no correlation was found between binding affinities at sigma1- or sigma2-binding sites and potency to inhibit K+-currents, suggesting that these effects on K+-channels are not directly related to occupancy of sigma binding sites.

How opioids inhibit GABA-mediated neurotransmission.

The midbrain region periaqueductal grey (PAG) is rich in opioid receptors and endogenous opioids and is a major target of analgesic action in the central nervous system. It has been proposed that the analgesic effect of opioids on the PAG works by suppressing the inhibitory influence of the neurotransmitter GABA (gamma-aminobutyric acid) on neurons that form part of a descending antinociceptive pathway. Opioids inhibit GABA-mediated (GABAergic) synaptic transmission in the PAG and other brain regions by reducing the probability of presynaptic neurotransmitter release, but the mechanisms involved remain uncertain. Here we report that opioid inhibition of GABAergic synaptic currents in the PAG is controlled by a presynaptic voltage-dependent potassium conductance. Opioid receptors of the mu type in GABAergic presynaptic terminals are specifically coupled to this potassium conductance by a pathway involving phospholipase A2, arachidonic acid and 12-lipoxygenase. Furthermore, opioid inhibition of GABAergic synaptic transmission is potentiated by inhibitors of the enzymes cyclooxygenase and 5-lipoxygenase, presumably because more arachidonic acid is available for conversion to 12-lipoxygenase products. These mechanisms account for the analgesic action of cyclooxygenase inhibitors in the PAG and their synergism with opioids.

Actions of the ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro.

The actions of the endogenous ORL1-receptor ligand nociceptin on the membrane properties and synaptic currents in rat periaqueductal gray (PAG) neurons were examined by the use of whole-cell patch-clamp recording in brain slices. Nociceptin produced an outward current in all neurons tested, with an EC50 of 39 +/- 7 nM. The outward current was unaffected by naloxone. Outward currents reversed polarity at -110 +/- 3 mV in 2.5 mM extracellular potassium, and the reversal potential increased when the extracellular potassium concentration was raised (slope = 66.3 mV/log[K+]o mM). Thus, the nociceptin-induced outward current was attributable to an increased K+ conductance. Nociceptin inhibited evoked fast GABAergic (IP-SCs) and glutamatergic (EPSCs) postsynaptic currents and increased paired-pulse facilitation in a subpopulation of PAG neurons. Nociceptin inhibited evoked IPSCs and EPSCs in approximately 50% of neurons throughout the PAG, except in the ventrolateral PAG, where nociceptin inhibited evoked IPSCs in most neurons. Nociceptin decreased the frequency of spontaneous miniature postsynaptic currents (mIPSCs and mEPSCs) in a subpopulation of PAG neurons but had no effect on their amplitude distributions. Thus, nociceptin had a presynaptic inhibitory effect on transmitter release. These findings suggest that nociceptin, via its pre- and postsynaptic actions, has the potential to modulate the analgesic, behavioral, and autonomic functions of the PAG.

Cortistatin increase of a potassium conductance in rat locus coeruleus in vitro.

1. In this study we examined the effects of cortistatin, a putative endogenous ligand for somatostatin (SRIF) receptors, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, by use of intracellular and whole cell patch clamp recording. We have compared the actions of cortistatin with those of SRIF and the SRIF analogue D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP). 2. When LC neurones were voltage clamped to -60 mV, application of cortistatin caused an outward current in all cells examined (n = 44), with a pEC50 of 6.62. SRIF also caused an outward current in all cells examined (n = 43), with a pEC50 of 6.93. 3. The outward currents caused by cortistatin in 2.5 mM extracellular K+ reversed polarity at -106 mV, very close to the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 10.5 mM resulted in a shift of the reversal potential of +38 mV, a shift consistent with a K+ conductance. The conductance activated by cortistatin showed mild inward rectification. 4. Continuous application of a high concentration of SRIF (1 microM) resulted in a decrease of the outward current to a steady level of 49% of the maximum response, with a t1/2 of 131 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the SRIF response did not result in any further outward current. Continuous application of a high concentration of cortistatin (10 microM) resulted in a decrease of the outward current to a steady level of 42% of the maximum response with a t1/2 of 114 s. Application of a high concentration of SRIF (3 microM) during the desensitized portion of the cortistatin response produced only a small outward current. 5. Continuous application of cortistatin (3 microM) also resulted in a decrease of the outward current (by 43%, t1/2 of 136 s) and application of a high concentration of CTOP (10 microM) during the desensitized portion of the cortistatin response did not produce any outward current. Continuous application of a high concentration of CTOP (10 microM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 143 s. Application of a high concentration of cortistatin (3 microM) during the desensitized portion of the CTOP response did not result in any further outward current. 6. The actions of cortistatin (300 nM-10 microM) were not affected by the opioid antagonist naloxone (10 microM). Application of met-enkephalin during the desensitized portion of the response to a high concentration of cortistatin (3 microM) produced an outward current similar to that produced by metenkephalin application alone. 7. Thus cortistatin efficaciously activates an inwardly rectifying K+ conductance in LC neurones. These actions appear to be mediated by a population of SRIF receptors, at which CTOP is also an agonist. Cortistatin does not appear to be a ligand for mu-opioid receptors in rat LC neurons.

Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons.

1. Whole-cell patch-clamp recordings were made from dissociated guinea-pig nodose and trigeminal ganglion neurons in culture to study second messenger mechanisms of the hyperpolarization-activated current (Ih) modulation. 2. Prostaglandin E2 (PGE2) and forskolin modulate Ih in primary afferents by shifting the activation curve in the depolarizing direction and increasing the maximum amplitude. 3. The cAMP analogues, RP-cAMP-S (an inhibitor of protein kinase A (PKA)) and SP-cAMP-S (an activator of PKA), both shifted the activation curve of Ih to more depolarized potentials and occluded the effects of forskolin. These results suggest that Ih is modulated by a direct action of the cAMP analogues. 4. Superfusion of other cyclic nucleotide analogues (8-Br-cAMP, 8-(4-chlorophenylthio)-cAMP and 8-Br-cGMP) mimicked the actions of forskolin and PGE2, but dibutyryl cGMP, 5'-AMP and adenosine had no effect on Ih. 8-Br-cAMP and 8-Br-cGMP had similar concentration response profiles, suggesting that Ih has little nucleotide selectivity. 5. The inhibitor peptide (PKI), the catalytic subunit of PKA (C subunit) and phosphatase inhibitors (microcystin and okadaic acid) had no effect on forskolin modulation of Ih. 6. These results indicate that Ih is regulated by cyclic nucleotides in sensory neurons. Positive regulation of Ih by prostaglandins produced during inflammation may lead to depolarization and facilitation of repetitive activity, and thus contribute to sensitization to painful stimuli.

Potentiation of rabbit trigeminal responses to capsaicin in a low pH environment.

The sensitivity of 35 adult rabbit trigeminal ganglion neurons to low pH (pH 6.0), 10 microM capsaicin (CAP) and 10 microM capsaicin at low pH (CAP@pH6.0) was studied using voltage-clamp whole-cell recording techniques. Neurons responded to pH 6.0 with a transient inward current, followed by a more slowly activating (sustained) net inward current. Responses to capsaicin showed only a sustained current. Capsaicin caused an increase in membrane conductance, whereas responses to low pH were associated with either a net increase or decrease in conductance. A subset of neurons (n = 14) responded to CAP@pH6.0 with a sustained current which exceeded the sum of the peak sustained currents evoked by CAP and pH 6.0 applied singularly by approximately a factor of 4. The current was associated with a substantial increase in membrane conductance. The present results indicate that, in addition to a direct conductance activating effect, protons have the ability to enhance the current evoked by capsaicin.

Opioid inhibition of Ih via adenylyl cyclase.

Opioids are coupled through G proteins to both ion channels and adenylyl cyclase. This study describes opioid modulation of the voltage-dependent cation channel, Ih, in cultured guinea pig nodose ganglion neurons. Forskolin, PGE2, and cAMP analogs shifted the voltage dependence of activation of Ih to more depolarized potentials and increased the inward current at -60 mV. Opioids had no effect on Ih alone, but reversed the effect of forskolin on Ih. This action of opioids was blocked by naloxone. Opioids had no effect on Ih in the presence of cAMP analogs, suggesting that modulation occurs at the level of adenylyl cyclase. The shift in the voltage dependence of Ih by agents that induce inflammation (i.e., PGE2) is one potential mechanism to mediate an increased excitability. Opioid inhibition of adenylyl cyclase and subsequent inhibition of Ih may be a mechanism by which opioids inhibit primary afferent excitability and relieve pain.

Responses of cultured adult monkey trigeminal ganglion neurons to capsaicin.

The sensitivity of adult primate (Macaca mulatta) trigeminal ganglion neurons to capsaicin was studied using whole-cell recording techniques. Neurons responding to capsaicin (9 out of 14) generated inward currents of up to 3.0 nA (median = 0.23 nA; interquartile range = 1.19 nA) upon drug application measured at -60 mV. Capsaicin-sensitive neurons had longer action potential (AP) durations than capsaicin-insensitive neurons.