Mark Connor

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 (γ-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 μ 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.

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1 In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid‐like receptor ORL1, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2 When locus coeruleus neurones were voltage clamped to −60 mV, application of nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 μm) or thiorphan (2 μm). 3 The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at −123 mV, more negative than the predicted K+ reversal potential of −105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4 Application of a high concentration of nociceptin (3 μm) occluded the current produced by simultaneous application of high concentrations of Met‐enkephalin (10 μm), (3 μm) somatostatin and UK 14304 (3 μm), indicating that nociceptin activated the same conductance as μ‐opioid and somatostatin receptors and α2‐adrenoceptors. 5 The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb′s estimated from 2 cells of −4.23 and −4.33. The μ‐opioid antagonist, CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Pen‐Thr‐NH2, 1 μm), the opioid antagonist, nalorphine (30 μm), or the somatostatin antagonist, CPP (cyclo(7‐aminoheptanoyl‐Phe‐D‐Tip‐Lys‐Thr[Bzl]) 3 μm) did not affect the nociceptin‐induced current. 6 Dynorphin A (3 μm), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM‐1 μm). 7 Continuous application of nociceptin (3 μm) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met‐enkephalin (30 μm) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met‐enkephalin in the same cells. Conversely, continuous application of Met‐enkephalin (30 μm) resulted in a decrease of Met‐enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 μm) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8 Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.

The effect of nociceptin on Ca2+ channel current and intracellular Ca2+ in the SH-SY5Y human neuroblastoma cell line.

The human neuroblastoma cell line SH‐SY5Y expresses the ‘orphan’ opioid receptor (ORL1). We have demonstrated that nociceptin, the putative endogenous ligand for ORL1, produces a concentration‐dependent inhibition of the N‐type calcium channel current in these cells (IC50 42 nM). In addition, in the presence of carbachol, nociceptin increased the intracellular concentration of Ca2+ (EC50 60 nM). Both effects of nociceptin were blocked by pertussis toxin pretreatment but not by the opioid antagonists CTAP (1 μm), naltrindole (1 μm) and naloxone (10 μm).

Opioid receptor signalling mechanisms.

1. Three pharmacological types of opioid receptors, μ, δ and κ, and their corresponding genes have been identified. Although other types of opioid receptors have been suggested, their existence has not been established unequivocally. A fourth opioid receptor, ORL1, which is genetically closely related to the others, has also been isolated. ORL1 responds to the endogenous agonist nociceptin (orphanin FQ) and displays a pharmacological profile that differs greatly from μ, δ and κ receptors.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide ω-atracotoxin-HV1 (ω-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded ω-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible β-hairpin protruding from a disulphide-bonded globular core comprising four β-turns. The three intramolecular disulphide bonds form a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, ω-ACTX-HV1 displays significant structural homology with the ω-agatoxins and ω-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that ω-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Gingerols: A novel class of vanilloid receptor (VR1) agonists

Gingerols, the pungent constituents of ginger, were synthesized and assessed as agonists of the capsaicin‐activated VR1 (vanilloid) receptor. [6]‐Gingerol and [8]‐gingerol evoked capsaicin‐like intracellular Ca2+ transients and ion currents in cultured DRG neurones. These effects of gingerols were blocked by capsazepine, the VR1 receptor antagonist. The potency of gingerols increased with increasing size of the side chain and with the overall hydrophobicity in the series. We conclude that gingerols represent a novel class of naturally occurring VR1 receptor agonists that may contribute to the medicinal properties of ginger, which have been known for centuries. The gingerol structure may be used as a template for the development of drugs acting as moderately potent activators of the VR1 receptor.

Capsaicin activation of glutamatergic synaptic transmission in the rat locus coeruleus in vitro

The vanilloid receptor protein (VR1) is a well‐characterised integrator of noxious stimuli in peripheral sensory neurones. There is evidence for the presence of VR1 in the central nervous system, but little information as to its role there. In this study we have examined the actions of agonists for VR1 receptors in the rat locus coeruleus (LC), using whole‐cell patch‐clamp recordings from acutely isolated neurones and neurones in slices. Superfusion with capsaicin resulted in a concentration‐dependent increase in the frequency of isolated miniature excitatory postsynaptic currents (mEPSCs) in LC neurones. The mean amplitude of the mEPSCs was not affected by capsaicin. The effects of capsaicin (1 μM) were abolished by the VR1 receptor antagonists capsazepine (10 μM) and iodoresiniferatoxin (300 nm). Removal of extracellular Ca2+ abolished the capsaicin‐induced increase in frequency of mEPSCs. Capsaicin superfusion had no consistent effects on evoked excitatory postsynaptic currents. Capsaicin superfusion also resulted in the release of an adrenoceptor agonist in the LC but did not affect the membrane currents of acutely isolated LC neurones. These data demonstrate that the VR1 receptor appears to be located presynaptically on afferents to the LC, and that activation of VR1 may serve to potentiate the release of glutamate and adrenaline/noradrenaline in this brain region.

μ-Opioid receptor desensitization: Is morphine different?

Opioid tolerance and dependence are important phenomena. The contribution of acute μ‐opioid receptor regulatory mechanisms to the development of analgesic tolerance or physical dependence are unknown, and even the mechanisms underlying relatively rapid receptor desensitization in single cells are unresolved. To a large degree, the uncertainty surrounding the mechanisms and consequences of short‐term regulation of μ‐opioid receptors in single cells arises from the limitations in the experimental design in many of the studies that have investigated these events. Receptor overexpression and use of assays in which regulatory mechanisms are likely to blunt control determinations have led to measurements of opioid receptor activity that are likely to be insensitive to receptor uncoupling. Together with uncertainties concerning molecular details of μ‐opioid receptor interactions with potential regulatory molecules such as G protein‐coupled receptor kinases and arrestins, we are left with an incomplete picture crudely copied from the well‐worked‐out regulatory schema for β2‐adrenoceptors. As a consequence, suggestions that clinically relevant μ‐opioid receptor agonists may have different propensities to produce tolerance and dependence that arise from their differential recruitment of regulatory mechanisms are premature, and have not yet been appropriately assessed, nor explained in the context of a thoroughly established regulatory scheme. In this commentary, we outline the experimental limitations that have given rise to conflicting ideas about how μ‐opioid receptors are regulated, and identify the issues we feel still need to be addressed before we can understand why morphine promotes receptor trafficking differently to other opioids.

Modulation of Ca2+ channel currents of acutely dissociated rat periaqueductal grey neurons

1 The actions of the neuropeptide nociceptin on the calcium channel currents (IBa) of acutely dissociated rat periaqueductal grey (PAG) neurons were examined using whole‐cell patch clamp techniques. These effects were compared with those of opioid receptor agonists and the GABAB receptor agonist baclofen. 2 Neurons from young adult rats (23 to 56 days old) expressed predominantly ω‐conotoxin GVIA (N‐type)‐ and ω‐agatoxin IVA (P/Q‐type)‐sensitive IBa, together with smaller amounts of nimodipine‐sensitive current and current resistant to all three blockers. There was proportionately more N‐type IBa in neurons from female rats and proportionately more resistant current in neurons from male rats. 3 Nociceptin (EC50, 5 nm) and baclofen (EC50, 0.8 μm) inhibited IBa in all PAG neurons, while the opioid agonist methionine enkephalin (met‐enkephalin; 300 nm‐10 μm) inhibited IBa in 40 % of neurons. The effects of met‐enkephalin were reversed by the μ‐opioid antagonist CTAP, and mimicked by the μ‐opioid agonist DAMGO (300 nm‐3 μm). The δ‐opioid agonists DPDPE and deltorphin II, and the κ‐opioid agonist U69593, did not affect IBa in any neuron. The actions of nociceptin were not mimicked or blocked by the opioid antagonist naloxone or the nociceptin analogue [desPhe1]‐nociceptin. 4 The effects of nociceptin and baclofen on IBa were blocked by pretreatment of the neurons with pertussis toxin (500 ng ml−1, 8 h). 5 Nociceptin predominantly inhibited the N‐type (EC50, 2 nm; maximum inhibition, 50 %) and P/Q‐type (EC50, 7 nm; maximum inhibition, 33 %) IBa while having little effect on the L‐type and R‐type IBa. 6 These results are consistent with the previously described actions of nociceptin, baclofen and μ‐opioids in PAG slices, whereby they couple to increases in an inwardly rectifying K+ conductance. These agonists thus have the potential to modulate the function of PAG neurons via a number of different cellular effectors.