enkephalin--ala(2)-mephe(4)-gly(5)- and resiniferatoxin

Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia.

Topics: Analgesics, Opioid; Animals; Calcium; Capsaicin; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Diterpenes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Fentanyl; Ganglia, Spinal; Humans; Hyperalgesia; Male; Membrane Potentials; Morphine; Mutagenesis, Site-Directed; Protein Binding; Rats; Receptors, Opioid, mu; Sensory Receptor Cells; Substance Withdrawal Syndrome; Tritium; TRPV Cation Channels

Opioids remain the mainstay of treatment for severe pain, but the associated hyperalgesia and tolerance are significant impediments to achieving adequate pain relief with opioids. Here we show that in the spinal cord, brief application of the mu-opioid receptor agonist (D-Ala(2),N-Me-Phe(4),Gly-ol(5))-enkephalin (DAMGO) at 1 mum, but not at 1-10 nm, caused an initial decrease followed by a large and long-lasting increase in the amplitude of monosynaptic EPSCs evoked from the dorsal root in approximately 50% of lamina I and II neurons. However, postsynaptic dialysis of the G-protein inhibitor had no effect on DAMGO-induced initial inhibition and long-term potentiation (LTP) in either lamina I or II neurons. DAMGO-induced LTP was associated with an increase in the paired-pulse depression ratio. Furthermore, DAMGO application and washout induced an initial decrease followed by a persistent increase in the frequency of miniature EPSCs. Bath application, but not postsynaptic dialysis, of an NMDA receptor antagonist or a calcium chelator abolished DAMGO-induced LTP. Strikingly, ablation of TRPV1-expressing primary afferents not only eliminated DAMGO-induced LTP but also prolonged DAMGO-induced inhibition of the miniature and evoked EPSCs (i.e., long-term depression). Thus, our study strongly suggests that TRPV1-expressing primary afferents play a prominent role in opioid-induced presynaptic LTP, which challenges a previous report suggesting the postsynaptic nature of this opioid-induced LTP. This excitatory effect of opioids on primary afferents can counteract the inhibitory effect of opioids on synaptic transmission at the spinal level and is likely involved in opioid-induced hyperalgesia and tolerance.

Topics: Analgesics, Opioid; Animals; Biophysics; Calcium; Chelating Agents; Diterpenes; Dizocilpine Maleate; Dose-Response Relationship, Drug; Egtazic Acid; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Guanosine Diphosphate; In Vitro Techniques; Lectins; Long-Term Potentiation; Male; Neurons; Patch-Clamp Techniques; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Spinal Cord; Thionucleotides; TRPV Cation Channels; Valine

The transient receptor potential vanilloid-1 (TRPV1) receptor is involved in peripheral and spinal nociceptive processing and is a therapeutic target for pain. We have shown previously that TRPV1 in the ventrolateral periaqueductal gray (VL-PAG) tonically contributes to brain stem descending antinociception by stimulating glutamate release into the rostral ventromedial medulla and off neuron activity. Because both opioid and vanilloid systems integrate and transduce pain sensation in these pathways, we studied the potential interaction between TRPV1 and mu-opioid receptors in the VL-PAG-rostral ventromedial medulla (RVM) system. We found that the TRPV1 agonist, capsaicin, and the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin, when coadministered into the ventrolateral-PAG at doses nonanalgesic per se, produce 1) antinociception in tests of thermal nociception; 2) stimulation of glutamate release into the RVM; and 3) inhibition of on neuron activity in the RVM. These effects were all antagonized by the TRPV1 and opioid receptor antagonists 5'-iodo-resiniferatoxin and naloxone, respectively, thus suggesting the existence of a TRPV1-mu-opioid interaction in the VL-PAG-RVM system. By using double immunofluorescence techniques, we found that TRPV1 and mu-opioid receptors are coexpressed in several neurons of the VL-PAG. These findings suggest that mu-receptor activation not only acts on inhibitory neurons to disinhibit PAG output neurons but also interacts with TRPV1 activation at increasing glutamate release into the RVM, possibly by acting directly on PAG output neurons projecting to the RVM.

Topics: Action Potentials; Animals; Capsaicin; Diterpenes; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glutamic Acid; Hyperalgesia; Male; Medulla Oblongata; Microinjections; Naloxone; Pain Measurement; Pain Threshold; Periaqueductal Gray; Rats; Rats, Wistar; Reaction Time; Receptors, Opioid, mu; TRPV Cation Channels

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic mu-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of mu-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of mu-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.

Topics: Animals; Diterpenes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Postsynaptic Potentials; Glutamic Acid; Male; Naloxone; Pain Threshold; Plant Lectins; Potassium Channels, Inwardly Rectifying; Rats; Rats, Sprague-Dawley; Spinal Cord; TRPV Cation Channels

Systemic administration of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, removes transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons and impairs thermal but not mechanical nociception in adult animals. In this study, we determined how loss of TRPV1-expressing sensory neurons alters the antinociceptive effect of mu opioids and mu opioid receptors in the spinal cord. The effect of morphine and (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) was measured by testing the paw mechanical withdrawal threshold in rats treated with RTX or vehicle. RTX treatment deleted TRPV1-immunoreactive dorsal root ganglion neurons and nerve terminals in the spinal dorsal horn. Also the mu opioid receptor immunoreactivity was markedly reduced in the superficial dorsal horn of RTX-treated rats. However, RTX treatment did not affect the dorsal horn neurons labeled with both TRPV1- and mu opioid receptor-immunoreactivity. Surprisingly, intrathecal morphine or DAMGO produced a greater increase in the withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the effect of intrathecal morphine and DAMGO in RTX-treated rats was also profoundly increased. Furthermore, the antinociceptive effect of systemic morphine was significantly potentiated in RTX-treated rats. The B(MAX) (but not K(D)) of [3H]-DAMGO binding and DAMGO-stimulated [35S]GTPgammaS activity in the dorsal spinal cord were significantly reduced in the RTX group. This study provides novel information that loss of TRPV1 afferent neurons eliminates presynaptic mu opioid receptors present on TRPV1-expressing afferent neurons but paradoxically potentiates the analgesic effect of mu opioid agonists. Mechano-nociception, transmitted through non-TRPV1 sensory neurons, is subject to potent modulation by mu opioid agonists.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Diterpenes; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fluorescent Antibody Technique; Ganglia, Spinal; Gene Expression Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Lectins; Male; Morphine; Neurons, Afferent; Pain Measurement; Protein Binding; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid, mu; Spinal Cord; Sulfur Isotopes; Tritium; TRPV Cation Channels

Possible correlation of release of endogenous glutamate (Glu) and aspartate (Asp) with stimulation parameters used to activate primary sensory neurons was examined using the rat spinal cord slice--dorsal root ganglion preparation and high performance liquid chromatography with fluorimetric detection. Selective activation of the low-threshold (A beta) primary afferent fibers resulted in a two-fold increase in the rate of basal outflow of Asp and a smaller increase in the outflow of Glu from the rat spinal dorsal horn slices into the superfusing medium. The activation of both the low (A beta)- and the high-threshold (A delta + C) primary afferents elicited also a significant increase in the outflow of Asp and Glu relative to control. Glu and Asp are released in significant amounts following superfusion of the dorsal root ganglia with capsaicin or resiniferatoxin. DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin), an agonist at mu-opioid receptors, attenuated the high-intensity stimulation-evoked outflow of Asp and Glu in a naloxone-sensitive manner. Our results have provided further evidence in support of the contention that Glu and Asp act as excitatory synaptic transmitters in the spinal dorsal horn. A role for mu-opioid receptors in modulation of spinal processing of somatosensory information is indicated.

Topics: Afferent Pathways; Animals; Aspartic Acid; Capsaicin; Diterpenes; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Ganglia, Spinal; Glutamates; Glutamic Acid; Glutamine; In Vitro Techniques; Kinetics; Naloxone; Nerve Fibers; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Spinal Cord; Time Factors