Management of pain by opioid analgesics is confounded by central adverse effects that limit clinical dosages. Consequently, there is considerable interest to understand peripheral analgesic effects of opioids. The actions of opioids on peripheral sensory neurons have been difficult to study because of a general lack of effect of opioid agonists on nociceptor function in culture despite documented presence of opioid receptors. In this study, the micro-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), did not alter guanosine 5'-O-(3-[(35)S]thio)-triphosphate (GTPgamma[(35)S]) binding, adenylyl cyclase activity, or neuropeptide release in primary cultures of rat trigeminal ganglion (TG). However, after brief exposure to bradykinin (BK), DAMGO stimulated GTPgamma[(35)S] binding and inhibited both prostaglandin E(2) (PGE(2))-stimulated adenylyl cyclase activity and BK/PGE(2)-stimulated neuropeptide release. The effect of BK was blocked by the B(2) antagonist HOE 140 [D-Arg[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin], but not by the B(1) antagonist, Lys-[Leu8]des-Arg9-BK, and was mimicked by the protease-activated receptor-2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-NH(2), and by activation of protein kinase C (PKC) or by administration of arachidonic acid (AA). The enhanced responsiveness of micro-opioid receptor signaling by BK priming was blocked by both cyclooxygenase and PKC inhibitors; however, the effect of AA was blocked only by a cyclooxygenase inhibitor. The results indicate that micro-opioid receptor signaling in primary sensory TG neurons is enhanced by activation of phospholipase C-coupled receptors via a cyclooxygenase-dependent AA metabolite that is downstream of PKC.

The role of protease activated receptor-2 (PAR-2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR-2 activate the capsaicin-sensitive subclass of trigeminal nociceptors in a PLC-PKC-dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR-2 agonist (SL-NH2) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch-clamp. The specificity of SL-NH2 and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR-2. The in vivo role of PAR-2 activation was determined measuring SL-NH2 and trypsin-evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL-NH2/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL-NH2 (100 microM) and trypsin (1-600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 microM). In cultures treated with siRNA against PAR-2, both SL-NH2 and trypsin responses were significantly diminished. Both SL-NH2 and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL-NH2 rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR-2 can also induce functional competence in DOR.

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4-5 months) and aged (26-27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Chemotherapy-induced peripheral neuropathy (CIPN) arises from collateral damage to peripheral afferent sensory neurons by anticancer pharmacotherapy, leading to debilitating neuropathic pain. No effective treatment for CIPN exists, short of dose-reduction which worsens cancer prognosis. Here, we report that stimulation of nicotinamide phosphoribosyltransferase (NAMPT) produced robust neuroprotection in an aggressive CIPN model utilizing the frontline anticancer drug, paclitaxel (PTX). Daily treatment of rats with the first-in-class NAMPT stimulator, P7C3-A20, prevented behavioral and histologic indicators of peripheral neuropathy, stimulated tissue NAD recovery, improved general health, and abolished attrition produced by a near maximum-tolerated dose of PTX. Inhibition of NAMPT blocked P7C3-A20-mediated neuroprotection, whereas supplementation with the NAMPT substrate, nicotinamide, potentiated a subthreshold dose of P7C3-A20 to full efficacy. Importantly, P7C3-A20 blocked PTX-induced allodynia in tumored mice without reducing antitumoral efficacy. These findings identify enhancement of NAMPT activity as a promising new therapeutic strategy to protect against anticancer drug-induced peripheral neurotoxicity.

Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca(2+) accumulation, diacylglycerol production, and protein kinase C activation and are rapidly desensitized via a G-protein receptor kinase 2-dependent mechanism. However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca(2+) influx and Ca(2+) release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca(2+) channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca(2+) influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca(2+) signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca(2+) increases and abridged nocifensive responses.

Opioid overdose is a leading cause of death in the United States. The only treatment available currently is the competitive antagonist, naloxone (Narcan® ). Although naloxone is very effective and has saved many lives, as a competitive antagonist it has limitations. Due to the short half-life of naloxone, renarcotization can occur if the ingested opioid agonist remains in the body longer. Moreover, because antagonism by naloxone is surmountable, renarcotization can also occur in the presence of naloxone if a relatively larger dose of opioid agonist is taken. In such circumstances, a long-lasting, non-surmountable antagonist would offer an improvement in overdose treatment. Methocinnamox (MCAM) has been reported to have a long duration of antagonist action at mu opioid receptors in vivo. In HEK cells expressing the human mu opioid receptor, MCAM antagonism of mu agonist-inhibition of cAMP production was time-dependent, non-surmountable and non-reversible, consistent with (pseudo)-irreversible binding. In vivo, MCAM injected locally into the rat hindpaw antagonized mu agonist-mediated inhibition of thermal allodynia for up to 96 h. By contrast, antagonism by MCAM of delta or kappa agonists in HEK cells and in vivo was consistent with simple competitive antagonism. Surprisingly, MCAM also shifted the concentration-response curves of mu agonists in HEK cells in the absence of receptor reserve in a ligand-dependent manner. The shift in the [D-Ala2 ,N-MePhe4 ,Gly-ol5 ]-enkephalin (DAMGO) concentration-response curve by MCAM was insensitive to naloxone, suggesting that in addition to (pseudo)-irreversible orthosteric antagonism, MCAM acts allosterically to alter the affinity and/or intrinsic efficacy of mu agonists.

The neuropeptide bradykinin (BK) sensitizes nociceptor activation following its release in response to inflammatory injury. Thereafter, the bioactivity of bradykinin is controlled by the enzymatic activities of circulating peptidases. One such enzyme, the metalloendopeptidase EC3.4.24.15 (EP24.15), is co-expressed with bradykinin receptors in primary afferent neurons. In this study, using approaches encompassing pharmacology, biochemistry, cell biology, and behavioral animal models, we identified a crucial role for EP24.15 and the closely related EP24.16 in modulating bradykinin-mediated hyperalgesia. Pharmacological analyses indicated that EP24.15 and EP24.16 inhibition significantly enhances bradykinin type-2 receptor activation by bradykinin in primary trigeminal ganglia cultures. In addition, bradykinin-induced sensitization of TRPV1 activation was increased in the presence of the EP24.15/16 inhibitor JA-2. Furthermore, behavioral analyses illustrated a significant dose-response relationship between JA-2 and bradykinin-mediated thermal hyperalgesia. These results indicate an important physiological role for the metallopeptidases EP24.15 and EP24.16 in regulating bradykinin-mediated sensitization of primary afferent nociceptors.