Armen N. Akopian

The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel α subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X2/3 heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious ‘warming’ stimuli.

Transient receptor potential TRPA1 channel desensitization in sensory neurons is agonist dependent and regulated by TRPV1-directed internalization

The pharmacological desensitization of receptors is a fundamental mechanism for regulating the activity of neuronal systems. The TRPA1 channel plays a key role in the processing of noxious information and can undergo functional desensitization by unknown mechanisms. Here we show that TRPA1 is desensitized by homologous (mustard oil; a TRPA1 agonist) and heterologous (capsaicin; a TRPV1 agonist) agonists via Ca2+‐independent and Ca2+‐dependent pathways, respectively, in sensory neurons. The pharmacological desensitization of TRPA1 by capsaicin and mustard oil is not influenced by activation of protein phosphatase 2B. However, it is regulated by phosphatidylinositol‐4,5‐bisphosphate depletion after capsaicin, but not mustard oil, application. Using a biosensor, we establish that capsaicin, unlike mustard oil, consistently activates phospholipase C in sensory neurons. We next demonstrate that TRPA1 desensitization is regulated by TRPV1, and it appears that mustard oil‐induced TRPA1 internalization is prevented by coexpression with TRPV1 in a heterologous expression system and in sensory neurons. In conclusion, we propose novel mechanisms whereby TRPA1 activity undergoes pharmacological desensitization through multiple cellular pathways that are agonist dependent and modulated by TRPV1.

A new member of the acid-sensing ion channel family

Acid-sensing ion channels (ASICs) are members of the epithelial sodium channel (ENaC)-degenerin family of two-pass transmembrane segment protein subunits which form multi-meric cation channels. Members of the ENaC-degenerin family are gated by stimuli as diverse as protons, peptides and mechanical distension. Here we describe a new member of the family, SPASIC or ASIC 4 (spinal cord ASIC) which is expressed throughout the central nervous system in an overlapping population of neurons that also express the ASIC subunit MDEG2. ASIC-4 which shows 44% identify with ASIC is developmentally regulated and expressed in a subset of sensory neurons as well as in the CNS. However, despite the strong homology with ASIC, the ASIC-4 transcript does not encode a proton gated cation channel.

Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia

Transient receptor potential vanilloid 1 (TRPV1) plays a major role in hyperalgesia and allodynia and is expressed both in the peripheral and central nervous systems (CNS). However, few studies have evaluated mechanisms by which CNS TRPV1 mediates hyperalgesia and allodynia after injury. We hypothesized that activation of spinal cord systems releases endogenous TRPV1 agonists that evoke the development of mechanical allodynia by this receptor. Using in vitro superfusion, the depolarization of spinal cord triggered the release of oxidized linoleic acid metabolites, such as 9-hydroxyoctadecadienoic acid (9-HODE) that potently activated spinal TRPV1, leading to the development of mechanical allodynia. Subsequent calcium imaging and electrophysiology studies demonstrated that synthetic oxidized linoleic acid metabolites, including 9-HODE, 13-HODE, and 9 and 13-oxoODE, comprise a family of endogenous TRPV1 agonists. In vivo studies demonstrated that intrathecal application of these oxidized linoleic acid metabolites rapidly evokes mechanical allodynia. Finally, intrathecal neutralization of 9- and 13-HODE by antibodies blocks CFA-evoked mechanical allodynia. These data collectively reveal a mechanism by which an endogenous family of lipids activates TRPV1 in the spinal cord, leading to the development of inflammatory hyperalgesia. These findings may integrate many pain disorders and provide an approach for developing analgesic drugs.

TRPA1-mediated responses in trigeminal sensory neurons: interaction between TRPA1 and TRPV1

The transient receptor potential (TRP)A1 channel is involved in the transduction of inflammation‐induced noxious stimuli from the periphery. Previous studies have characterized the properties of TRPA1 in heterologous expression systems. However, there is little information on the properties of TRPA1‐mediated currents in sensory neurons. A capsaicin‐sensitive subset of rat and mouse trigeminal ganglion sensory neurons was activated with TRPA1‐specific agonists, mustard oil and the cannabinoid WIN55,212. Mustard oil‐ and WIN55,212‐gated currents exhibited marked variability in their kinetics of activation and acute desensitization. TRPA1‐mediated responses in neurons also possess a characteristic voltage dependency with profound outward rectification that is influenced by extracellular Ca2+ and the type and concentration of TRPA1‐specific agonists. Examination of TRPA1‐mediated responses in TRPA1‐containing cells indicated that the features of neuronal TRPA1 are not duplicated in cells expressing only TRPA1 and, instead, can be restored only when TRPA1 and TRPV1 channels are coexpressed. In summary, our results suggest that TRPA1‐mediated responses in sensory neurons have distinct characteristics that can be accounted for by the coexpression of the TRPV1 and TRPA1 channels.

Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents

The transient receptor potential vanilloid 1 (TRPV1) channel is the principal detector of noxious heat in the peripheral nervous system. TRPV1 is expressed in many nociceptors and is involved in heat-induced hyperalgesia and thermoregulation. The precise mechanism or mechanisms mediating the thermal sensitivity of TRPV1 are unknown. Here, we have shown that the oxidized linoleic acid metabolites 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE) are formed in mouse and rat skin biopsies by exposure to noxious heat. 9- and 13-HODE and their metabolites, 9- and 13-oxoODE, activated TRPV1 and therefore constitute a family of endogenous TRPV1 agonists. Moreover, blocking these substances substantially decreased the heat sensitivity of TRPV1 in rats and mice and reduced nociception. Collectively, our results indicate that HODEs contribute to the heat sensitivity of TRPV1 in rodents. Because oxidized linoleic acid metabolites are released during cell injury, these findings suggest a mechanism for integrating the hyperalgesic and proinflammatory roles of TRPV1 and linoleic acid metabolites and may provide the foundation for investigating new classes of analgesic drugs.

Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain

Increased voltage-gated sodium channel activity may contribute to the hyperexcitability of sensory neurons in inflammatory and neuropathic pain states. We examined the levels of the transcript encoding the tetrodotoxin-resistant sodium channel SNS in dorsal root ganglion neurons in a range of inflammatory and neuropathic pain models in the rat. Local Freunds adjuvant or systemic nerve growth factor-induced inflammation did not substantially alter the total levels of SNS mRNA. When NGF-treated adult rat DRG neurons in vitro were compared with NGF-depleted control neurons, SNS total mRNA levels and the levels of membrane-associated immunoreactive SNS showed a small increase (17 and 25%, respectively), while CGRP levels increased fourfold. SNS expression is thus little dependent on NGF even though SNS transcript levels dropped by more than 60% 7-14 days after axotomy. In the streptozotocin diabetic rat SNS levels fell 25%, while in several manipulations of the L5/6 tight nerve ligation rat neuropathic pain model, SNS levels fell 40-80% in rat strains that are either susceptible or relatively resistant to the development of allodynia. Increased expression of SNS mRNA is thus unlikely to underlie sensory neuron hyperexcitability associated with inflammation, while lowered SNS transcript levels are associated with peripheral nerve damage.

LPS Sensitizes TRPV1 via Activation of TLR4 in Trigeminal Sensory Neurons

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked aconcentration-dependent increase in intracellular calcium accumulation (Ca2+)i and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca2+)i, release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections.

Modulation of trigeminal sensory neuron activity by the dual cannabinoid–vanilloid agonists anandamide, N‐arachidonoyl‐dopamine and arachidonyl‐2‐chloroethylamide

Peripheral cannabinoids have been shown to suppress nociceptive neurotransmission in a number of behavioral and neurophysiological studies. It is not known, however, whether cannabinoids exert this action through direct interactions with nociceptors in the periphery and/or if other processes are involved. To gain a better understanding of the direct actions of cannabinoid‐vanilloid agonists on sensory neurons, we examined the effects of these compounds on trigeminal ganglion (TG) neurons in vitro. AEA (EC50=11.0 μM), NADA (EC50=857 nM) and arachidonyl‐2‐chloroethylamide ACEA (EC50=14.0 μM) each evoked calcitonin gene‐related peptide (CGRP) release from TG neurons. The TRPV1 antagonists iodo‐resiniferatoxin (I‐RTX) and capsazepine (CPZ) each obtunded AEA‐, NADA‐, ACEA‐ and capsaicin (CAP)‐evoked CGRP release with individually equivalent IC50s for each of the compounds (I‐RTX IC50 range=2.6–4.0 nM; CPZ IC50 range=523–1140 μM). The pro‐inflammatory mediator prostaglandin E2 significantly increased the maximal effect of AEA‐evoked CGRP release without altering the EC50. AEA, ACEA and CAP stimulated cAMP accumulation in TG neurons in a calcium‐ and TRPV1‐dependent fashion. Moreover, the protein kinase inhibitor staurosporine significantly inhibited AEA‐ and CAP‐evoked CGRP release. The pungency of AEA, NADA, ACEA and CAP in the rat eye‐wipe assay was also assessed. Interestingly, when applied intraocularly, NADA or CAP each produced nocifensive responses, while AEA or ACEA did not. Finally, the potential inhibitory effects of these cannabinoids on TG nociceptors were evaluated. Neither AEA nor ACEA decreased CAP‐evoked CGRP release. Furthermore, neither of the cannabinoid receptor type 1 antagonists SR141716A nor AM251 had any impact on either basal or CAP‐evoked CGRP release. AEA also did not inhibit 50 mM K+‐evoked CGRP release and did not influence bradykinin‐stimulated inositol phosphate accumulation. We conclude that the major action of AEA, NADA and ACEA on TG neurons is excitatory, while, of these, only NADA is pungent. These findings are discussed in relation to our current understanding of interactions between the cannabinoid and vanilloid systems and nociceptive processing in the periphery.