Boyi Liu

The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl– channels

Bradykinin (BK) is an inflammatory mediator and one of the most potent endogenous pain-inducing substances. When released at sites of tissue damage or inflammation, or applied exogenously, BK produces acute spontaneous pain and causes hyperalgesia (increased sensitivity to potentially painful stimuli). The mechanisms underlying spontaneous pain induced by BK are poorly understood. Here we report that in small nociceptive neurons from rat dorsal root ganglia, BK, acting through its B2 receptors, PLC, and release of calcium from intracellular stores, robustly inhibits M-type K+ channels and opens Ca2+-activated Cl- channels (CaCCs) encoded by Tmem16a (also known as Ano1). Summation of these two effects accounted for the depolarization and increase in AP firing induced by BK in DRG neurons. Local injection of inhibitors of CaCC and specific M-channel openers both strongly attenuated the nociceptive effect of local injections of BK in rats. These results provide a framework for understanding spontaneous inflammatory pain and may suggest new drug targets for treatment of such pain.

TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis

Allergic contact dermatitis is a common skin disease associated with inflammation and persistent pruritus. Transient receptor potential (TRP) ion channels in skin‐innervating sensory neurons mediate acute inflammatory and pruritic responses following exogenous stimulation and may contribute to allergic responses. Genetic ablation or pharmacological inhibition of TRPA1, but not TRPV1, inhibited skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine‐resistant scratching behavior in mice exposed to the haptens, oxazolone and urushiol, the contact allergen of poison ivy. Hapten‐challenged skin of TRPA1‐deficient mice contained diminished levels of inflammatory cytokines, nerve growth factor, and endogenous pruritogens, such as substance P (SP) and serotonin. TRPA1‐deficient sensory neurons were defective in SP signaling, and SP‐induced scratching behavior was abolished in Trpa1–/– mice. SP receptor antagonists, such as aprepitant inhibited both hapten‐induced cutaneous inflammation and scratching behavior. These findings support a central role for TRPA1 and SP in the integration of immune and neuronal mechanisms leading to chronic inflammatory responses and pruritus associated with contact dermatitis.—Liu, B., Escalera, J., Balakrishna, S., Fan, L., Caceres, A. I., Robinson, E., Sui, A., McKay, M. C., McAlexander, M. A., Herrick, C. A., Jordt, S. E., TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis. FASEB J. 27, 3549–3563 (2013). www.fasebj.org

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold‐sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant‐sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthols effects were reversed by a TRPM8 antagonist, AMTB. Menthols effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking‐related morbidities.—Willis, D. N., Liu, B., Ha, M. A., Jordt, S.‐E., Morris, J. B. Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants. FASEB J. 25, 4434–4444 (2011). www.fasebj.org

TRPM8 is the Principal Mediator of Menthol-induced Analgesia of Acute and Inflammatory Pain

Summary The analgesic mechanism of menthol remains controversial. In behavioral and imaging tests in mice, we identify TRPM8 as the major mediator of menthol analgesia, and TRPM8‐selective agents as novel analgesics. Abstract Menthol, the cooling natural product of peppermint, is widely used in medicinal preparations for the relief of acute and inflammatory pain in sports injuries, arthritis, and other painful conditions. Menthol induces the sensation of cooling by activating TRPM8, an ion channel in cold‐sensitive peripheral sensory neurons. Recent studies identified additional targets of menthol, including the irritant receptor, TRPA1, voltage‐gated ion channels and neurotransmitter receptors. It remains unclear which of these targets contribute to menthol‐induced analgesia, or to the irritating side effects associated with menthol therapy. Here, we use genetic and pharmacological approaches in mice to probe the role of TRPM8 in analgesia induced by L‐menthol, the predominant analgesic menthol isomer in medicinal preparations. L‐menthol effectively diminished pain behavior elicited by chemical stimuli (capsaicin, acrolein, acetic acid), noxious heat, and inflammation (complete Freund’s adjuvant). Genetic deletion of TRPM8 completely abolished analgesia by L‐menthol in all these models, although other analgesics (acetaminophen) remained effective. Loss of L‐menthol–induced analgesia was recapitulated in mice treated with a selective TRPM8 inhibitor, AMG2850. Selective activation of TRPM8 with WS‐12, a menthol derivative that we characterized as a specific TRPM8 agonist in cultured sensory neurons and in vivo, also induced TRPM8‐dependent analgesia of acute and inflammatory pain. L‐menthol– and WS‐12–induced analgesia was blocked by naloxone, suggesting activation of endogenous opioid‐dependent analgesic pathways. Our data show that TRPM8 is the principal mediator of menthol‐induced analgesia of acute and inflammatory pain. In contrast to menthol, selective TRPM8 agonists may produce analgesia more effectively, with diminished side effects.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

NGF Inhibits M/KCNQ Currents and Selectively Alters Neuronal Excitability in Subsets of Sympathetic Neurons Depending on their M/KCNQ Current Background

M/KCNQ currents play a critical role in the determination of neuronal excitability. Many neurotransmitters and peptides modulate M/KCNQ current and neuronal excitability through their G protein–coupled receptors. Nerve growth factor (NGF) activates its receptor, a member of receptor tyrosine kinase (RTK) superfamily, and crucially modulates neuronal cell survival, proliferation, and differentiation. In this study, we studied the effect of NGF on the neuronal (rat superior cervical ganglion, SCG) M/KCNQ currents and excitability. As reported before, subpopulation SCG neurons with distinct firing properties could be classified into tonic, phasic-1, and phasic-2 neurons. NGF inhibited M/KCNQ currents by similar proportion in all three classes of SCG neurons but increased the excitability only significantly in tonic SCG neurons. The effect of NGF on excitability correlated with a smaller M-current density in tonic neurons. The present study indicates that NGF is an M/KCNQ channel modulator and the characteristic modulation of the neuronal excitability by NGF may have important physiological implications.

Activation of Epidermal Growth Factor Receptor Inhibits KCNQ2/3 Current through Two Distinct Pathways: Membrane PtdIns(4,5)P2 Hydrolysis and Channel Phosphorylation

KCNQ2/3 currents are the molecular basis of the neuronal M currents that play a critical role in neuron excitability. Many neurotransmitters modulate M/KCNQ currents through their G-protein-coupled receptors. Membrane PtdIns(4,5)P2 hydrolysis and channel phosphorylation are two mechanisms that have been proposed for modulation of KCNQ2/3 currents. In this study, we studied regulation of KCNQ2/3 currents by the epidermal growth factor (EGF) receptor, a member of another family of membrane receptors, receptor tyrosine kinases. We demonstrate here that EGF induces biphasic inhibition of KCNQ2/3 currents in human embryonic kidney 293 cells and in rat superior cervical ganglia neurons, an initial fast inhibition and a later slow inhibition. Additional studies indicate that the early and late inhibitions resulted from PtdIns(4,5)P2 hydrolysis and tyrosine phosphorylation, respectively. We further demonstrate that these two processes are mutually dependent. This study indicates that EGF is a potent modulator of M/KCNQ currents and provides a new dimension to the understanding of the modulation of these channels.

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy

Significance In the United States, the most common cause of allergic contact dermatitis (ACD) is contact with poison ivy. Severe itch and skin inflammation are the major manifestations of poison ivy-induced ACD. In this study, we have established a critical role of IL-33/ST2 (interleukin 33/growth stimulation expressed gene 2) signaling in both itch and skin inflammation of poison ivy-induced ACD and revealed a previously unidentified interaction of IL-33/ST2 signaling with primary sensory neurons that may underlie the pruritic mechanisms of poison ivy-induced ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy dermatitis and, possibly, other chronic itch conditions in which IL-33/ST2 signaling may participate. Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2. Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

Transient Receptor Potential Cation Channel Subfamily M Member 8 channels mediate the anti-inflammatory effects of eucalyptol

Eucalyptol (1,8‐cineol), the major ingredient in the essential oil of eucalyptus leaves and other medicinal plants, has long been known for its anti‐inflammatory properties. Eucalyptol interacts with the TRP cation channels among other targets, but it is unclear which of these mediates its anti‐inflammatory effects.

Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition

The M-type potassium channel, of which its molecular basis is constituted by KCNQ2-5 homo- or heteromultimers, plays a key role in regulating neuronal excitability and is modulated by many G protein-coupled receptors. In this study, we demonstrate that histamine inhibits KCNQ2/Q3 currents in human embryonic kidney (HEK)293 cells via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis mediated by stimulation of H(1) receptor and phospholipase C (PLC). Histamine inhibited KCNQ2/Q3 currents in HEK293 cells coexpressing H(1) receptor, and this effect was totally abolished by H(1) receptor antagonist mepyramine but not altered by H(2) receptor antagonist cimetidine. The inhibition of KCNQ currents was significantly attenuated by a PLC inhibitor U-73122 but not affected by depletion of internal Ca(2+) stores or intracellular Ca(2+) concentration ([Ca(2+)](i)) buffering via pipette dialyzing BAPTA. Moreover, histamine also concentration dependently inhibited M current in rat superior cervical ganglion (SCG) neurons by a similar mechanism. The inhibitory effect of histamine on KCNQ2/Q3 currents was entirely reversible but became irreversible when the resynthesis of PIP(2) was impaired with phosphatidylinsitol-4-kinase inhibitors. Histamine was capable of producing a reversible translocation of the PIP(2) fluorescence probe PLC(delta1)-PH-GFP from membrane to cytosol in HEK293 cells by activation of H(1) receptor and PLC. We concluded that the inhibition of KCNQ/M currents by histamine in HEK293 cells and SCG neurons is due to the consumption of membrane PIP(2) by PLC.