Anoctamin-1 Cl(-) channels in nociception: activation by an N-aroylaminothiazole and capsaicin and inhibition by T16A[inh]-A01.

BACKGROUND: Anoctamin 1 (ANO1 or TMEM16A) Ca(2+)-gated Cl(-) channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl(-) concentrations ([Cl(-)]i) sensory neuronal Cl(-) channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca(2+) ([Ca(2+)]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl(-)]i and (2) by lowering sensory neuronal [Cl(-)]i to switch ANO1 to be inhibitory. RESULTS: ANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl(-)]i (40 mM), E-act (10 µM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca(2+) activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin-B) current-clamp technique. Subsequently, at physiological [Cl(-)]i, capsaicin (15 µM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 µM). Under conditions of low [Cl(-)]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 µM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM). CONCLUSIONS: An ANO1-activator (E-act) induced [Cl(-)]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia.

A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma.

Asthma is an inflammatory disorder caused by airway exposures to allergens and chemical irritants. Studies focusing on immune, smooth muscle, and airway epithelial function revealed many aspects of the disease mechanism of asthma. However, the limited efficacies of immune-directed therapies suggest the involvement of additional mechanisms in asthmatic airway inflammation. TRPA1 is an irritant-sensing ion channel expressed in airway chemosensory nerves. TRPA1-activating stimuli such as cigarette smoke, chlorine, aldehydes, and scents are among the most prevalent triggers of asthma. Endogenous TRPA1 agonists, including reactive oxygen species and lipid peroxidation products, are potent drivers of allergen-induced airway inflammation in asthma. Here, we examined the role of TRPA1 in allergic asthma in the murine ovalbumin model. Strikingly, genetic ablation of TRPA1 inhibited allergen-induced leukocyte infiltration in the airways, reduced cytokine and mucus production, and almost completely abolished airway hyperreactivity to contractile stimuli. This phenotype is recapitulated by treatment of wild-type mice with HC-030031, a TRPA1 antagonist. HC-030031, when administered during airway allergen challenge, inhibited eosinophil infiltration and prevented the development of airway hyperreactivity. Trpa1(-/-) mice displayed deficiencies in chemically and allergen-induced neuropeptide release in the airways, providing a potential explanation for the impaired inflammatory response. Our data suggest that TRPA1 is a key integrator of interactions between the immune and nervous systems in the airways, driving asthmatic airway inflammation following inhaled allergen challenge. TRPA1 may represent a promising pharmacological target for the treatment of asthma and other allergic inflammatory conditions.

TRPA1 is a major oxidant sensor in murine airway sensory neurons.

Sensory neurons in the airways are finely tuned to respond to reactive chemicals threatening airway function and integrity. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway reflexes can provoke severe complications in patients affected by inflammatory airway conditions like rhinitis and asthma. Here, we showed that both hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a reactive oxygen species, activated Ca(2+) influx and membrane currents in an oxidant-sensitive subpopulation of chemosensory neurons. These responses were absent in neurons from mice lacking TRPA1, an ion channel of the transient receptor potential (TRP) gene family. TRPA1 channels were strongly activated by hypochlorite and hydrogen peroxide in primary sensory neurons and heterologous cells. In tests of respiratory function, Trpa1(-/-) mice displayed profound deficiencies in hypochlorite- and hydrogen peroxide-induced respiratory depression as well as decreased oxidant-induced pain behavior. Our results indicate that TRPA1 is an oxidant sensor in sensory neurons, initiating neuronal excitation and subsequent physiological responses in vitro and in vivo.

Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases.

The release of methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation, and edema. Similar responses are elicited by chemicals used as tear gases. Despite frequent exposures, the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca(2+) imaging and electrophysiology to show that the noxious effects of isocyanates and those of all major tear gas agents are caused by activation of Ca(2+) influx and membrane currents in mustard oil-sensitive sensory neurons. These responses are mediated by transient receptor potential ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate- and tear gas-induced nocifensive behavior after both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. Treatment with TRPA1 antagonists may prevent and alleviate chemical irritation of the eyes, skin, and airways and reduce the adverse health effects of exposures to a wide range of toxic noxious chemicals.

Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle.

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.

Sensory detection and responses to toxic gases: mechanisms, health effects, and countermeasures.

The inhalation of reactive gases and vapors can lead to severe damage of the airways and lung, compromising the function of the respiratory system. Exposures to oxidizing, electrophilic, acidic, or basic gases frequently occur in occupational and ambient environments. Corrosive gases and vapors such as chlorine, phosgene, and chloropicrin were used as warfare agents and in terrorist acts. Chemical airway exposures are detected by the olfactory, gustatory, and nociceptive sensory systems that initiate protective physiological and behavioral responses. This review focuses on the role of airway nociceptive sensory neurons in chemical sensing and discusses the recent discovery of neuronal receptors for reactive chemicals. Using physiological, imaging, and genetic approaches, Transient Receptor Potential (TRP) ion channels in sensory neurons were shown to respond to a wide range of noxious chemical stimuli, initiating pain, respiratory depression, cough, glandular secretions, and other protective responses. TRPA1, a TRP ion channel expressed in chemosensory C-fibers, is activated by almost all oxidizing and electrophilic chemicals, including chlorine, acrolein, tear gas agents, and methyl isocyanate, the highly noxious chemical released in the Bhopal disaster. Chemicals likely activate TRPA1 through covalent protein modification. Animal studies using TRPA1 antagonists or TRPA1-deficient mice confirmed the role of TRPA1 in chemically induced respiratory reflexes, pain, and inflammation in vivo. New research shows that sensory neurons are not merely passive sensors of chemical exposures. Sensory channels such as TRPA1 are essential for maintenance of airway inflammation in asthma and may contribute to the progression of airway injury following high-level chemical exposures.

Breathtaking TRP channels: TRPA1 and TRPV1 in airway chemosensation and reflex control.

New studies have revealed an essential role for TRPA1, a sensory neuronal TRP ion channel, in airway chemosensation and inflammation. TRPA1 is activated by chlorine, reactive oxygen species, and noxious constituents of smoke and smog, initiating irritation and airway reflex responses. Together with TRPV1, the capsaicin receptor, TRPA1 may contribute to chemical hypersensitivity, chronic cough, and airway inflammation in asthma, COPD, and reactive airway dysfunction syndrome.

TRPA1 mediates the noxious effects of natural sesquiterpene deterrents.

Plants, fungi, and animals generate a diverse array of deterrent natural products that induce avoidance behavior in biological adversaries. The largest known chemical family of deterrents are terpenes characterized by reactive alpha,beta-unsaturated dialdehyde moieties, including the drimane sesquiterpenes and other terpene species. Deterrent sesquiterpenes are potent activators of mammalian peripheral chemosensory neurons, causing pain and neurogenic inflammation. Despite their wide-spread synthesis and medicinal use as desensitizing analgesics, their molecular targets remain unknown. Here we show that isovelleral, a noxious fungal sesquiterpene, excites sensory neurons through activation of TPRA1, an ion channel involved in inflammatory pain signaling. TRPA1 is also activated by polygodial, a drimane sesquiterpene synthesized by plants and animals. TRPA1-deficient mice show greatly reduced nocifensive behavior in response to isovelleral, indicating that TRPA1 is the major receptor for deterrent sesquiterpenes in vivo. Isovelleral and polygodial represent the first fungal and animal small molecule agonists of nociceptive transient receptor potential channels.

TRPM7 channel is sensitive to osmotic gradients in human kidney cells.

TRPM7 (transient-receptor-potential melastatin 7) is an ion channel with alpha-kinase function. TRPM7 is divalent-selective and regulated by a range of receptor-stimulated second messenger pathways, intracellular Mg-nucleotides, divalent and polyvalent cations and pH. TRPM7 is ubiquitously found in mammalian cells, including kidney, the responsible organ for osmolyte regulation, posing the question whether the channel is osmosensitive. Recent reports investigated the sensitivity of native TRPM7-like currents to cell swelling with contradictory results. Here, we assess the sensitivity of TRPM7 to both hypo- and hyperosmotic conditions and explored the involvement of the channel's kinase domain. We find that hypotonicity facilitates TRPM7 at elevated intracellular magnesium and Mg.ATP (3-4 mm), but has no effect in the absence of these solutes. Hypertonic conditions, in contrast, inhibit TRPM7 with an IC(50) of 430 mosmol l(-1). This inhibitory effect is maintained in the complete absence of intra- and extracellular divalent ions, although shifted to higher osmolarities (IC(50) = 510 mosmol l(-1)). TRPM7 senses osmotic gradients rather than ionic strength and this is independent of cAMP or not affected by cytochalasin D treatment. Furthermore, the kinase-domain deletion mutant of TRPM7 shows a similar behaviour to osmolarity as the wild-type protein, both in the presence and absence of divalent ions. This indicates that at least part of the osmosensitivity resides in the channel domain. Physiologically, TRPM7 channels do not seem to play an active role in regulatory volume changes, but rather those volume changes modulate TRPM7 activity through changes in the cytosolic concentrations of free Mg, Mg-nucleotides and a further unidentified factor. We conclude that TRPM7 senses osmotically induced changes primarily through molecular crowding of solutes that affect channel activity.