TRPV1 and TRPA1 Mediate Peripheral Nitric Oxide-Induced Nociception in Mice

Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1

TRPV1 is activated by both acidic and basic pH.

Maintaining physiological pH is required for survival, and exposure to alkaline chemicals such as ammonia (smelling salts) elicits severe pain and inflammation through unknown mechanisms. TRPV1, the capsaicin receptor, is an integrator of noxious stimuli including heat and extracellular acidic pH. Here, we report that ammonia activates TRPV1, TRPA1 (another polymodal nocisensor), and other unknown receptor(s) expressed in sensory neurons. Ammonia and intracellular alkalization activate TRPV1 through a mechanism that involves a cytoplasmic histidine residue, not used by other TRPV1 agonists such as heat, capsaicin or low pH. Our studies show that TRPV1 detects both acidic and basic deviations from homeostatic pH

Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels

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NO donor activates DRG neurons via TRPV1 and TRPA1.

<p>Summary of the percent of responsive cultured DRG neurons to SNAP (3 mM), capsaicin (10 µM), and mustard oil (0.5 mM) from wildtype (with or without antagonists), TRPV1^−/−, TRPA1^−/−, and TRPV1^−/−TRPA1^−/− (V1^−/− A1^−/−) animals.</p>*<p>Ruthenium Red (10 µM), BCTC (1 µM), or AP-18 (100 µM) were applied 3 min prior to SNAP application until SNAP washout.</p>**<p>ATP (100 µM) was applied in the last of the experiment as a positive control. Approximately 20% of total DRG neurons were activated by application of ATP.</p

NO donors induce TRPV1-dependent thermal hyperalgesia and nocifensive behavior upon sensitization via TRPV1 and TRPA1.

<p>(A) Normalized changes in paw withdrawal latency are shown in response to a heat stimulus (average of three stimuli) 30 min after SNAP injection (25 mM in PBS, 1% DMSO). Decreases in paw withdrawal latency (%) were normalized to the value of SNAP-injected control mice (left columns) for each grouping (the larger the percentage, the faster the withdrawl to a heat stimulus). The first grouping represents SNAP- or vehicle- (1% DMSO) injected C57BL6 mice. The next three groupings show SNAP-induced thermal hyperalgesia in littermates with genotypes shown at bottom. Latencies of control animals were similar; 2.9±0.4 (SNAP-injected C57BL6), 2.7±0.3 (TRPV1^+/+), 3.5±1.0 (TRPA1^+/+), 3.4±0.6 (A1^+/− V1^+/−). (B, C) Nocifensive response duration (flicking, licking, and lifting injected paw) recorded over 10 min after injection of NO donors in the hindpaw. N≥10 mice were tested (N≥6 for vehicle controls). Mice were first injected with forskolin and m-3M3FBS (1 mM each) followed by NOC7 (25 mM) (B) or NOR3 (1.5 mM) (C) injection. Genotype of animals is indicated (A1: TRPA1, V1: TRPV1). Vehicle-injected control mice ((B): A1^+/− V1^+/− + vehicle: vehicle injection 5 min after first injection, (C):CTRL+NOR3 vehicle: vehicle injection 15 min after first injection, and CTRL+FSK/3M3FBS vehicle: first injected with vehicle for forskolin and m-3M3FBS followed by NOR3 injection 15 min after first injection) are also shown. CTRL mice in (C) share the same genetic background and carry at least one copy of wildtype TRPV1 and TRPA1; the first four columns represent results from littermates.</p

PKA and PLC pathways sensitize TRPA1 to NO donor.

<p>Summary of the percent of responsive “sensitized” DRG neurons to SNAP (3 mM), capsaicin (10 µM), and mustard oil (0.5 mM) from mice of the indicated genotypes (V1 represents TRPV1 and A1 represents TRPA1). DRG neurons were incubated with forskolin (100 µM) and m-3M3FBS (50 µM) for 10 min prior to SNAP application.</p>*<p>ATP (100 µM) was applied in the last of the experiment as a positive control. Approximately 20% of total DRG neurons were activated by application of ATP.</p