Activation of TRPC channels contributes to OA‐NO₂‐induced responses in guinea‐pig dorsal root ganglion neurons

Abstract

Key pointsr Nitro-oleic acid (OA-NO 2 ) and related nitroalkenes are electrophilic fatty acid derivatives that function as metabolic and anti-inflammatory signalling mediators and contribute to resolution of inflammation.r OA-NO 2 is known to activate transient receptor potential (TRP) A1 and V1 channels in rat and mouse primary sensory neurons.r However, in guinea-pig dorsal root ganglion neurons OA-NO 2 activated TRPC channels and elicited a transient excitatory effect followed by a long-lasting inhibitory effect.r Our data challenge the prevailing view that electrophilic compounds act specifically on TRPA1 and suggest that OA-NO 2 can exert a modulatory effect on sensory mechanisms via activation of TRPC channels.Abstract Effects of nitro-oleic acid (OA-NO 2 ) on TRP channels were examined in guinea-pig dissociated dorsal root ganglia (DRG) neurons using calcium imaging and patch clamp techniques. OA-NO 2 increased intracellular Ca 2+ in 60-80% DRG neurons. 1-Oleoyl-2acetyl-sn-glycerol (OAG), a TRPC agonist, elicited responses in 36% of OA-NO 2 -sensitive neurons while capsaicin (TRPV1 agonist) or allyl-isothiocyanate (AITC, TRPA1 agonist) elicited responses in only 16% and 10%, respectively, of these neurons. A TRPV1 antagonist (diarylpiperazine, 5 μM) in combination with a TRPA1 antagonist (HC-030031, 30 μM) did not change the amplitude of the Ca 2+ transients or percentage of neurons responding to OA-NO 2 ; however, a reducing agent DTT (50 mM) or La 3+ (50 μM) completely abolished OA-NO 2 responses. OA-NO 2 also induced a transient inward current associated with a membrane depolarization followed by a prolonged outward current and hyperpolarization in 80% of neurons. The reversal potentials of inward and outward currents were approximately −20 mV and −60 mV, respectively. Inward current was reduced when extracellular Na + was absent, but unchanged by niflumic acid (100 μM), a Cl − channel blocker. Outward current was abolished in the absence of extracellular Ca 2+ or a combination of two Ca 2+ -activated K + channel blockers (iberiotoxin, 100 nM and apamin, 1 μM). BTP2 (1 or 10 μM), a broad spectrum TRPC antagonist, or La 3+ (50 μM) completely abolished OA-NO 2 currents. RT-PCR performed on mRNA extracted from DRGs revealed the expression of all seven subtypes of TRPC channels. These results support the hypothesis that OA-NO 2 activates TRPC channels other than the TRPV1 and TRPA1 channels already known to be targets in rat and mouse sensory neurons and challenge the prevailing view that electrophilic compounds act X. Zhang and J. Beckel contributed equally to this paper.

Discovery of bioactive nitrated lipids and nitro-lipid-protein adducts using mass spectrometry-based approaches

“…There are several proteins reported to be targets of NO 2 -FA electrophilic reactivity, for example, the p65 subunit of NF-κB [1], [23], [92], heme oxygenase-1 (HO-1) [17], [19], [22], [67], [89], [93], mitogen-activated protein kinase (MAPK) phosphatase 1 (MPK-1) [92], Kelch-like ECH-associated protein 1 (Keap 1) [17], [22], [46], [88], metalloproteinases (MMP-7 and MMP-9) [75], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [42], [94], protein disulfide isomerase (PDI) [95], and transient receptor potential (TRP) channels [96], [97], [98], [99] (Table 3). NO 2 -FA can also conduct their biological signaling roles by a receptor-dependent signaling action and peroxisome proliferator-activated receptor gamma (PPARγ) is one of the main targets, which is a significant route for the anti-inflammatory effect associated with NO 2 -FA derivatives [6], [23], [47], [65], [93], [100], [101], [102].…”

Nitro-fatty acids in cardiovascular regulation and diseases characteristics and molecular mechanisms

“…Herein, we have outlined those relevant to cardiovascular, pulmonary and renal diseases. Further evidences on the anti-inflammatory actions of NO 2 -FAs are included in Table 1 and include inflammatory bowel disease (101), chronic obstructive pulmonary disease (102) or the regulation of transient reaction potential (TRP) A1, C and V1 by NO 2 -FAs in nociceptive neuronal cells, centrally involved in the regulation of symptoms of inflammation and pain control by the central nervous system (103–105). …”

The effect of the electrophilic fatty acid nitro-oleic acid on TRP channel function in sensory neurons

“…Calcium imaging and patch clamp recording revealed that another TRP channel mediates the effects of NO 2 -OA on sensory neurons in the guinea pig [30]. NO 2 -OA increases intracellular Ca 2+ (EC 50 , 8.8 µM, Fig.…”

“…3C,D). 1-oleoyl-2acetyl-sn-glycerol (OAG), a TRPC 3/6/7 agonist, elicits responses in 36% of NO 2 -OA sensitive neurons while capsaicin or AITC elicit responses in only 16% and 10%, respectively, of these neurons [30]. A TRPV1 antagonist (DPA, 5 µM,) in combination with a TRPA1 antagonist (HC-030031, 30 µM) does not change the amplitude of the Ca 2+ transients or the percentage of neurons responding to NO 2 -OA; however, DTT (50 mM) or La 3+ (50 µM), which is known to block TRPC channels, completely abolishes the NO 2 -OA responses (Fig.…”

“…Because nitrated fatty acids may induce some of their biological effects by the release of NO [31], the potential role of NO 2 -OA as an NO donor was also examined. The NO donor SNAP does not elicit detectable increases in intracellular calcium and pretreatment with the NO scavenger, cPTIO, does not prevent the NO 2 -OA induced calcium transients [30], suggesting that the effects of NO 2 -OA are not due to NO mediated nitrosylation of TRP channels, which has also been shown to occur when stimulated directly with NO donors [32].…”