Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

Abstract

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.

Keywords: NIPSNAP1; Nocistatin; TRPA1; nociception; peripheral sensory neurons; sensitization.

Figure 1.

Nocistatin specifically sensitizes TRPA1-mediated calcium response in DRG neurons. (A) Representative averaged traces of ratiometric calcium imaging in a population of cultured DRG neurons (1 day in vitro, DIV) upon application of indicated stimuli. Averaged changes in intracellular calcium levels are represented by the ratio of the fluorescence intensity at 340nm over 380nm. Traces represent averaged changes in intracellular calcium from a population of neurons (n > 20 neurons in this example) including responding neurons and non-responders of both conditions. As a quality control for neuronal health, only neurons responding to 50 µM MO at the end of the experiment are considered. Note that application of 10 μM Nocistatin or Vehicle (N/V) alone (first stimulus) does not elicit apparent calcium changes. (B,C) Quantification of the percentage of responding neurons and mean amplitude of neuronal calcium changes upon stimulation with (B) 10 μM Nocistatin or Vehicle (N/V) along with 25 μM MO (percentage of responding neurons: V: 11.5 ± 2.4 %; N: 24.4 ± 2.2 %; p = 0.017, 2-tailed Student's t-test), and (C) upon application of 50 μM MO at the end of the experiment (ns, 2-tailed Student's t-test). (D) Quantification of the percentage of responding neurons and mean amplitude of neuronal calcium changes upon stimulation with 10 μM Nocistatin or Vehicle (N/V) along with 0.1 μM Capsaicin (CAPS). Nocistatin neither affects the percentage of responders nor the mean amplitude of responses (ns, 2-tailed Student's t-test). For each condition we analyzed n > 300 neurons from N = 3 independent cultures. All data are represented as mean ± SEM.

Figure 2.

NIPSNAP1 knock-down does not affect Nocistatin-induced TRPA1 sensitization. (A, B) The extent of NIPSNAP1 knock-down (KD) after transfection with NIPSNAP1 siRNA as measured by (A) qPCR (relative Nipsnap1 mRNA levels: 0.22 ± 0.05 compared to GAPDH used as reference, i.e. approx. 78 % knock-down, p = 0.0041; 2-tailed Student's t-test) and (B) immunocytochemistry (p = 0.0019; 2-tailed Student's t-test; ≥700 neurons analyzed). N = 2–3 independent cultures each. (C) Representative images and quantification of immunohistochemistry on cryosections of mouse DRG co-labeled for NIPSNAP1 and Peripherin. White arrows highlight neurons co-expressing NIPSNAP1 and Peripherin. Scale bar, 50 μm. ≥12000 neurons analyzed. (D, E) DRG cultures were transfected with NIPSNAP1-siRNA or AllStar Negative control (Qiagen) and used for ratiometric calcium imaging 3 days later (3 DIV). Quantification of the percentage of responding neurons of neuronal calcium changes upon stimulation with 10 μM Nocistatin or Vehicle (N/V) along with (D) 25 μM MO (p = 0.0354, one way ANOVA followed by Fisher's LSD test) or (E) 50 μM MO at the end of the experiment (ns, one way ANOVA followed by Fisher's LSD test). Mean response amplitudes to either MO concentration were not altered among conditions (data not shown). For each condition we analyzed a minimum of n ≥ 150 neurons from N = 5 independent cultures. Please note that presented values cannot be compared to values shown in Figure 1 due to differences in culture conditions and transfection (experiments were performed 1 DIV in Figure 1, while 3 DIV after transfection in this figure). All data are represented as mean ± SEM.