Transient receptor potential melastatin-3 (TRPM3) mediates nociceptive-like responses in Hydra vulgaris

The ability of mammals to feel noxious stimuli lies in a heterogeneous group of primary somatosensory neurons termed nociceptors, which express specific membrane receptors, such as the Transient Receptor Potential (TRP) family. Here, we show that one of the most important nociceptive-like pathways is conserved in the freshwater coelenterate Hydra vulgaris, the most primitive organism possessing a nervous system. In particular, we found that H. vulgaris expresses TRPM3, a nociceptor calcium channel involved in the detection of noxious heat in mammals. Furthermore, we detected that both heat shock and TRPM3 specific agonist (i.e., pregnenolone sulfate) induce the modulation of the heat shock protein 70 (HSP70) and the nitric oxide synthase (NOS), two genes activated by TRP-mediated heat painful stimuli in mammals. As expected, these effects are inhibited by a TRPM3 antagonist (i.e., mefenamicacid). Interestingly, the TRPM3 agonist and heat shock also induce the expression of nuclear transcription erythroid 2-related factor (Nrf2) and superoxide dismutase (SOD), known markers of oxidative stress; noteworthy gene expression was also inhibited by the TRPM3 antagonist. As a whole, our results demonstrate the presence of conserved molecular oxidative/nociceptive-like pathways at the primordial level of the animal kingdom

Identification, characterization and localization of <i>Hydra</i> TRPM3.

<p>(A) Expression of TRPM3 protein in whole body of <i>H</i>. <i>vulgaris</i>. Western blot is representative of six independent experiments. (B) Immunofluorescence of Hydra whole mounts with the anti-TRPM3 antibody. Signals for TRPM3 are clearly extracellular and appear mainly in tentacles and foot. Specimens were examined in the confocal microscope. Scale bars, 100 μm.</p

Effect of heat shock (HS; 34°C) and pregnenolone sulfate (PS; 10 μM) on HyCuZnSOD mRNA expression in <i>H</i>. <i>vulgaris</i>.

<p>Both HS (A) and TRPM3 agonist PS (C) induce an increase of SOD mRNA level, the maximal peak being observed after 24 h. A pretreatment with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits the effect relative to HS (B) or PS (D). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 h) ± SEM (n = 9). (A) * p≤0.01 vs 0; (B) ^ p≤0.05 vs CTRL and ^^ p≤0.05 vs HS; (C) ^ p≤0.05 vs 0; (D) ^ p≤0.05 vs CTRL and ^^ p≤0.05 vs PS.</p

Action of heat shock (HS; 34°C) and pregnenolone sulfate (PS; 10 μM) on Nrf2 mRNA expression in <i>H</i>. <i>vulgaris</i>.

<p>HS (A) and the TRPM3 agonist PS (C) time-dependently induce Nrf2 with a peak observed after 1.5 h. A pretreatment for 10 min with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits the effect relative to both HS (B) and PS (D). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0 h) ± SEM (n = 9). (A) * p≤0.01 vs 0 and ^ p≤0.05 vs 0; (B) * p≤0.01 vs CTRL and ^^ p≤0.05 vs HS; (C) * p≤0.01 vs 0 and ^ p≤0.05 vs 0; (D) * p≤0.01 vs CTRL and ** p≤0.01 vs PS.</p

Common responses to mechanical solicitations in untreated (17°C) and HS-treated (34°C) animals.

<p>a) untreated polyp without mechanical solicitations: open tentacles, elongated body and foot adhered to the substrate; b) untreated polyp after mechanical solicitation: contraction of body and tentacles; c) HS-treated polyp followed by mechanical solicitation: open tentacles, elongated body and lack of adhesion to the substrate; d) HS-treated polyp was placed at 17°C for 5 min before mechanical solicitations: partial recovery of functions (e.g., tentacles reactivity); e) HS-treated polyp was placed at 17°C for 10 min before mechanical solicitations: total recovery of functional features. The table shows physiological variables analyzed during the HS time points; + means responsiveness,—means loss of responsiveness. Te: tentacles; af: adhesive foot; co: column.</p

Role of TRPM3 on HSP70 mRNA expression in <i>H</i>. <i>vulgaris</i>.

<p>(A) A pretreatment for 10 min with the TRPM3 antagonist mefenamic acid (MFA; 20 μM) inhibits HSP70 gene expression induced by heat shock (HS; 34°C). (B) TRPM3 agonist pregnenolone sulfate (PS; 10 μM) increase HSP70 gene expression, the peak being observed after 1.5 h. (C) MFA (20 μM) inhibits HSP70 gene expression induced by PS (10 μM). Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (T = 0) ± SEM (n = 9). (A) * p≤0.01 vs CTRL and ^^ p≤0.05 vs HS; (B) * p≤0.01 vs 0; (C) * p≤0.01 vs CTRL and ^^ p≤0.05 vs PS.</p

Effect of heat shock (HS; 34°C) and pregnenolone sulfate (PS; 10 μM) on NOS gene expression in <i>H</i>. <i>vulgaris</i>.

<p>HS (A) and PS (C) induce an increase of NOS mRNA expression with a maximal effect at 24 h. When animals are pretreated with mefenamic acid (MFA; 20 μM), NOS expression induced by HS (B) or PS (D) is abolished. Data are calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (T = 0) ± SEM (n = 9). (A) * p≤0.01 vs 0; (B) * p≤0.01 vs CTRL and ** p≤0.01 vs HS; (C) * p≤0.01 vs 0; (D) * p≤0.01 vs CTRL and ** p≤0.01 vs PS.</p