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. 2022 Jul 21:31:101315.
doi: 10.1016/j.bbrep.2022.101315. eCollection 2022 Sep.

Functional analysis of thermo-sensitive TRPV1 in an aquatic vertebrate, masu salmon (Oncorhynchus masou ishikawae)

A Yoshimura  1 , S Saito  2   3 , C T Saito  2   3 , K Takahashi  1   4 , M Tominaga  3 , T Ohta  1   4
Affiliations

Functional analysis of thermo-sensitive TRPV1 in an aquatic vertebrate, masu salmon (Oncorhynchus masou ishikawae)

A Yoshimura et al. Biochem Biophys Rep. .

Abstract

Transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in nociceptive primary sensory neurons and acts as a sensor for heat and capsaicin. The functional properties of TRPV1 have been reported to vary among species and, in some cases, the species difference in its thermal sensitivity is likely to be associated with thermal habitat conditions. To clarify the functional properties and physiological roles of TRPV1 in aquatic vertebrates, we examined the temperature and chemical sensitivities of TRPV1 in masu salmon (Oncorhynchus masou ishikawae, Om) belonging to a family of salmonids that generally prefer cool environments. First, behavioral experiments were conducted using a video tracking system. Application of capsaicin, a TRPV1 agonist, induced locomotor activities in juvenile Om. Increasing the ambient temperature also elicited locomotor activity potentiated by capsaicin. RT-PCR revealed TRPV1 expression in gills as well as spinal cord. Next, electrophysiological analyses of OmTRPV1 were performed using a two-electrode voltage-clamp technique with a Xenopus oocyte expression system. Heat stimulation evoked an inward current in heterologously expressed OmTRPV1. In addition, capsaicin produced current responses in OmTRPV1-expressing oocytes, but higher concentrations were needed for its activation compared to the mammalian orthologues. These results indicate that Om senses environmental stimuli (heat and capsaicin) through the activation of TRPV1, and this channel may play important roles in avoiding environments disadvantageous for survival in aquatic vertebrates.

Keywords: Capsaicin; HEPES, N-2-hydroxyethyl-piperazine-2-ethanonesufonic acid; Om, Oncorhynchus masou ishikawae; Salmonids; Species differences; TRPV1, Transient receptor potential vanilloid 1; Thermo-sensor.

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Conflict of interest statement

We have no conflict-of-interest to declare.

Figures

Fig. 1
Fig. 1
Behavioral responses to capsaicin in Oncorhynchus masou ishikawae. (A) Left: Diagram of the behavioral experiment setting. Each Oncorhynchus masou ishikawae (Om) was released into a plastic case filled with water in the presence or absence of capsaicin, and the locomotor activity of Om was monitored using a video camera. At the same time, water temperature was monitored by using a temperature sensor. These data were stored on the hard disk of a PC in conjunction with an AD converter. Right: Changes in water temperature for 15 min in the water case during the analysis. Non-treated water was used as a control (gray line). Capsaicin was added to water at 100 μM (red line). (B) Typical changes in the moving distance of Om in non-treated water (upper: control, gray line) and capsaicin (100 μM)-added water (lower: red line). Moving distances were plotted every 1 s for 15 min. The arrowhead (▼) indicates the unmeasurable point, at which data could not be collected because the fish moved erratically. (C) Summarized effects of capsaicin on the total moving distance for 15 min. Columns with vertical lines show mean ± SEM (control [water alone]: n = 7, capsaicin 10 μM: n = 7, 30 μM: n = 7, 100 μM: n = 7, vehicle, ethanol 0.1%, equivalent to 100 μM capsaicin: n = 5). *, p < 0.05, **, p < 0.01 by one-way ANOVA with the Tukey-Kramer test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Potentiation of behavioral responses to heat by capsaicin in Oncorhynchus masou ishikawae. (A) Typical changes in the moving distance of Om with increasing water temperature (gray line) for 10 min in the absence (upper: control) or presence of 30 μM capsaicin (lower). The arrowhead (▼) shows the unmeasurable point as described in Fig. 1. Note that the onset of swimming induced by heat became earlier in the presence of capsaicin than in its absence. (B) Summarized changes in the moving distance caused by a heat ramp with various concentrations of capsaicin were plotted against temperature. Symbols with vertical lines show mean ± SEM (control [water alone]: n = 7, capsaicin 10 μM: n = 7, 30 μM: n = 7, 100 μM: n = 7). (C) Dose-dependent effects of capsaicin in the threshold temperatures for swimming in Om. Threshold temperatures were defined as temperatures when Om started to swim. Symbols with vertical lines show mean ± SEM. (D) Capsaicin decreases the threshold temperature eliciting behavioral changes. Increasing water temperature induced behavioral changes in the order of “start swimming”, disequilibrium such as “overturn” and “inversion”, and finally “death” of Om. Threshold temperatures were defined as temperatures at which a behavioral change occurred. Columns with vertical lines show mean ± SEM. These behavioral changes were determined by visual observations in addition to the software analysis shown in B. *, p < 0.05, **, p < 0.01 by nonparametric Steel-Dwass multiple-comparison test.
Fig. 3
Fig. 3
TRPV1 expression in the gills and spinal cord in Om. (A) mRNA expression of TRPV1 in gills and spinal cord (SC) of Om. RT-PCR was performed using cDNA obtained from each tissue with [RT(+)] or without [RT(−)] reverse transcriptase. RT-PCR was also carried out using the primer pair of β-actin as a control. (B) Summarized ΔCt values of TRPV1 mRNA in the gills and spinal cord by RT-qPCR analysis (n = 5). β-actin was used as an internal control. Columns with vertical lines show mean ± SEM. NS; Not significant by the unpaired Student's t-test (p = 0.130).
Fig. 4
Fig. 4
Heat sensitivity of OmTRPV1 expressed in X. laevis oocytes. (A, B) Representative current (upper) and temperature (lower) traces for heat stimulation in Xenopus oocytes injected with (A) OmTRPV1 cRNA and (B) distilled water (DW). (C) An Arrhenius plot of the current elicited by heat stimulation in A. (D) Summarized temperature thresholds for the activation by heat stimulation in OmTRPV1. The average temperature threshold for the activation, shown with a line, was 28.2 ± 0.6 °C (n = 25). The box indicates the upper and lower quartile range, and a whisker represents the highest or lowest non-outlier observations estimated using 1.5 times interquartile range. (E) A representative current trace for acid (pH 5.0) and heat stimulations in Xenopus oocytes injected with OmTRPV1 cRNA (n = 5).
Fig. 5
Fig. 5
Capsaicin sensitivity of OmTRPV1 expressed in Xenopus oocytes. (A) A representative current trace for capsaicin (Cap, 30 μM) in Xenopus oocytes injected with OmTRPV1 cRNA. (B) A dose-dependent activation of OmTRPV1 to capsaicin stimulation. The current amplitudes were obtained from Xenopus oocytes at 4 days post OmTRPV1 cRNA injection, and datapoints were fitted with a sigmoidal function to obtain the EC50 value (10.4 μM). The each datapoint represents mean ± SEM (capsaicin 1 μM; n = 4, 3 μM; n = 5, 10 μM; n = 11, 30 μM; n = 11, 100 μM; n = 11). (C) Amino acid sequence alignments of vertebrate TRPV1s related to capsaicin sensitivity within transmembrane domain 3 (TM3) and TM4 are shown. Arrows indicate two amino acids associated with capsaicin sensitivity highlighted in boxes.
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