Cutaneous TRPV4 Channels Activate Warmth-Defense Responses in Young and Adult Birds

Abstract

Transient receptor potential vanilloid 4 (TRPV4) channels are sensitive to warm ambient temperatures (T_as), triggering heat loss responses in adult rats in a T_as range of ∼26-30°C. In birds, however, the thermoregulatory role of TRPV4 has never been shown. Here, we hypothesized that stimulation of TRPV4 induces thermolytic responses for body temperature (T_b) maintenance in birds, and that this function is already present in early life, when the T_a range for TRPV4 activation does not represent a warm condition for these animals. We first demonstrated the presence of TRPV4 in the dorsal and ventral skin of chickens (Gallus gallus domesticus) by immunohistochemistry. Then, we evaluated the effects of the TRPV4 agonist, RN1747, and the TRPV4 antagonists, HC067047 and GSK2193874, on T_b and thermoeffectors at different T_as in 5-day-old chicks and 60-day-old adult chickens. For the chicks, RN1747 transiently reduced T_b both in thermoneutrality (31°C) and in a cold T_a for this phase (26°C), which relied on huddling behavior inhibition. The TRPV4 antagonists alone did not affect T_b or thermoeffectors but blocked the T_b decrease and huddling inhibition promoted by RN1747. For the adults, TRPV4 antagonism increased T_b when animals were exposed to 28°C (suprathermoneutral condition for adults), but not to 19°C. In contrast, RN1747 decreased T_b by reducing metabolic rate and activating thermal tachypnea at 19°C, a T_a below the activation range of TRPV4. Our results indicate that peripheral TRPV4 receptors are functional in early life, but may be inhibited at that time when the range of activation (∼26-30°C) represents cold T_a for chicks, and become physiologically relevant for T_b maintenance when the activation T_a range for TRPV4 becomes suprathermoneutral for adult chickens.

Keywords: Metabolism; body temperature; chicken; huddling; peripheral thermoreceptor; thermolysis; thermoregulation.

FIGURE 1

Representative photomicrographs of immunostaining for TRPV4 in the skin of adult chickens: ventral (A) and dorsal (B) cut of the skin; negative control without the primary antibody (C). TRPV4 is seen as a green staining. Arrow: Stratum corneum; 1: Epidermis; 2: Dermis; 3: Muscle.

FIGURE 2

(A,B) Effect of pre-treatment with the TRPV4 antagonist HC067047 (intramuscular injection, i.m.; 50 μg kg⁻¹; vehicle is 90% saline +10% ethanol; 1 ml kg⁻¹) on the body temperature change induced by the TRPV4 agonist RN1747 (topical application on the dorsal skin; 0.5 mg ml⁻¹; vehicle is 100% propyleneglycol; 3.75 ml kg⁻¹) in 5-day-old chicks under thermoneutral condition (T_a = 31°C; A) or exposed to cold (T_a = 26°C; B). Tbi, initial body temperature. C) Influence of the pre-treatment with HC067047 on the effect of RN1747 on the huddling behavior induced by cold exposure (T_a = 26°C) in 5-day-old chicks, as assessed by the number of single chicks (top plot) and the area occupied by the group of five chicks (bottom plot). D) Representative images of each treatment showing the area occupied by the animals at the time 180 min from the graphs in C (2 hours after the agonist application). All values are expressed as mean ± SEM. The continuous arrow indicates the moment of the antagonist injection and the dashed arrow indicates the time of the topical application of the agonist. The number of animals in each group is shown in parentheses. *p < 0.05, significant difference from the vehicle group at the same time point (two-way ANOVA; Sidak post-hoc test).

FIGURE 3

Effect of pre-treatment with the TRPV4 antagonist, GSK2193874 (intramuscular injection, i.m.; 50 μg kg⁻¹; vehicle is DMSO 1%; 1  ml kg⁻¹), on change in body temperature (delta; A), oxygen consumption ( $\dot{\mathrm{V}}$ O₂; B), ventilation ( $\dot{\mathrm{V}}$ _E; C), tidal volume (V_T; D) and breathing frequency (f; E) induced by treatment with the TRPV4 agonist, RN1747 (topical application on the dorsal skin; 0.5 mg ml⁻¹; vehicle is 100% propylene glycol; 3.75 ml kg⁻¹), in 5-day-old chicks under thermoneutral condition (T_a = 31°C). T_bi, initial body temperature. All values are expressed as mean ± SEM. The continuous arrow indicates the moment of the antagonist injection and the dashed arrow indicates the time of the topical application of the agonist. The number of animals in each group is shown in parentheses. *p < 0.05, significant difference from the vehicle group at the same time point (two-way ANOVA; Sidak post-hoc test).

FIGURE 4

Effect of intramuscular injection (i.m.; 1 ml kg⁻¹) of the TRPV4 antagonist, GSK2193874 (100 and 500 μg kg⁻¹), or vehicle (1% DMSO) on body temperature (T_b; A), oxygen consumption ( $\dot{\mathrm{V}}$ O₂; B), ventilation ( $\dot{\mathrm{V}}$ _E; C), tidal volume (V_T; D) and breathing frequency (f; E) of 60-day-old chickens under warm condition (T_a = 28°C; graphs in the left). Effect of the i. m. injection of GSK2193874 (500 μg kg⁻¹) or 1% DMSO (1 ml kg⁻¹) on the same respective variables in 60-day-old chicken under mild cold condition (T_a = 19°C; graphs on the right). Tbi, initial body temperature. The arrow indicates the moment of injection. Number of animals in each group is shown in parentheses. All the animals were pre-exposed to a hot condition (30°C) for inducing cutaneous vasodilation and facilitate the distribution of drugs throughout the peripheral sites of receptors (Almeida et al., 2012). *p < 0.05, significant difference from the vehicle group at the same time point (two-way ANOVA; Sidak post-hoc test).

FIGURE 5

Effect of topical application (both dorsal and the ventral skin surfaces; 3.75 ml kg⁻¹; ∼6 ml each side) of the TRPV4 agonist, RN1747 (0.5 mg ml⁻¹), or vehicle (100% propylene glycol) on body temperature (T_b; A), oxygen consumption ( $\dot{\mathrm{V}}$ O₂; B), ventilation ( $\dot{\mathrm{V}}$ _E; C), tidal volume (V_T; D) and breathing frequency (f; E) in 60-day-old chickens under mild cold (T_a = 19°C). T_bi, initial body temperature. The arrow indicates the moment of application. The number of animals in each group is shown in parentheses. All the animals were pre-exposed to a hot condition (30°C) for inducing cutaneous vasodilation and facilitate the distribution of drugs throughout the peripheral sites of receptors (Almeida et al., 2012). *p < 0.05, significant difference from the vehicle group at the same time point (two-way ANOVA; Sidak post-hoc test).

FIGURE 6

The cartoon illustrates the thermoregulatory effects of stimulation of cutaneous TRPV4 in chicken at different ages, 5 and 60 days old. In 5-day-old chicks, chemical stimulation of the TRPV4 receptors induces hypothermia, and specific antagonists inhibit this response. At this age, 30–32°C is considered thermoneutral, while 26°C or less and 35°C or more are below and above thermoneutrality, respectively. In 60-day-old chickens (adults), thermal stimulation (warm exposure; ∼28°C) of the TRPV4 receptors avoids hyperthermia. Moreover, at this age, chemical stimulation of TRPV4, even in cold condition (below 20°C), activates heat loss (warmth-defense) responses.