Involvement of galanin receptors 1 and 2 in the modulation of mouse vagal afferent mechanosensitivity

Abstract

It is established that the gut peptide galanin reduces neuronal excitability via galanin receptor subtypes GALR1 and GALR3 and increases excitability via subtype GALR2. We have previously shown that galanin potently reduces mechanosensitivity in the majority of gastro-oesophageal vagal afferents, and potentiates sensitivity in a minority. These actions may have implications for therapeutic inhibition of gut afferent signalling. Here we investigated which galanin receptors are likely to mediate these effects. We performed quantitative RT-PCR on RNA from vagal (nodose) sensory ganglia, which indicated that all three GALR subtypes were expressed at similar levels. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of galanin receptor ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors which respond only to mucosal stroking. Galanin induced potent inhibition of mechanosensitivity in both types of afferents. This effect was totally lost in mice with targeted deletion of Galr1. The GALR1/2 agonist AR-M961 caused inhibition of mechanosensitivity in Galr1+/+ mice, but this was reversed to potentiation in Galr1-/- mice, indicating a minor role for GALR2 in potentiation of vagal afferents. We observed no functional evidence of GALR3 involvement, despite its expression in nodose ganglia. The current study highlights the complex actions of galanin at different receptor subtypes exhibiting parallels with the function of galanin in other systems.

Figure 1. Transcript expression and relative expression of galanin receptors in whole mouse nodose ganglia

A, RT-PCR products separated on a 3% agarose gel to confirm the presence of GALR1, GALR2 and GALR3 transcripts. The sizes of the amplified products were confirmed showing intense single bands corresponding to the predicted sizes of GALR1, GALR2, GALR3 and β-actin transcripts. In addition, this validated the products obtained during quantitative analysis. B, quantitative RT-PCR revealed there to be no significant difference in transcript expression between galanin receptor subtypes (P > 0.05, Mann–Whitney U test). Experiments were performed at least in triplicate. GALR1, 2 and 3 receptor expression are calculated relative to β-actin mRNA levels.

Figure 2. The effect of galanin on mouse gastro-oesophageal vagal tension receptors

Stimulus–response functions of tension receptors to circumferential tension from Galr1+/+ (A, n = 7) and Galr1−/− (B, n = 13) mice. The responses are before (•) and after exposure to galanin (1 (○), 3 (▪) and 10 nm (□)). ***Significant difference from control using a two-way ANOVA (P < 0.001). C, original recording of a tension receptor response in Galr1+/+ animal to circular tension with a 3 g weight before (Ca), during (Cb) and after washout (Cc) of galanin (10 nm). D, original recording in Galr1−/− animal of a tension receptor response to circular tension with a 3 g weight before (Da) and during (Db) exposure to galanin (10 nm).

Figure 3. The effect of galanin on mouse gastro-oesophageal vagal mucosal receptor

Stimulus–response functions from wild-type (A, n = 7) and Galr1−/− (B, n = 8) mice of mucosal receptors to mucosal stroking with calibrated von Frey hairs (10–1000 mg). The responses are before (•) and after exposure to galanin (1 (○), 3 (▪) and 10 nm (□)). Asterisks indicate significant difference from control using a two-way ANOVA (*P < 0.05, **P < 0.01 and ***P < 0.001). C, original recording of a mucosal receptor response from wild-type mouse to mucosal stroking with a 200 mg von Frey hair before (Ca) and during (Cb) exposure to galanin (10 nm). D, original recording from Galr1−/− mouse of a mucosal receptor response to mucosal stroking with a 200 mg von Frey hair before (Da) and during (Db) exposure to galanin (10 nm).

Figure 4. The effect of galanin and SNAP 37889 on the mechanosensitivity of gastro-oesophageal vagal afferents

Stimulus–response functions of tension (A, n = 5) and mucosal (B, n = 5) receptors to circumferential tension and mucosal stroking. The responses are before (•), after exposure to galanin (○: A, 1 nm; B, 10 nm) and after exposure to galanin and SNAP 37889 (▪: A, 10 nm; B, 100 nm). Asterisks indicate significant difference from control using a two-way ANOVA (**P < 0.01 and ***P < 0.001).

Figure 5. The effect of galanin and AR-M961 on the mechanosensitivity of gastro-oesophageal vagal tension receptors

Stimulus–response functions of tension receptors to circumferential tension from wild-type (A; n = 5) and GalR1−/− (B; n = 6) mice. A, the responses of wild-type tension receptors before (•), after exposure to galanin (○; 100 nm), after washout of galanin (▪), after exposure to AR-M961 (□; 10 nm) and finally after exposure to AR-M961 (▴; 100 nm). B, the responses of Galr1−/− tension receptors before (•) and after exposure to AR-M961 (○; 100 nm). Asterisks indicate significant difference from control using a two-way ANOVA (**P < 0.01 and ***P < 0.001).