TRPV1 function in mouse colon sensory neurons is enhanced by metabotropic 5-hydroxytryptamine receptor activation

Abstract

Using whole-cell patch-clamp methods, we examined the hypothesis that serotonin [5-hydroxytryptamine (5-HT)] receptor activation enhances TRPV1 function in mouse colon sensory neurons in lumbosacral dorsal root ganglia, which were identified by retrograde labeling with DiI (1,1'-dioctadecyl-3,3,3',3-tetramethlindocarbocyanine methanesulfonate) injected into multiple sites in the wall of the descending colon. 5-HT increased membrane excitability at a temperature below body temperature in response to thermal ramp stimuli in colon sensory neurons from wild-type mice, but not from TRPV1 knock-out mice. 5-HT significantly enhanced capsaicin-, heat-, and proton-evoked currents with an EC50 value of 2.2 microm. 5-HT (1 microm) significantly increased capsaicin-evoked (100 nm) and proton-evoked (pH 5.5) currents 1.6- and 4.7-fold, respectively, and significantly decreased the threshold temperature for heat current activation from 42 to 38 degrees C. The enhancement of TRPV1 by 5-HT was significantly attenuated by selective 5-HT2 and 5-HT4 receptor antagonists, but not by a 5-HT3 receptor antagonist. In support, 5-HT2 and 5-HT4 receptor agonists mimicked the facilitating effects of 5-HT on TRPV1 function. Downstream signaling required G-protein activation and phosphorylation as intracellularly administered GDP-beta-S [guanosine 5'-O-(2-thiodiphosphate], protein kinase A inhibitors, and an A-kinase anchoring protein inhibitor significantly blocked serotonergic facilitation of TRPV1 function; 5-HT2 receptor-mediated facilitation was also inhibited by a PKC inhibitor. We conclude that the facilitation of TRPV1 by metabotropic 5-HT receptor activation may contribute to hypersensitivity of primary afferent neurons in irritable bowel syndrome patients.

Figure 1.

Temperature-dependent excitability of colon sensory neuron from wild-type and TRPV1^-/- mice. a, Representative responses in current-clamp recording mode to a temperature ramp. Thermal stimuli evoked depolarization and action potentials. b, c, Representative records from wild-type (b) and TRPV1^-/- (c) neurons at different temperatures show action potential generation at temperatures above 42°C in wild-type neurons and above 50°C in TRPV1^-/- neurons. d, Mean ± SEM action potential frequency of colon afferent neurons in response at 45°C in wild-type (black bar; n = 8) and TRPV1^-/- (gray bar; n = 6; t = 3.49; ^**p < 0.01) mice.

Figure 2.

Characteristics of TRPV1 function in colon afferent neurons. a, Representative records of heat-evoked (I_heat) and capsaicin-evoked (CAP; 10 μm; filled circle) currents. The right panel shows the threshold temperature for heat activation using heat ramps as illustrated in the left panel (see Materials and Methods for details). b, Repeat application of capsaicin (CAP; 1 μm; 5 sec, 1 min interval; n = 6; F = 50.11; p < 0.001) and acid (H⁺; pH 4.5; n = 5; F = 2.96; p < 0.05) in the presence of extracellular calcium are associated with significant desensitization of current responses as shown in samples tracings (left); data are summarized in a graph (right). Acid-evoked currents did not desensitize significantly in calcium-free solutions (-Ca²⁺). c, Representative records of acid-evoked currents, pH 4.5, in the presence of vehicle, capsazepine (CZP; 10 μm), or amilorlide (AMI; 200 μm). Data are summarized for vehicle (n = 6), capsazepine (n = 9; t = 5.81; ^**p < 0.01), and amiloride (n = 9; t = 0.93; not significant) in d and e, respectively. f, Representative records of acid-evoked sustained currents in wild-type (left) and TRPV1^-/- (middle) mice. None of the four TRPV1^-/- neurons tested responded to capsaicin (CAP; bottom record; middle). Data for current responses to different proton concentrations are summarized in the right panel for wild-type mice (filled circles; n = 8) and TRPV1^-/- mice (unfilled circles; n = 11).

Figure 3.

Immunohistochemical evidence of TRPV1expression in colon DRG neurons. a, The left panel shows a DiI-labeled neuron visualized with fluorescent light at an excitation wavelength of 546 nm. When excited with fluorescent light with a wavelength of 488 nm, two cells with TRPV1-like IR can be seen in the same field (middle). The merged image in the right panel demonstrates the colocalization of the retrograde label DiI and TRPV1-like IR in one neuron marked by the arrow. TRPV1-like IR (b) was not detected in the absence of the primary antibody [1 °Ab(-); c] or after preincubation of the primary antibody with the blocking peptide [BP(+); d]. Scale bar, 30 μm.

Figure 4.

5-HT enhances excitability of a colon sensory neuron in response to thermal stimulation. Representative records from wild-type (a) and TRPV1^-/- (b) neurons at different temperatures in the presence of 5-HT (1 μm pretreatment for 2 min) are shown. Action potentials in neurons from wild-type mice were generated at significantly lower temperatures (36°C) in the presence of 5-HT (c; compare with Fig. 1b). In neurons from TRPV1^-/- mice, action potentials were generated only at temperatures >45°C in the presence of 5-HT. Temperature-dependent action potential firing after exposure to vehicle (unfilled symbols) or 1 μm 5-HT (filled symbols) is summarized for wild-type (c) and TRPV1^-/- mice (d). Compared with vehicle, 5-HT significantly shifted the response to temperature in neurons from wild-type mice (5-HT, n = 8; vehicle, n = 8; F = 19.40; p < 0.001). The dotted vertical line in c indicates the normal core temperature of wild-type mice.

Figure 5.

5-HT enhances capsaicin-, heat-, and proton-evoked currents in colon sensory neurons. a, Representative records of the current evoked by capsaicin (100 nm) after exposure to 5-HT (1 μm; left). Summary data for the second response to capsaicin, normalized to the initial response, are shown to the right (filled bar: 1 μm 5-HT, n = 7; unfilled bar: vehicle, n = 6; t = 2.46; ^*p < 0.05). b, Representative heat-evoked current after 2 min pretreatment with 5-HT (1 μm). The middle record shows the threshold temperature determined in this neuron; data are summarized in the right panel for 5-HT (filled bar; n = 7) and vehicle (unfilled bar; n = 6; t = 2.50; ^*p < 0.05, 5-HT vs vehicle). The dotted vertical line indicates the normal core temperature of wild-type mice. c, Representative records of acid-evoked currents before and after exposure to 5-HT (1 μm). Compared with exposure to vehicle (right; unfilled bar; n = 8), the TRPV1 antagonist capsazepine (CPZ; 10 μm; filled bar) significantly attenuated serotonergic facilitation of the acid-evoked current (n = 3; t = 6.86; ^***p < 0.001). d, The normalized current in response to protons, pH 5.5, is plotted as a function of 5-HT concentration before the second acid stimulus and fitted to the Hill equation (data from 47 cells). MDL72222 (3 μm) was added in experiments with 10 μm of 5-HT. e, The time course of changes in the acid-evoked current after 1 min exposure to 5-HT (1 μm; filled circles; n = 7) or vehicle (unfilled circles; n = 7) (^*p < 0.05). f, pH dependence of the acid-evoked current after 1 min exposure to 5-HT (1 μm; filled circles; n = 7) or vehicle (unfilled circles; n = 8). The data were fitted to the Hill equation; the difference in pH₅₀ between the groups was significant (t = 5.06; p < 0.001).

Figure 6.

5-HT₃ channel is activated by 5-HT in colon afferent neurons but does not contribute to serotonergic facilitation of TRPV1 function. a, Representative currents activated by increasing 5-HT concentrations (0.1, 1, 3, and 10 μm). The data were normalized to cell capacitance and are summarized in the right panel (0.1 μm, n = 6; 1 μm, n = 9; 3 μm, n = 8; 10 μm, n = 8). b, The selective 5-HT₃ antagonist MDL-72222 (600 nm) reversibly blocked the inward current triggered by 10 μm 5-HT (n = 6; t = 25.3; p < 0.001). c, The 5-HT₃ antagonist MDL7222 did not block facilitation by 5-HT (1 μm) of the acid-evoked current. The data summarized to the right for vehicle (unfilled bar; n = 11) and MDL72222 (filled bar; n = 8; t =-0.52; not significant) treatments are shown.

Figure 7.

Metabotropic 5-HT receptors are involved in serotonergic facilitation of TRPV1 activation in colon afferent neurons. a, Representative records of 5-HT-produced facilitation of acid-evoked currents before (cont) and after treatment with selective 5-HT₂ or 5-HT₄ receptor antagonists. Ketanserin (KET; 10 μm), SB204070 (10 μm), or GR113808 (10 μm) were added 5 sec before application of 5-HT (1 μm). b, Representative records of acid-elicited currents before (cont) and after treatment with vehicle or selective 5-HT₂ or 5-HT₄ receptor agonists. DOI (1 μm), αMe-5-HT (1 μm), cisapride (1 μm), or 5-MeOT (1 μm) were given for 50 sec before the second acid stimulation. The data for antagonists (c) and agonists (d) are summarized as responses normalized to the amplitude of the initial proton-evoked current [n = 6-11; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs 5-HT (c) or vehicle (d); 1-way ANOVA with Bonferroni t test].

Figure 8.

Intracellular signal transduction involved in serotonergic facilitation of TRPV1 activation in colon afferent neurons. a, Representative records of acid-elicited current before (cont) and after treatment with 5-HT in the absence or presence of inhibitors of intracellular signaling pathways. GDP-βS (GDPβS; 0.5 mm), H-89 (1 μm), protein kinase inhibitor 6-22 amide (PKI_6-22; 5 μm), or calphostin C (CalpC; 1 μm) was added to the intracellular solution. b, c, Representative records of acid-evoked currents before (cont) and after treatment with DOI (b) or 5-MeOT (c) in the absence or presence of intracellular pathway inhibitors. PKI_6-22 (5 μm), CalpC (1 μm), or AKAP inhibitor St-Ht31 (5 μm) was added to the intracellular solution. The data are summarized for effects on 5-HT (d), DOI (e), and 5-MeOT (f) (n = 6-11; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001; 1-way ANOVA with Bonferroni t test).