P2Y2 receptors mediate ATP-induced resensitization of TRPV1 expressed by kidney projecting sensory neurons

Abstract

The transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated cation channel expressed by sensory nerves. P2Y receptors are G protein-coupled receptors that are also expressed by TRPV1-positive sensory neurons. Therefore, we studied interactions between P2Y receptors and TRPV1 function on kidney projecting sensory neurons. Application of Fast Blue (FB) to nerves surrounding the renal artery retrogradely labeled neurons in dorsal root ganglia of rats. Whole cell recording was performed on FB-labeled neurons maintained in primary culture. Capsaicin was used to activate TRPV1. Four types of kidney projecting neurons were identified based on capsaicin responses: 1) desensitizing (35%), 2) nondesensitizing (29%), 3) silent (3%), and 4) insensitive (30%). Silent neurons responded to capsaicin only after ATP (100 microM) pretreatment. ATP reversed desensitization in desensitizing neurons. Insensitive neurons never responded to capsaicin. UTP, a P2Y purinoceptor 2 (P2Y(2))/P2Y(4) receptor agonist, reversed capsaicin-induced TRPV1 desensitization. 2-methyl-thio-ATP (2-Me-S-ATP), a P2Y(1) receptor agonist, did not change desensitization. MRS 2179 and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), drugs that block P2Y(1) receptors, did not block ATP-induced resensitization of TRPV1. Suramin, a P2Y(2) receptor antagonist, blocked resensitization caused by UTP. Immunocytochemical studies showed that FB-labeled neurons coexpressed P2Y(2) receptors and TRPV1. We conclude that P2Y(2) receptor activation can maintain TRPV1 function perhaps during sustained episodes of activity of kidney projecting sensory neurons.

Fig. 1.

Isolated dorsal root ganglion (DRG) neurons retrogradely labeled by Fast Blue (FB). A: DRG neurons under bright field illumination 1 wk after FB application. B: FB-labeled DRG neurons revealed using UV epifluorescence illumination, same field as A. Arrows indicate FB-labeled neurons. C: overlay of A and B.

Fig. 2.

Colocalization of transient receptor potential vanilloid 1 (TRPV1) and CGRP on kidney projecting sensory neurons using fluorescence microscopy. A: kidney projecting sensory neuron labeled by FB (blue). B: TRPV1- immunostaining (green) on the same neuron. C: CGRP-immunostaining (red) on the same neuron. D: overlay picture combining A, B, and C.

Fig. 3.

Four types of kidney projecting sensory neurons based on capsaicin reactivity. Traces are representative recordings obtained from each type of neuron. A: repeated applications of capsaicin (CAP; 1 μM) cause TRPV1 desensitization. B: in the second type of neuron, TRPV1 is not desensitized by repeated applications of capsaicin. C: in the third type of neuron, capsaicin does not induce a TRPV1 response until after pretreatment with ATP (100 μM). D: capsaicin does not cause a TRPV1 response, even after ATP pretreatment. Responses are representative of recordings obtained from 189 kidney projecting sensory neurons.

Fig. 4.

Concentration response curves for capsaicin-induced activation of TRPV1 on kidney projecting sensory neurons. A: concentration-response curve in neurons (n = 16 neurons from 9 rats) exhibiting nondesensitizing TRPV1 responses. B: concentration response obtained from neurons (n = 9 neurons from 7 rats), exhibiting desensitizing TRPV1 responses. Data are expressed as means ± SE and are currents normalized to the maximum response obtained in each neuron.

Fig. 5.

Capsaicin-induced TRPV1 desensitization and reversal caused by ATP on kidney projecting sensory neurons. A: representative trace shows that repeated capsaicin applications induce TRPV1 desensitization. B: mean data of experiments illustrated in A. Data are expressed as means ± SE; n = 6 neurons from 4 rats. C: representative trace shows that ATP (100 μM) reversed capsaicin-induced TRPV1 desensitization. D: mean data of experiments illustrated in C. Data are expressed as means ± SE; n = 7 neurons from 6 rats. CAP1, CAP2, and CAP3 represent the sequence of capsaicin applications *P < 0.01 vs. first capsaicin application. #P < 0.01 vs. second capsaicin application.

Fig. 6.

Colocalization of TRPV1 and P2Y receptor subtypes on kidney projecting sensory neurons. A, E: kidney projecting sensory neurons labeled by FB. B, F: TRPV1-immunostaining in the same neurons shown in A and E. C: P2Y₁ receptor immunostaining in the same neuron shown in A and B. G: P2Y₂ receptor immunostaining in the same neuron shown in E and F. D, H: Overlay pictures.

Fig. 7.

ATP-induced TRPV1 resensitization is mediated by P2Y₂ receptors on kidney projecting sensory neurons. A: representative recordings showing that the P2Y₁ receptor agonist, 2-Me-S-ATP (100 μM) does not reverse capsaicin-induced TRPV1 desensitization. B: mean data of experiments illustrated in A. Data are expressed as means ± SE; n = 6 neurons from 4 rats. C: representative trace shows that UTP (50 μM) reverses capsaicin-induced TRPV1 desensitization. D: mean data of experiments illustrated in C. Data are expressed as means ± SE; n = 6 neurons from 6 rats. *P < 0.01 vs. first application of capsaicin. #P < 0.01 vs. second application of capsaicin.

Fig. 8.

ATP-induced TRPV1 resensitization is not blocked by P2Y₁ receptor or non-P2Y₂ receptor antagonist. A: representative recordings showing that the P2Y₁ receptor antagonist, MRS 2179 (10 μM), does not block ATP-induced TRPV1 resensitization. B: mean data of experiments illustrated in A. Data are expressed as means ± SE; n = 6 neurons from 3 rats. C: representative trace shows that pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) (10 μM) does not block ATP-induced TRPV1 resensitization. D: mean data of experiments illustrated in C. Data are expressed as means ± SE; n = 4 neurons from 2 rats. *P < 0.01 vs. first application of capsaicin. #P < 0.01 vs. second application of capsaicin.

Fig. 9.

Suramin (SUR), a P2Y₂ receptor antagonist, blocks UTP-induced resensitization of TRPV1. A: representative recording showing that suramin (30 μM) blocks the resensitization of TRPV1 caused by UTP. B: mean data of experiments illustrated in A. Data are expressed as means ± SE; n = 6 neurons from 5 rats. *P < 0.01 vs. first application of capsaicin.