Possible involvement of P2Y2 metabotropic receptors in ATP-induced transient receptor potential vanilloid receptor 1-mediated thermal hypersensitivity

Abstract

The capsaicin receptor transient receptor potential V1 (TRPV1; also known as vanilloid receptor 1) is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that extracellular ATP potentiates the TRPV1 currents evoked by capsaicin or protons and reduces the temperature threshold for its activation through metabotropic P2Y receptors in a PKC-dependent pathway, suggesting that TRPV1 activation could trigger the sensation of pain at normal body temperature in the presence of ATP. Here, we show that ATP-induced thermal hyperalgesia was abolished in mice lacking TRPV1, suggesting the functional interaction between ATP and TRPV1 at a behavioral level. However, thermal hyperalgesia was preserved in P2Y1 receptor-deficient mice. Patch-clamp analyses using mouse dorsal root ganglion neurons indicated the involvement of P2Y2 rather than P2Y1 receptors. Coexpression of TRPV1 mRNA with P2Y2 mRNA, but not P2Y1 mRNA, was determined in the rat lumbar DRG using in situ hybridization histochemistry. These data indicate the importance of metabotropic P2Y2 receptors in nociception through TRPV1.

Figure 1.

TRPV1 is essential for the development of ATP-induced thermal hypersensitivity in vivo, and P2Y receptors are predominantly involved. A, Wild-type (•), TRPV1 ^-/- (○), or P2Y₁^-/- mice (□) were injected intraplantarly with ATP (100 nmol), and the response latency to radiant heating of the hindpaw was measured at various time points after injection. Values are expressed as mean ± SE; n = 6 for each group. ^*p < 0.05 and ^**p < 0.01 versus wild-type and P2Y₁^-/- mice; two-tailed unpaired t test. B, Behavioral analyses in response to αβ meATP (20 nmol) injection similar to A in wild-type (•,▴) or TRPV1 ^-/- mice (○). In some experiments, mice were pretreated with TNP–ATP (50 nmol) (▴). Values are expressed as mean ± SE; n = 6 for each group. ^*p < 0.05 and ^**p < 0.01 versus wild type without TNP–ATP pretreatment; two-tailed unpaired t test. C, Behavioral analyses in response to ATP (100 nmol) injection similar to A in wild-type mice pretreated with TNP–ATP (50 nmol) (▵) or saline (•). Values are expressed as mean ± SE; n = 6 for each group.

Figure 2.

Extracellular ATP or UTP potentiates capsaicin-evoked currents in DRG neurons from wild-type or P2Y₁-deficient mice. A–C, Representative traces of increases in the capsaicin (CAP)-activated currents by extracellular ATP (100 μm) in DRG neurons from wild-type (WT) (B) or P2Y₁-deficient (C) mice. A shows a control trace without ATP pretreatment. Holding potential was -60 mV. D, A representative trace of increases in the capsaicin-activated currents by UTP (100 μm) in DRG neurons from wild-type mice. E, Effects of ATP, UTP, or UTP plus suramin (50 μm) on the capsaicin-activated currents. Currents were normalized to the currents initially evoked by capsaicin (100 nm) in the absence of the additives. Normalized currents in the absence of ATP in wild-type mice, in the presence of ATP in wild-type mice, in the presence of ATP in P2Y₁-deficient mice, in the presence of UTP in wild-type mice, or in the presence of UTP plus suramin in wild-type mice were 1.07 ± 0.26 (n = 3), 4.01 ± 0.92 (n = 8), 4.37 ± 0.74 (n = 4), 6.24±1.58 (n = 4), or 0.95 ± 0.57 (n = 3), respectively. ^*p < 0.05 and ^**p < 0.01 versus absence of ATP in wild type; ^#p < 0.05 versus presence of UTP and suramin in wild type; two-tailed unpaired t test. WT, Wild type.

Figure 3.

P2Y₂ mRNA, but not P2Y₁ mRNA, is coexpressed with TRPV1 mRNA in DRG. A, PCR amplification of P2Y₁ (a) and P2Y₂ (b) cDNA fragments from the RNAs of mouse DRG neurons. The expected sizes of the DNA fragments for mouse P2Y₁ and P2Y₂ are 651 and 781 bp, respectively. N, Negative control. B, Coexpression of TRPV1 mRNA with P2Y₁ and P2Y₂ mRNAs in rat DRG neurons. Double in situ hybridization histochemistry was performed on the sections. b and d are dark-field photomicrographs of a and c, respectively. The TRPV1 mRNA-expressing neurons were hybridized by a DIG-labeled antisense probe and visualized as DAB staining (brown cells in a and c). The P2Y₁ mRNA-expressing (b) and P2Y₂ mRNA-expressing (d) neurons were hybridized by the appropriate ³⁵S-labeled antisense probe and visualized as clusters of silver grains. p, Positively labeled neurons. Arrowheads indicate P2Y₁ mRNA-expressing neurons that do not express TRPV1 mRNA, whereas arrows indicate the neurons expressing both TRPV1 and P2Y₂ mRNAs.

Figure 4.

UTP induces thermal hyperalgesia in mice. Behavioral analyses in response to UTP (100 nmol) injection similar to Figure 1 in wild-type mice pretreated with TNP–ATP (50 nmol) (□) or saline (•). Values are expressed as mean ± SE; n = 6 for each group.