TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase

Abstract

Histamine provokes itching and is a major skin disease complaint. Histamine is known to excite a subset of sensory neurons, predominantly C-fibers. Although histamine is pruritogenic, its signaling pathways that excite sensory neurons have not been identified. Because the metabolic products of lipoxygenases (LOs) activate transient receptor potential vanilloid receptor-1 (TRPV1) in sensory neurons, we hypothesized that histamine excites sensory neurons by activating TRPV1 via phospholipase A2 (PLA2) and LO stimulation. In cultured sensory neurons, histamine evoked inward currents that were reduced by capsazepine, a TRPV1 blocker. Moreover, histamine provoked inward currents when histamine receptor subtype 1 (H1R) and TRPV1 were expressed heterologously, but not when H1R or TRPV1 was expressed alone. In addition, histamine caused Ca2+ influxes in sensory neurons in wild-type mice but not in TRPV1-/- mice. Furthermore, histamine caused a 2.5-fold increase in the production of 12-hydroxyeicosatetraenoic acid, a metabolite of LO, in cultured sensory neurons. When injected subcutaneously into the necks of mice, histamine caused bouts of scratching, which were greatly reduced by pretreatment with capsazepine, a TRPV1 blocker, and by inhibitors of PLA2, LO, and H1R. Furthermore, mice lacking TRPV1 markedly reduced histamine-induced scratching compared with wild type. Together, these results indicate that TRPV1 plays a key role in mediating the pruritogenic action of histamine via the PLA2/LO pathway.

Figure 1.

Histamine induced currents in DRG neurons and in HEK 293T cells transfected with TRPV1 and H1R that were blocked by inhibitors or blockers of PLA₂, LO, TRPV1, and H1R. A, In rat neonatal DRG neurons, application of 100 μm histamine (His) evoked an inward current that was blocked by treatment with a TRPV1 antagonist, capsazepine (CPZ; 10 μm). Holding potential was −60 mV. B, HEK293T cells were transfected with either H1R or TRPV1 alone or transfected with H1R and TRPV1. A current response to 10 μm histamine was observed only when cells were transfected with both H1R and TRPV1 (bottom). Application of 1 μm capsaicin (Cap) evoked inward currents when cells were transfected with TRPV1 alone (middle) or with H1R (bottom). C, Trace examples of the effects of inhibitors of PLA₂ [quinacrine (Qnc)] or LO (NDGA) on histamine-induced currents in HEK cells transfected with H1R and TRPV1. In HEK cells expressing H1R and TRPV1, a second histamine challenge induced a clear tachyphylaxis. Pretreatment with NDGA or quinacrine remarkably abolished this second histamine-evoked influx (middle and bottom). D, Summary of effects of NDGA (n = 9), quinacrine (n = 9), mepyramine (H1R antagonist; n = 7), and capsazepine (n = 7) on current responses to a second histamine challenge. Error bars indicate SEMs. *p < 0.001.

Figure 2.

A, B, Histamine (His; 100 μm) challenge elicited increased intracellular Ca²⁺ in DRG neurons of wild-type mice (A) but not in DRG neurons of TRPV1^−/− mice (B). C, Summary of Ca²⁺ influx induced by histamine application in cultured sensory neurons isolated from wild-type (WT; n = 75) or TRPV1^−/− (n = 80) mice. F₀ denotes initial fluorescence detected at time 0. D, mRNA levels of H1R, cytosolic PLA₂ (PLA₂), 12-LO, and TRPV1 in DRG cells isolated from TRPV1^−/− mice. Notice that no significant differences in mRNA levels were found except for TRPV1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Error bars indicate SEMs. Con, Control.

Figure 3.

Application of histamine (His) increased the levels of 12(S)-HETE in primary cultured DRG cells from wild-type (WT) C57BL/6J as well as TRPV1^−/− (filled bars) mice, as determined by immunoassay. Noticeable increase in 12(S)-HETE levels were observed after 10 μm histamine application versus nontreated controls in DRG cells of both types of mice (n = 6). This increase was blocked when 10 μm NDGA was cotreated with histamine (n = 6). Error bars indicate SEMs. Con, Control.

Figure 4.

Intradermal histamine injection in mice induced scratching, which is attenuated when factors in histamine signaling pathway are hampered. A, Dose–response curve of histamine-induced scratching in ICR mouse. Histamine (0.4 ∼ 100 nmol in 200 μl; filled squares; n = 5 ∼ 9) or saline (open diamond; n = 8) was injected to the dorsum of the neck intradermally of ICR mice. Bouts of scratching with hind limbs were counted for 20 min. B, Effects of blockers or inhibitors of the proposed histamine signaling pathway on histamine-induced scratching. Various blockers or inhibitors were first treated intraperitoneally 30 min before the histamine (8 nmol) injection (n = 7 ∼ 8). C, Intradermal histamine (His) induced scratching in wild-type C57BL/6J (n = 8) or TRPV1^−/− mice (n = 12) compared with saline injections (n = 5). Notice that TRPV1^−/− mice showed reduced histamine-induced scratching compared with the wild-type controls. *p < 0.001. Error bars indicate SEMs. Con, Control.