Nitric oxide enhances substance P-induced itch-associated responses in mice

Abstract

1 Substance P (SP) elicits itch and itch-associated responses in humans and mice, respectively. In mice, NK(1) tachykinin receptors are involved in SP-induced itch-associated responses, scratching, and mast cells do not play a critical role. The present study was conducted to elucidate the role of nitric oxide (NO) on SP-induced scratching in mice. 2 An intradermal injection of SP (100 nmol site(-1)) elicited scratching in mice, and it was suppressed by an intravenous injection of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), but not by its inactive enantiomer D-NAME. Intradermal injections of L-NAME (100 nmol site(-1)), another NOS inhibitor 7-nitroindazole (10 nmol site(-1)) and the NO scavenger haemoglobin (0.01-10 nmol site(-1)) also inhibited SP-induced scratching. 3 L-NAME (100 nmol site(-1)) did not affect scratching induced by an intradermal injection of 5-hydroxytryptamine (100 nmol site(-1)). 4 Intradermal injections of L-arginine (300 nmol site(-1)) and the NO donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 nmol site(-1)) increased scratching induced by SP. Intradermal injections of L-arginine (1-1000 nmol site(-1)) or NOR3 (1-100 nmol site(-1)) alone were without effects on scratching. 5 Intradermal injections of SP (10-100 nmol site(-1)) increased the intradermal concentration of NO in a dose-dependent manner in mice. An increase in NO levels induced by SP was inhibited by L-NAME and the NK(1) tachykinin receptor antagonist L-668,169, but not by the NK(2) tachykinin receptor antagonist L-659,877. 6 SP (1-10 micro M) elicited NO production in cultured human keratinocytes and the SP-induced NO production was inhibited by L-NAME and L-668,169. 7 We conclude that intradermal SP increases NO in the skin, possibly through the action on NK(1) tachykinin receptors on the epidermal keratinocytes and that NO enhances SP-induced itch-associated responses.

Figure 1

Scratching following an intradermal injection of substance P in mice. Mice were given an intradermal injection of saline or substance P (100 nmol site⁻¹), and the scratching of the injection site by the hind paws was counted at 10-min intervals. Values are the means and s.e.mean for eight animals. Two-way repeated measures analysis of variance, main effect of substance P, F(1,14)=19.83, P<0.001; group×time interaction, F(5,70)=6.26, P<0.0001.

Figure 2

Inhibitory effects of nitric oxide synthase inhibitor and haemoglobin on substance P-induced scratching in mice. Mice were given an intradermal injection of substance P (SP; 100 nmol site⁻¹) and scratching was counted for 1 h. L-NAME (0.1–10 mg kg⁻¹) and the inactive enantiomer D-NAME (10 mg kg⁻¹) were injected intravenously 5 min before SP, and L-arginine (L-Arg; 10 mg kg⁻¹) was injected together with L-NAME. Haemoglobin (Hb; 10 nmol site⁻¹) was injected intradermally together with SP. Values are the means and s.e.mean for eight animals. ^*P <0.05 vs SP control (Dunnett's test).

Figure 3

Inhibitory effects of nitric oxide synthase inhibitor and NK₁ receptor antagonist on substance P-induced scratching. Mice were given an intradermal injection of saline or substance P (100 nmol site⁻¹). L-NAME (100 nmol site⁻¹) and L-668,169 (50 nmol site⁻¹) were injected together with substance P. Values are the means and s.e.mean for eight animals. ^*P<0.05 vs substance P alone (Dunnett's test).

Figure 4

Enhancing effect of L-arginine (L-Arg) on substance P (SP)-induced scratching. L-Arg (300 nmol site⁻¹) and the isomer D-Arg (300 nmol site⁻¹) were injected alone or together with SP (10, 30 nmol site⁻¹). Values are the means and s.e.mean for eight animals. ^*P<0.05 vs SP plus saline control. #P<0.05 vs saline alone (Student-Newman-Keuls test).

Figure 5

Substance P (SP)-induced production of nitric oxide in the mouse skin. Procedures for the determination of cutaneous nitric oxide are described in Methods. (a) Time-course and dose-dependence of nitric oxide production following intradermal SP. SP (10–100 nmol site⁻¹) or saline was injected intradermally into the rostral back. n=5–6. (b) Effects of tachykinin receptor antagonists on SP-induced nitric oxide production. L-668,169 (L-668) and L-659,877 (L-659) were injected intradermally together with SP (100 nmol site⁻¹). n=8. ^*P<0.05 vs SP alone (CN). (c) Effects of L-NAME on SP-induced nitric oxide production. L-NAME and D-NAME were injected intravenously (i.v.) 5 min before or intradermally (i.d.) together with SP (100 nmol site⁻¹). n=6. ^*P<0.05 vs SP alone (CN).

Figure 6

Substance P-induced production of nitric oxide in cultured human keratinocytes. (a) Dose-dependence of nitric oxide production induced by substance P (SP). (b) Suppressive effects of L-668,169 on nitric oxide production induced by SP (10 μM). (c) Suppressive effects of L-NAME on nitric oxide production induced by SP (10 μM). L-668,169, L-NAME and D-NAME were applied together with SP. The concentration of nitric oxide was determined 5 min after SP application. Values are the means and s.e.mean for six samples. ^*P<0.05 when compared with SP alone.