Involvement of tachykinin receptors in Clostridium perfringens beta-toxin-induced plasma extravasation

Abstract

1 Clostridium perfringens beta-toxin causes dermonecrosis and oedema in the dorsal skin of animals. In the present study, we investigated the mechanisms of oedema induced by the toxin. 2 The toxin induced plasma extravasation in the dorsal skin of Balb/c mice. 3 The extravasation was significantly inhibited by diphenhydramine, a histamine 1 receptor antagonist. However, the toxin did not cause the release of histamine from mouse mastocytoma cells. 4 Tachykinin NK(1) receptor antagonists, [D-Pro(2), D-Trp(7,9)]-SP, [D-Pro(4), D-Trp(7,9)]-SP and spantide, inhibited the toxin-induced leakage in a dose-dependent manner. Furthermore, the non-peptide tachykinin NK(1) receptor antagonist, SR140333, markedly inhibited the toxin-induced leakage. 5 The leakage induced by the toxin was markedly reduced in capsaicin-pretreated mouse skin but the leakage was not affected by systemic pretreatment with a calcitonin gene-related peptide receptor antagonist (CGRP(8-37)). 6 The toxin-induced leakage was significantly inhibited by the N-type Ca(2+) channel blocker, omega-conotoxin MVIIA, and the bradykinin B(2) receptor antagonist, HOE140 (D-Arg-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-bradykinin), but was not affected by the selective L-type Ca(2+) channel blocker, verapamil, the P-type Ca(2+) channel blocker, omega-agatoxin IVA, tetrodotoxin (TTX), the TTX-resistant Na(+) channel blocker, carbamazepine, or the sensory nerve conduction blocker, lignocaine. 7 These results suggest that plasma extravasation induced by beta-toxin in mouse skin is mediated via a mechanism involving tachykinin NK(1) receptors.

Figure 1

Local plasma extravasation induced by beta-toxin in mouse dorsal skin. (A) Dose-dependence of beta-toxin-induced plasma extravasation. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected into the tail vein. After 5 min, the beta-toxin (5–100 ng) was injected i.d. (50 μl site⁻¹). Plasma extravasation was measured 60 min after the injection of beta-toxin. (B) Time-course of beta-toxin-induced plasma extravasation. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected in the tail vein. After 5 min, the beta-toxin (20 ng site⁻¹) was injected i.d. (50 μl site⁻¹). Plasma extravasation was measured various time after the injection of beta-toxin. Values are the mean±s.e.mean, n=6.

Figure 2

Effect of beta-toxin on mouse dermal tissue. Saline (A) or beta-toxin (50 ng site⁻¹) (B) was injected i.d. into the dorsal skin of mice. After 12 h, dermal tissues from the dorsal skin were fixed in formalin and sections were stained with haematoxylin and eosin.

Figure 3

Effect of diphenhydramine on plasma extravasation induced by beta-toxin or histamine in dorsal skin of mice. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected into the tail vein. After 5 min, beta-toxin (50 ng site⁻¹) or histamine (5 μg site⁻¹) and diphenhydramine (0.1 or 0.5 μg site⁻¹) were simultaneously injected i.d. into the dorsal skin of mice. Plasma extravasation was measured 60 min after the injection of beta-toxin. Values are the mean±s.e.mean, n=6. ^*P<0.01, compared with control,^**P<0.001, compared with saline.

Figure 4

Effect of tachykinin NK₁ receptor antagonists on plasma extravasation induced by beta-toxin in dorsal skin of mice. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected into the tail vein. After 5 min, pretreatments with various amounts of spantide and [D-Pro², D-Trp^{7, 9}]SP were performed 1 min before beta-toxin (50 ng site⁻¹) or septide (1 nmol site⁻¹) challenge. Plasma extravasation was measured 60 min after the injection of beta-toxin. Values are the mean±s.e.mean, n=6. ^*P<0.05, compared with control, ^**P<0.01, compared with control.

Figure 5

Effect of SR140333 treatment on plasma extravasation induced by beta-toxin in dorsal skin of mice. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected into the tail vein. Various doses of SR140333 were given as pretreatments i.d. or i.v. 5 min before i.d. injection of toxin. Beta-toxin (50 ng site⁻¹) and septide (1 nmole site⁻¹) were injected i.d.. Plasma extravasation was measured 60 min after the injection of beta-toxin. Values are the mean±s.e.mean, n=6. ^*P<0.05, compared with vehicle, ^**P<0.01, compared with saline.

Figure 6

Effect of capsaicin on plasma extravasation induced by beta-toxin in dorsal skin of mice. After the dorsal skin was shaved, capsaicin solution (20 μg ml⁻¹ in 10% ethanol solution containing 10% Tween 80) was painted twice a day for 4 days. As a control, the diluent alone was applied to the skin. A mixture of ¹²⁵I-BSA and Evans blue dye (0.1 ml of 2.5% solution) was injected into the tail vein. Afer 5 min, beta-toxin (50 μg site⁻¹) (A) and histamine (5 μg site⁻¹), compound 48/80 (20 μg site⁻¹) or septide (1 nmole site⁻¹) (B) were injected intradermally into the skin. Plasma extravasation was measured 60 min after the injection of beta-toxin or agents. Values are the mean±s.e.mean, n=6. ^*P<0.01, compared with vehicle.