Subcutaneous tissue and in perimysium internum 12 h after the injection. These final results suggested that betatoxin possessed two eects, indicating that the toxin induced early oedema formation and late necrosis in skin. The toxininduced extravasation was lowered by coinjection with diphenhydramine, a TMCB In Vitro histamine 1 receptor antagonist (Simons et al., 2001; Ishida et al., 2000) (0.1 mg site71, P50.01; 0.five mg site71, P50.001) inside a dosedependent manner, but was not completely diminished (Figure 3). The plasma extravasation induced by histamine was signi antly lowered by coinjection of diphenhydramine (Figure 3). It consequently is probably that the toxininduced plasma leakage is entirely associated with histamine release. To analyse the eect with the toxin on mast cells, mouse mastocytoma P815 cells (56108) had been treated together with the toxin (300 mg ml71) or compound 48/80 (50 mg ml71) for 30 min, plus the histamine in the supernatant was measured. The percentage of histamine release within the cells was as follows: PBS (car), four.51.eight ; betatoxin, five.12.2 ; compound 48/80, 72.56.eight (means.e.imply,Figure 1 Regional plasma extravasation induced by betatoxin in mouse N-Hydroxysulfosuccinimide In Vivo dorsal skin. (A) Dosedependence of betatoxininduced plasma extravasation. A mixture of 125IBSA and Evans blue dye (0.1 ml of two.five solution) was injected into the tail vein. Right after 5 min, the betatoxin (5 100 ng) was injected i.d. (50 ml site71). Plasma extravasation was measured 60 min after the injection of betatoxin. (B) Timecourse of betatoxininduced plasma extravasation. A mixture of 125IBSA and Evans blue dye (0.1 ml of 2.five option) was injected inside the tail vein. Following 5 min, the betatoxin (20 ng site71) was injected i.d. (50 ml site71). Plasma extravasation was measured different time following the injection of betatoxin. Values will be the means.e.imply, n=6.n=5; P50.01, compared with car). The result indicated that betatoxin cannot induce the release of histamine in the cells. Our previous report also showed that the toxin does not induce the release of histamine from rat mast cells (Sakurai Fujii, 1987). It for that reason seems that the toxin does not directly act on mast cells.The effect of tachykinin receptor antagonist and capsaicin around the toxininduced plasma extravasationTo test when the toxininduced plasma extravasation is associated with tachykinins, the eect of tachykinin NK1 antagonist, [DPro2, DTrp7,9]SP, [DPro4, DTrp7,9]SP and spantide around the toxininduced plasma leakage was investigated. Figure 4 shows that coinjection of these NK1 antagonists resulted inside a reduction in the toxininduced leakage in a dosedependent manner (five.0 10 mg site71). Intradermal injection of a selective NK1 receptor agonist, septide (1.0 nmol site71), induced plasma extravasation inside a doserelated manner. The extravasation induced by septide was signi antly reduced by coinjection of NK1 antagonists (Figure four). [DPro4, DTrp7,9]SP, an NK1 antagonist, exhibited the identical potency in inhibiting the toxin or septideinduced plasma leakage (information not shown).British Journal of Pharmacology vol 138 (1)M. Nagahama et alC. perfringens betatoxin and plasma extravasationFigure 2 Eect of betatoxin on mouse dermal tissue. Saline (A) or betatoxin (50 ng site71) (B) was injected i.d. in to the dorsal skin of mice. Immediately after 12 h, dermal tissues in the dorsal skin have been ed in formalin and sections were stained with haematoxylin and eosin.Figure three Eect of diphenhydramine on plasma extravasation induced by betatoxin or histamine in dorsal skin of mice. A m.