Evidence that TRPV1-mediated vasodilatation is associated with reactive oxygen species production

Starr, A. & Brain, S.D. Cardiovascular Division, Kings College London, SE1 1UL, UK.

Calcitonin gene related peptide (CGRP) and substance P (SP) are released from stimulated sensory neurones.

Recently, this group (Grant et al, 2002) established that vasodilatation in the mouse ear induced by the TRPV1 receptor agonist capsaicin, is only abolished when the vasodilator mechanisms of both CGRP and SP are blocked. We now realise that SP-induced vascular activity is independent of nitric oxide and vasodilator prostaglandins and is possibly mediated by reactive oxygen species (ROS). The aim of this study is to determine whether ROS are involved in mediating neurogenic vasodilatation in the microcirculation.

Neurogenic vasodilatation was induced using capsaicin (200 µg in 20 µl), applied topically to the ears of TRPV1 wildtype (WT) and knockout mice (KO, Gift from Merck, Sharpe and Dohme, both sexes, 25-30g) or CD1 mice (female, 25-30g) under urethane anaesthesia (2.5mg/g i.p.). Vehicle (ethanol) was applied to the contralateral ears. The hydrogen peroxide (H2O2) and superoxide scavengers catalase and superoxide dismutase (CAT, SOD, 25000U/kg each) or vehicle control (denatured enzyme) were administered i.p. prior to capsaicin treatment. Blood flow in the ear was then assessed for one hour using a laser Doppler blood flowmeter or imaged using a laser Doppler scanner. Results were expressed as the area under the recorded flux vs. time curve.Ear homogenates from these animals were assayed for H2O2 using the horseradish peroxidase oxidation of phenol red method ( Pick and Keisari, 1980). Results were expressed as concentration of H2O2 per gram of tissue. Statistical analysis was carried out by ANOVA followed by Bonferroni's test.

Capsaicin induces increased ear blood flow in TRPV1 WT mice compared to contralateral ears (82.5 ± 13.4 vs 26.7 ± 6.1 x103 ‘area under the flux vs. time curve’ units, mean ± s.e.m, n=8, P<0.001). This response was absent in KO mice. Combined administration of CAT and SOD to CD1 mice abolished increased blood flow compared to vehicle treated mice (120.7 ± 18.0 vs 22.0 ± 4.0 x103 ‘area under the flux vs. time curve’ units, mean ± s.e.m, n=6-10, P<0.001). Lower levels of H2O2 were observed in ear homogenates of TRPV1 KO than WT mice and also in CD1 mice given CAT and SOD than in vehicle treated mice (Table 1 ) . These data suggest that mechanisms of neurogenic vasodilatation in the mouse microvasculature are dependent on ROS.

Table 1. H2O2 in ear homogenates. Data expressed as H2O2/g tissue, mean ± s.e.m).

# P< 0.001 compared to TRPV1 wildtype mice.
* P< 0.05, ** P< 0.001 compared to vehicle treated mice.

Grant et al (2002) Br. J .Pharmacol 135, 356-362.
Pick & Kaiseri (1980) J. Immunol. Methods 38, 161-170.

We thank the BHF and BBSRC for support.