Proceedings of The Physiological Society
Cardiff University (2009) Proc Physiol Soc 17, PC43
Substance MCS-18 from Helleborus purpurascens inhibits capsaicin and acid induced activation of TRPV1
C. Neacsu1, A. C. Ciobanu1, O. Toader1, G. Szegli2, F. Kerek3, A. Babes1
1. Department of Animal Physiology and Biophysics, University of Bucharest, Bucharest, Romania. 2. Cantacuzino Institute for Microbiology and Immunology, Bucharest, Bucharest, Romania. 3. Donatur GmbH, Martinsried, Germany.
Extracts from the roots of the plant Helleborus purpurascens have been traditionally used in Balkan folk medicine for their anti-nociceptive activities. The aim of this work was to asses the effects of substance MCS-18, extracted and purified from Helleborus roots, on TRPV1 a well known polymodal receptor involved in nociception, in rat DRG neurons. DRG neurons were held in primary culture for 24 hours. TRPV1 was also expressed and tested in HEK293 cells. Neurons and HEK293 cells were analyzed with microfluorimetry, using the calcium indicator Calcium Green-1 AM and the patch-clamp technique. MCS-18 inhibited responses to capsaicin 300 nM when the agonist was applied five times, repeatedly (DF/F0 was 0.03 for the third response [MCS-18 was co-applied] compared to 0.21 for the 2nd response, n = 25; p < 0.0001). When MCS-18 was pre-applied it decreased the capsaicin responses from 0.55 ± 0.05 to 0.43 ± 0.06 (p < 0.05). Activation by acid pH (5.5) in DRG neurons, was also reduced by about 44% (0.14 ± 0.03 compared to 0.25 ± 0.02, n = 32, p < 0.01). Inhibition of pH responses was mediated by TRPV1, because MCS-18 had no effect on ASIC channels. Also, the substance had no effect on the increases in [Ca2+]i evoked by the TRPA1 agonist cinnamaldehyde. MCS-18 did not inhibit heat (45 oC) responses. Inhibition of capsaicin (2 µM) activation was dose-dependent as confirmed by whole-cell patch-clamp (from 5% inhibition at 0.01 µg/ml, to 90% inhibition at 10 µg/ml). Moreover capsaicin currents were also inhibited in excised outside-out patches in DRG neurons. When expressed in HEK293 cells, rTRPV1 responses to capsaicin are inhibited by MCS-18, and the magnitude of the inhibition was 84% as shown by calcium imaging experiments. The same effect was shown by patch-clamp data. The antagonistic effect of MCS-18 on TRPV1 activation by capsaicin and protons may be important in pain therapy.
Where applicable, experiments conform with Society ethical requirements