Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca2+ imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca2+ dependency in the signal transduction of capsaicin-induced desensitization. On the average, capsaicin increased cAMP, cGMP concentration and SP release in bell-shaped concentration-dependent manner, with the maximal responses at concentrations around 1 mu M, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca2+ concentration ([Ca2+](i)) increase depended on extracellular Ca2+ influx as an initial trigger. The Ca2+ influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca2+-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca2+-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca2+ removal, suggesting at least in part a Ca2+-independent pathway is involved. Further study showed that cAMP increase was totally abolished by G-protein and adenylate cyclase (AC) antagonist, suggesting a G-protein-dependent pathway in cAMP increase. However, SP release was blocked by inhibiting PKC, but not G-protein or AC, suggesting a G-protein independent pathway in SP release. These results suggest that both Ca2+-dependent and independent mechanisms are involved in the regulation of capsaicin on second messengers systems, which could be a novel mechanism underlying distinct desensitization of capsaicin and might provide additional opportunities in the development of effective analgesics in pain treatment. (c) 2012 Elsevier Ireland Ltd. All rights reserved.