ABSTRACT
Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca²âº imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca²âº 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 µM, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca²âº concentration ([Ca²âº](i)) increase depended on extracellular Ca²âº influx as an initial trigger. The Ca²âº influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca²âº-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca²âº-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca²âº removal, suggesting at least in part a Ca²âº-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 Ca²âº-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.
Subject(s)
Calcium/metabolism , Capsaicin/pharmacology , Neurons/metabolism , Second Messenger Systems/drug effects , TRPV Cation Channels/physiology , Animals , Capsaicin/metabolism , Cells, Cultured , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Male , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology , Substance P/metabolismABSTRACT
AIM: The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. METHODS: Single-channel and whole-cell patch-clamp techniques were used to record the K(ATP) current (I(KATP)) in acutely isolated rat ventricular myocytes. RESULTS: Oxidized glutathione (GSSG, 1 mmol/L) increased the I(KATP), while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I(KATP) during normoxia. To further corroborate the effect of the redox agent on the K(ATP) channel, we employed the redox couple DTT (1 mmol/L)/H2O2 (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia. H2O2 increased the I(KATP) in a concentration dependent manner, which was reversed by DTT (1 mmol/L). In addition, our results have shown that 15 min of hypoxia increased the I(KATP), while GSH (1 mmol/L) could reverse the increased I(KATP). Furthermore, in order to study the signaling pathways of the I(KATP) augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93. The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I(KATP) augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K(ATP) channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway. CONCLUSION: The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I(KATP).
