capsazepine and evodiamine

Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.

Topics: Alkaloids; Analgesics; Animals; Calcium; Calcium Channels; Capsaicin; Dose-Response Relationship, Drug; Evodia; Ganglia, Spinal; HEK293 Cells; Hot Temperature; Humans; Hyperalgesia; Male; Medicine, Chinese Traditional; Nerve Tissue Proteins; Neurons; Patch-Clamp Techniques; Plant Extracts; Quinazolines; Quinolines; Rats; Rats, Sprague-Dawley; Transient Receptor Potential Channels; TRPA1 Cation Channel; TRPV Cation Channels

We investigated the molecular mechanism underlying the role of transient receptor potential vanilloid type 1 (TRPV1), a Ca(2+)-permeable non-selective cation channel, in the activation of endothelial nitric oxide (NO) synthase (eNOS) in endothelial cells (ECs) and mice.. In ECs, TRPV1 ligands (evodiamine or capsaicin) promoted NO production, eNOS phosphorylation, and the formation of a TRPV1-eNOS complex, which were all abrogated by the TRPV1 antagonist capsazepine. TRPV1 ligands promoted the phosphorylation of Akt, calmodulin-dependent protein kinase II (CaMKII) and TRPV1, and increased the formation of a TRPV1-Akt-CaMKII complex. Removal of extracellular Ca(2+) abolished the ligand-induced increase in the phosphorylation of Akt and CaMKII, formation of a TRPV1-eNOS complex, and eNOS activation. Inhibition of PI3K and CaMKII suppressed the ligand-induced increase in TRPV1 phosphorylation, formation of a TRPV1-eNOS complex, and eNOS activation. TRPV1 activation increased the phosphorylation of Akt, CaMKII, and eNOS in the aortas of wild-type mice but failed to activate eNOS in TRPV1-deficient aortas. Additionally, TRPV1 ligand-induced angiogenesis was diminished in eNOS- or TRPV1-deficient mice. When compared with apolipoprotein E (ApoE)-deficient mice, ApoE/TRPV1-double-knockout mice displayed reduced phosphorylation of eNOS, Akt, and CaMKII in aortas but worsened atherosclerotic lesions.. TRPV1 activation in ECs may trigger Ca(2+)-dependent PI3K/Akt/CaMKII signalling, which leads to enhanced phosphorylation of TRPV1, increased TRPV1-eNOS complex formation, eNOS activation and, ultimately, NO production.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Capsaicin; Cattle; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinazolines; RNA Interference; Signal Transduction; Time Factors; Transfection; TRPV Cation Channels

Evodiamine, a quinozole alkaloid constituent of Evodia rutaecarpa, has been reported previously to induce several responses comparable to capsaicin in animal systems. Here, we characterize evodiamine as an agonist for rat TRPV1 expressed heterologously in CHO cells. Evodiamine bound to rat TRPV1 with a Ki of 5.95 +/- 0.87 microM, as measured by inhibition of [3H] RTX binding (capsaicin, Ki = 1.8 +/- 0.3 microM). Evodiamine was a full agonist for induction of 45Ca2+ uptake, with an EC50 of 856 +/- 43 nM (capsaicin, EC50 = 45 +/- 4 nM) and was competitively antagonized by capsazepine, as revealed by a Schild plot. The pattern of cellular response, as determined by calcium imaging, was similar to that with capsaicin and yielded an EC(50) of 1.03 +/- 0.21 [micro sign]M. Molecular modeling suggested a consistent pattern of overlap between evodiamine and TRPV1 agonists. We conclude that evodiamine represents a novel class of agonists for rat TRPV1, albeit 3-19-fold less potent than capsaicin, and thus represents a new potential class of lead molecules for drug development.

Topics: Animals; Calcium; Capsaicin; Cations, Divalent; CHO Cells; Cricetinae; Diterpenes; Ligands; Models, Biological; Molecular Conformation; Molecular Structure; Plant Extracts; Quinazolines; TRPV Cation Channels

Previous investigations have indicated that the pharmacological activities of evodiamine, a major alkaloidal component of the dried, unripe fruit of Evodia rutaecarpa Bentham (Rutaceae), are associated with the stimulation of calcitonin gene-related peptide (CGRP) release and that CGRP protects the myocardium against ischemia-reperfusion injury. In the present study, we have examined whether evodiamine protects against myocardial ischemia-reperfusion injury in rats and whether the protective effects of evodiamine are related to the activation of capsaicin-sensitive sensory nerves. Rats were pretreated with evodiamine 10 min before the experiment, and then the left main coronary artery of rat hearts was subjected to 60 min occlusion followed by 180 min reperfusion. Infarct size, the activity of serum creatine kinase, serum concentrations of TNF-alpha and plasma concentrations of CGRP were measured. Pretreatment with evodiamine (30 or 60 microg/kg, i.v.) markedly increased the content of CGRP in plasma concomitantly with a significant reduction in infarct size, the activity of serum creatine kinase, and TNF-alpha level, and the effects of evodiamine were completely abolished by capsazepine (5.0 mg/kg, s.c.), a competitive vanilloid receptor antagonist. These results suggest that evodiamine exerts a protection against myocardial ischemia-reperfusion injury in rats and that the protective effects of evodiamine are related to stimulation of CGRP release via activation of vanilloid receptors.

Topics: Animals; Calcitonin Gene-Related Peptide; Capsaicin; Cardiotonic Agents; Creatine Kinase; Evodia; Infusions, Intravenous; Male; Myocardial Reperfusion Injury; Myocardium; Phytotherapy; Plant Extracts; Quinazolines; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha