Abstract
Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy.
Highlights
The tuber of the species Aconitum is a crude drug that has been utilized from ancient times
It is well known that aconitine alkaloids in processed Aconiti tuber (PAT) such as mesaconitine[13], aconitine[14] and benzoylmesaconine[15] have analgesic activity, it remains unclear whether these aconitine alkaloids act on the opioid systems
It has been noted that the content of diester-type alkaloids such as mesaconitine and aconitine are converted to monoester alkaloids such as benzoylmesaconine and benzoylhypaconine by heating the Aconiti tuber
Summary
Pharmacokinetic study of the PAT-containing traditional medicine, GJG. A pharmacokinetic study of GJG was performed to identify the bioavailable molecules in PAT. The results showed that DAMGO inhibited the increase of intracellular cAMP induced by forskolin (Supplementary Figure S3). When added together with 100 nM DAMGO, ignavine showed an intriguing characteristic, with a concentration of 1 μM enhancing the activity of DAMGO in the early period (Fig. 4B) but a concentration of 10 μM inhibiting DAMGO activity in the later period (Fig. 4C) These data are in good accordance with the results of the receptor internalization assay. Morphine and β-funaltrexamine (β-FNA) were used for the simulation study as a representative MOR agonist and antagonist, respectively, and we constructed docking models of each ligand (Fig. 7). DAMGO induced vesicle formation (shown by red arrowheads) in the intracellular space, which represented receptor internalization, 20 min after administration. These simulation data suggest that the structural change caused by ignavine binding would be different to that caused by either the reference agonist (morphine) or the antagonist (β-FNA)
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