Lotus Seed Green Embryo Extract and a Purified Glycosyloxyflavone Constituent, Narcissoside, Activate TRPV1 Channels in Dorsal Root Ganglion Sensory Neurons.

Several studies have documented the broad-spectrum bioactivities of a lotus seed (Plumula nelumbinis [PN]) green embryo extract. However, the specific bioactive components and associated molecular mechanisms remain largely unknown. This study aimed to identify the ion channel-activating mechanisms of PN extracts. Using fluorometric imaging and patch-clamp recordings, PN extracts were screened for calcium channel activation in dorsal root ganglion (DRG) neurons. The TRPV1 channels in DRG neurons were strongly activated by the PN extract (mean amplitude of 131 ± 45 pA at 200 µg/mL) and its purified glycosyloxyflavone narcissoside (401 ± 271 pA at 100 µM). Serial treatment with a 200 µg/mL PN extract in TRPV1-overexpressing HEK293T cells induced robust desensitization to 10 ± 10% of the initial current amplitude. Thus, we propose that the PN extract and narcissoside function as TRPV1 agonists. This new finding may advance our knowledge regarding the traditional and scientific functions of PN in human health and disease.

Ghrelin receptor is activated by naringin and naringenin, constituents of a prokinetic agent Poncirus fructus.

ETHNOPHARMACOLOGICAL RELEVANCE: Poncirus fructus (PF), also known as a dried immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae), has long been traditionally used for the various gastrointestinal disorders in Eastern Asia. AIM OF STUDY: The aqueous extract of PF (PF-W) has the strong prokinetic effect, yet the underlying mechanism is still elusive. The present study investigated whether PF-W has any effect on motilin receptor or ghrelin receptor, since these receptors enhance intestinal motility when activated. MATERIALS AND METHODS: The effect of PF-W and its components on motilin or ghrelin receptor was determined by calcium imaging and whole-cell patch clamp methods. RESULTS: PF-W activates the ghrelin receptor, but not the motilin receptor, resulting in a transient increase of intracellular calcium levels. Furthermore, among various constituents of PF, only naringin and naringenin evoked the intracellular calcium augmentation via the ghrelin receptor. Moreover, cortistatin-8 - a ghrelin receptor inhibitor - specifically blocked naringin- and naringenin-induced calcium increases. In addition, naringin and naringenin induced inward currents in ghrelin receptor-expressing cells under whole-cell patch clamp configuration. CONCLUSION: PF-W activates the ghrelin receptor, and naringin and naringenin are key constituents responsible for the activation of ghrelin receptor. Therefore, the present study suggests that the ghrelin receptor is a molecular entity responsible for the strong prokinetic activity of PF-W.

20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginseng, inhibits colon cancer growth by targeting TRPC channel-mediated calcium influx.

Abnormal regulation of Ca(2+) mediates tumorigenesis and Ca(2+) channels are reportedly deregulated in cancers, indicating that regulating Ca(2+) signaling in cancer cells is considered as a promising strategy to treat cancer. However, little is known regarding the mechanism by which Ca(2+) affects cancer cell death. Here, we show that 20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol (20-GPPD), a metabolite of ginseng saponin, causes apoptosis of colon cancer cells through the induction of cytoplasmic Ca(2+). 20-GPPD decreased cell viability, increased annexin V-positive early apoptosis and induced sub-G1 accumulation and nuclear condensation of CT-26 murine colon cancer cells. Although 20-GPPD-induced activation of AMP-activated protein kinase (AMPK) played a key role in the apoptotic death of CT-26 cells, LKB1, a well-known upstream kinase of AMPK, was not involved in this activation. To identify the upstream target of 20-GPPD for activating AMPK, we examined the effect of Ca(2+) on apoptosis of CT-26 cells. A calcium chelator recovered 20-GPPD-induced AMPK phosphorylation and CT-26 cell death. Confocal microscopy showed that 20-GPPD increased Ca(2+) entry into CT-26 cells, whereas a transient receptor potential canonical (TRPC) blocker suppressed Ca(2+) entry. When cells were treated with a TRPC blocker plus an endoplasmic reticulum (ER) calcium blocker, 20-GPPD-induced calcium influx was completely inhibited, suggesting that the ER calcium store, as well as TRPC, was involved. In vivo mouse CT-26 allografts showed that 20-GPPD significantly suppressed tumor growth, volume and weight in a dose-dependent manner. Collectively, 20-GPPD exerts potent anticarcinogenic effects on colon carcinogenesis by increasing Ca(2+) influx, mainly through TRPC channels, and by targeting AMPK.

An aqueous extract of Poncirus fructus activates the prokinetic activity of 5-HT receptor subtype 4 without hERG interaction.

AIM OF THE STUDY: Poncirus fructus (PF)--also known as the dried, immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae)--is a natural substance that has long been used for various gastrointestinal disorders in eastern Asia. An aqueous extract of PF (PF-W) has particularly potent gastroprokinetic effects, but its molecular mechanism was not well understood. Identification of the underlying prokinetic mechanism of PF-W was pursued in the present study. MATERIALS AND METHODS: Changes in in vitro cAMP levels and in vivo intestinal transit rate (ITR) caused by PF-W were measured after pretreatment with GR125487, an antagonist for serotonin receptor subtype 4 (5-HT4R). An [(3)H] astemizole binding assay and electrophysiology experiments were performed to determine if PF-W has any interaction with the human ether-à-go-go related gene (hERG) potassium channel. RESULTS: PF-W induced an increase in intracellular cAMP in 5-HT4R-expressing HEK293T cells, indicating that PF-W does activate 5-HT4R. Moreover, pretreatment with GR125487 successfully blocked the increase, suggesting that the response was 5-HT4R-specific. More importantly, pretreatment of GR125487 in rats inhibited the elevation of ITR by PF-W, indicating that the prokinetic effect of PF-W was indeed exerted via 5-HT4R. On the other hand, both [(3)H]-astemizole binding assay and electrophysiological experiments revealed that PF-W did not interfere at all with the hERG channel. CONCLUSION: It was found that PF-W exerts its prokinetic activity through a 5-HT4R-mediated pathway, with no interaction with hERG channels. Therefore, PF-W is a good candidate that might be developed as a prokinetic agent with fewer expected cardiac side effects.

Hydroxy-alpha-sanshool activates TRPV1 and TRPA1 in sensory neurons.

Sanshools are major active ingredients of Zanthoxylum piperitum and are used as food additives in East Asia. Sanshools cause irritant, tingling and sometimes paresthetic sensations on the tongue. However, the molecular mechanism underlying the pungent or tingling sensation induced by sanshools is not known. Because many transient receptor potential (TRP) channels are responsible for the sensations induced by various spices and food additives, we expressed 17 TRP channels in human embryonic kidney (HEK) cells and investigated their activation by hydroxy-alpha-sanshool (HalphaSS) or hydroxy-beta-sanshool (HbetaSS) isolated from Zanthoxylum piperitum. It was found that HalphaSS, but not HbetaSS, depolarized sensory neurons with concomitant firing of action potentials and evoked inward currents. Among 17 TRP channels expressed in HEK cells, HalphaSS caused Ca(2+) influx in cells transfected with TRPV1 or TRPA1, and evoked robust inward currents in cells transfected with TRPV1 or TRPA1. In primary cultured sensory neurons, HalphaSS induced inward currents and Ca(2+) influx in a capsazepine-dependent manner. Moreover, HalphaSS-induced currents and Ca(2+) influx were greatly diminished in TRPV1(-/-) mice. HalphaSS evoked licking behavior when injected into a single hind paw of wild-type mice, but this was much reduced in TRPV1-deficient mice. These results indicate that TRPV1 and TRPA1 are molecular targets of HalphaSS in sensory neurons. We conclude that the activations of TRPV1 and TRPA1 by HalphaSS explain its unique pungent, tingling sensation.

Agonist recognition sites in the cytosolic tails of vanilloid receptor 1.

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N- and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [(3)H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket.