ABSTRACT
Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3 (TRPV3) channel cause Olmsted syndrome characterized by severe itching and keratoderma, indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases. However, currently available TRPV3 tool inhibitors are either nonselective or less potent, thus impeding the validation of TRPV3 as therapeutic target. Using whole-cell patch-clamp and single-channel recordings, we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A (IAA) and isochlorogenic acid B (IAB) that selectively inhibit TRPV3 currents with IC50 values of 2.7 ± 1.3 and 0.9 ± 0.3 μmol/L, respectively, and reduce the channel open probability to 3.7 ± 1.2% and 3.2 ± 1.1% from 26.9 ± 5.5%, respectively. In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus. Furthermore, the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol. Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers. Taken together, our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology, but also holds developmental potential for treatment of dermatitis and chronic pruritus.
ABSTRACT
OBJECTIVE To explore the effect of total flavonoids of Rhododendra simsii (TFR) on improving cerebral ischemia/reperfusion injury (CIRI) and its relationship with STIM/Orai-regulated operational Ca2+influx (SOCE) pathway. METHODS Oxygen-glucose deprivation/reoxygenation (OGD/R) PC12 cells were used to simulate CIRI in vitro, and the intracellular Ca2+ concentration and apoptosis rate of PC12 cells were detected by laser confocal microscope and flow cytometry, respectively. The regulation of STIM/Orai on SOCE was analyzed by STIM/Orai gene silencing and STIM/Orai gene overexpression. The CIRI model was established by MCAO in SD rats. The activities of inflammatory cyto?kines IL-1, IL-6 and TNF-αin serum were detected by ELISA. The pathological changes of ischemic brain tissue and the infarction of rat brain tissue were detected by HE staining and TTC staining. The protein and mRNA expression levels of STIM1, STIM2, Orai1, caspase-3 and PKB in brain tissue were detected by Western blotting and RT-qPCR, respectively. RESULTS The results of in vitro experiment showed that the fluorescence intensity of Ca2+ and apoptosis rate in PC12 cells treated with TFR were significantly lower than those in OGD/R group, and this trend was enhanced by SOCE antagonist 2-APB. STIM1/STIM2/Orai1 gene silencing significantly reduced apoptosis and Ca2+overload in OGD/R model, while TFR combined with overexpression of STIM1/STIM2/Orai1 aggravated apoptosis and Ca2+overload. In the in vivo experiment, TFR significantly reduced the brain histopathological damage, infarction of brain tissue, the contents of IL-1, IL-6 and TNF-α in the serum in MCAO rats and down-regulated the expression of STIM1, STIM2, Orai1 and caspase-3 protein and mRNA in the brain tissue, and up-regulated the expression of PKB. The above effects were enhanced by the addition of 2-APB. CONCLUSION The above results indicate that TFR may reduce the contents of inflammatory factors and apoptosis, decrease Ca2+ overload and ameliorate brain injury by inhibiting SOCE pathway mediated by STIM and Orai, suggesting that it has a protective effect against subacute CIRI.
ABSTRACT
Inositol 1,4,5-trisphosphate receptors (InsP3Rs) modulate Ca2+ release from intracellular Ca2+ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP3Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca2+ release from intracellular Ca2+ stores and Ca2+ entry through store-operated Ca2+ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca2+ entry, but significantly inhibited carbamylcholine (CCh)-induced Ca2+ release. In contrast, 2-APB did not block CCh-induced Ca2+ release, but remarkably blocked SOC-mediated Ca2+ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced Ca2+ release, but 2-APB at lower concentration, which effectively blocked Ca2+ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP3-induced Ca2+ release and direct stimulation of Ca2+ release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP3R, and 2-APB at lower concentration is considered a blocker of Ca2+ entry through SOC channels in the pancreatic acinar cell.
Subject(s)
Animals , Mice , Acinar Cells , Boron Compounds , Caffeine , Calcium , Carbachol , Inositol 1,4,5-Trisphosphate Receptors , Membranes , ReticulumABSTRACT
BACKGROUND AND OBJECTIVES: Elevated intracellular calcium level is known to play important roles in the apoptotic pathway. IP3 receptor (ligand-gated channels that release Ca2+ from intracellular stores) is emerging as a key site for regulation of apoptosis. 2-Aminoethoxydiphenyl borate (2-APB) is one of the reliable IP3 receptor antagonists. We examined the effect of 2-APB on gentamicin ototoxicity in vitro, using the HEI-OC1 cell line. MATERIALS AND METHOD: HEI-OC1 cells were trWWeated with 100micrometer gentamicin. Using a CaspACE assay, we measured the caspases-3 activity in the gentamicin treated hair cells with and without 2-APB pre-incubation. We also observed intra-cellular calcium concentrations in HEI-OC1 cells using a confocal microscopy (calcium green-1 stain). Live cell imaging was performed by using fluorescence video-time lapse system. RESULTS: Cytosolic calcium elevation by gentamicin was remarkably inhibited by 2-APB. Caspases-3 activities of gentamicin treated cells were higher than those of the control. After incubation with 2-APB, caspases-3 activities and cell death of gentamicin treated cells were shown to decrease. CONCLUSION: 2-APB reduces Caspases-3 activity in the gentamicin treated HEI-OC1 cells by inhibition of cytosolic calcium increase.