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  • br Materials and methods br Results We examined the effects

    2019-12-02


    Materials and methods
    Results We examined the effects of curcumin in Fluo-4 loaded SH-EP1 (±)-Bay K 8644 stably transfected with human α7-nACh receptor. Application of curcumin alone in the concentrations up to 100 μM for 30 sec. did not induce any detectable change in intracellular Ca2+ levels in (n = 14 from 3 separate experiments). Application of choline (1 mM), a selective agonist for α7-nACh receptor (Albuquerque et al., 2009) for 30 sec. induced a rapid increase in intracellular Ca2+ concentrations (Fig. 1A, control). Application of non-selective cholinergic agonist, ACh was also found to produce a similar rise in intracellular Ca2+ (n = 12; data not shown). Calcium transients induced by choline was completely inhibited by 10 min pre-incubation with 10 μM methyllycaconitine, a selective antagonist for α7-nACh receptor (Fig. 1A and B). Pre-incubation with 1 μM curcumin (for 10 min) was associated with a significant potentiation (2.2 fold, n = 12, ANOVA, P = .001) of the choline-induced Ca2+ transient in the cells (Fig. 1A and B). The effects of curcumin were concentration-dependent showing an IC50 value of 133 nM (Fig. 1C). Notably, 10 min. applications of 10 μM curcumin, did not alter the magnitudes of Ca2+ transients induced by the application of high-K+ in the bath (60 mM KCl, n = 14, ANOVA, P = .579) or Ca2+ transients induced by 200 μM ACh in Fluo-4 AM loaded SH-EP1 cells expressing human α4β2-nACh receptor (n = 14; ANOVA, P = .078; Fig. 1D). Entrance of Ca2+ through endogenously expressed voltage-gated Ca2+ channels in SH-EP1 cells can contribute to Ca2+ transients induced by choline. Therefore, we have tested effects of curcumin in the presence of L-type Ca2+ channel antagonists nifedipine (1 μM) and verapamil (1 μM), N-type Ca2+ channel antagonist ω-conotoxin MVIIC (1 μM), and nonselective Ca2+ channel antagonist, bepridil (10 μM). In the presence of these antagonists (n = 15, P = .068 for nifedipine and n = 19, P = .086 for verapamil; n = 11, P = .074 for ω-conotoxin and n = 14, P = .083 for bepridil) curcumin (10 μM) continued to potentiate choline-induced Ca2+ transients (Fig. 2A and 2B). In the concentrations used in this study, these Ca2+ channel antagonists themselves did not have any effect on choline-induced Ca2+ transients (n = 11–14, ANOVA, P > .05). Co-application of curcumin and choline, without pre-incubation with curcumin, was not associated with a rise in the choline-induced Ca2+ transients (n = 18, ANOVA, P = .512) suggesting that the effects of curcumin on nACh receptor Ca2+ signal require receptor priming by additional cellular mechanisms such as kinases. We therefore investigated the involvement of protein kinases A, C, and Ca2+-calmodulin dependent kinase (CaM-kinase), which are known to regulate functions of ligand-gated ion channels (Zhang et al., 1995, Talwar and Lynch, 2014), in curcumin\'s effect on α7-nACh receptor Ca2+ response. A 30 min. pretreatment with 3 μM Go-6 983 (Go; protein kinase C specific inhibitor; Young et al., 2005), 10 μM KT-5 720 (KT; protein kinase A specific inhibitor; Cabell and Audesirk, 1993), or 10 μM KN-62 (KN; CaM kinase II specific inhibitor, Tokumitsu et al., 1990) was found to have no effect on curcumin (10 μM) mediated potentiation of the choline (1 mM)-induced Ca2+ response (ANOVA; n = 18–21; P = .001 for Go, KT, and KN groups). The results of these experiments were presented in Fig. 3A. After 30 min. pretreatment, protein kinase inhibitors (Go, KT, KN) at the concentrations used in this study did not cause any alterations on the choline-induced Ca2+ transients (n = 12–14). In vivo curcumin is known to be metabolized to compounds such as tetrahydrocurcumin (THC), demethylcurcumin (DMC), and didemethylcurcumin (DDMC) (Anand et al., 2007). We tested the effects of these curcumin metabolites on choline-induced Ca2+ transients in SH-EP1 cells. Our findings indicate that these curcumin metabolites (at 10 μM concentrations) can also significantly (ANOVA; n = 12–16; P = .001 for THC, DMC, and DDMC groups) potentiate choline-induced Ca2+ transients in this cell line (Fig. 3B).