The 'Calcium Antagonist' Tmb-8 [345-Trimethoxybenzoic Acid 8- (Diethylamino)Octyl Ester] Is A Potent Non-Competitive Functional Antagonist At Diverse Nicotinic Acetylcholine Receptor Subtypes



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[3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester] (TMB-8) has seen wide use as an 'intracellular Ca2+ antagonist.' However, this study shows that TMB-8 acts as a noncompetitive, functional antagonist at diverse nicotinic acetylcholine receptor (nAChR) subtypes with potencies that exceed those for other reported effects of TMB-8, including inhibition of intracellular Ca2+ mobilization. TMB-8 is a potent inhibitor (IC50 ~400 nM) of agonist-stimulated ion flux mediated by functional human muscle nAChR or ganglionic α3β4-nAChR subtypes expressed by TE671/RD or SH-SY5Y cells. TMB-8 is also a potent inhibitor (IC50 ~500 nM) of a functional, central nervous system nAChR subtype that mediates nicotinic agonist-stimulated [3H]dopamine release from rat brain synaptosomes. TMB-8 is much less potent (IC50 ~30-200 μM) as an inhibitor of high-affinity 3H-labeled acetylcholine or 125I-labeled α-bungarotoxin binding to human muscle nAChR, ganglionic α3β4-nAChR, or ganglionic α7-nAChR subtypes. Moreover, functional inhibition by TMB-8 of muscle-type nAChR is due to a reduction in agonist efficacy, but not potency, and is proportionately stronger with increasing agonist concentration, thereby suggesting that TMB-8 acts as a noncompetitive inhibitor. Similar effects are observed for local anesthetics such as tetracaine and procaine (functional IC50 values of ~5 and ~50 μM, respectively), although TMB-8 is the most potent of these agents. Studies with TMB-8 or BAPTA [1,2-bis(2-aminophenoxy)ethane N,N,N',N'- tetraacetic acid] analogues indicate that the amino group of TMB-8 is essential and that Ca2+ chelation is not required for inhibition of nAChR function. Other studies indicate that TMB-8 is a much less potent inhibitor of tetrodotoxin-sensitive, voltage-gated Na+-channel function in TE671/RD or SH-SY5Y cells (IC50 values of ~70-200 μM) or of [3H]dopamine release from rat brain synaptosomes stimulated by high extracellular KCl concentrations (IC50 ~10 μM). By contrast, tetracaine has more comparable potencies as a blocker of voltage-gated ion channels (IC50 ~20 μM) or of nAChR function. Collectively, these results and examination of the structure of TMB-8 suggest the hypothesis that it might act like a local anesthetic to effect nAChR open channel block. Moreover, the potency of TMB-8-mediated nAChR functional blockade suggests that caution should be applied when using this compound in studies of intracellular Ca2+ mobilization.

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Journal of Pharmacology and Experimental Therapeutics







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