THE CALCIUM-ANTAGONIST TMB-8 [3,4,5-TRIMETHOXYBENZOIC ACID 8-(DIETHYTAMINO)OCTYL ESTER] IS A POTENT, NONCOMPETITIVE, FUNCTIONAL ANTAGONIST AT DIVERSE NICOTINIC ACETYLCHOLINE-RECEPTOR SUBTYPES

[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 pot encies that exceed those for other reported effects of TMB-8, includin g inhibition of intracellular Ca2+ mobilization. TMB-8 is a potent inh ibitor (IC50 similar to 400 nM) of agonist-stimulated ion flux mediate d by functional human muscle nAChR or ganglionic alpha 3 beta 4-nAChR subtypes expressed by TE671/RD or SH-SY5Y cells. TMB-8 is also a poten t inhibitor (IC50 similar to 500 nM) of a functional, central nervous system nAChR subtype that mediates nicotinic agonist-stimulated [H-3]d opamine release from rat brain synaptosomes. TMB-8 is much less potent (IC50 similar to 30-200 mu M) as an inhibitor of high-affinity H-3-la beled acetylcholine or I-125-labeled alpha-bungarotoxin binding to hum an muscle nAChR, ganglionic alpha 3 beta 4-nAChR, or ganglionic alpha 7-nAChR subtypes, Moreover, functional inhibition by TMB-8 of muscle-t ype 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. Sim ilar effects are observed for local anesthetics such as tetracaine and procaine (functional IC50 values of similar to 5 and similar to 50 mu M, 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'-t etraacetic acid] analogues indicate that the amino group of TMB-8 is e ssential and that Ca2+ chelation is not required for inhibition of nAC hR function. Other studies indicate that TMB-8 is a much less potent i nhibitor of tetrodotoxin-sensitive, voltage-gated Na+-channel function in TE671/RD or SH-SY5Y cells (IC50 values of similar to 70 - 200 mu M ) or of [H-3]dopamine release from rat brain synaptosomes stimulated b y high extracellular KCI concentrations (IC50 similar to 10 mu M), By contrast, tetracaine has more comparable potencies as a blocker of vol tage-gated ion channels (IC50 similar to 20 mu M) 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-mediat ed nAChR functional blockade suggests that caution should be applied w hen using this compound in studies of intracellular Ca2+ mobilization.