8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca2+ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of ''Ca2+ signalling'' in single fura-2 loaded HT29 colonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mu mol/l intracellular Ca2+ ([Ca2+](i)) transient with an IC50 of 20 mu mol/l. However, [Ca2+](i), transients induced by other phospholipase C coupled agonists ATP (10 mu mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mu mol/l). The agonist-induced [Ca2+](i) transients remained equally unaffected by 100 mu mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mu mol/l for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca2+](i) transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mu mol/l) alone induced a small [Ca2+](i) increase (Delta[Ca2+](i): 40 +/- 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization (Delta pH: 0.1 +/- 0.02, n = 7) occurring simultaneously with the increase in [Ca2+](i). From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the ''tool'' TMB-8 as an ''intracellular Ca2+ antagonist''; (2) TMB-8 acts a muscarinic receptor antagonist at the M(3) receptor; (3) TMB-8 does not influence the release of Ca2+ from intracellular stores when IP3 signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca2+](i) transients at higher concentrations.