title: 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29.
abstract: 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 mumol/l intracellular Ca2+ ([Ca2+]i) transient with an IC50 of 20 mumol/l. However, [Ca2+]i transients induced by other phospholipase C coupled agonists ATP (10 mumol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mumol/l). The agonist-induced [Ca2+]i transients remained equally unaffected by 100 mumol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mumol/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 mumol/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 M3 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.
authors.affiliation: Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Leipziger
authors.forename: J
authors.initials: J
authors.lastname: Thomas
authors.forename: P
authors.initials: P
authors.lastname: Rubini-Illes
authors.forename: R
authors.initials: R
authors.lastname: Nitschke
authors.forename: R
authors.initials: R
authors.lastname: Greger
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D016257:Fura-2 / Q000737:chemistry; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D018727:Muscarinic Antagonists / Q000378:metabolism / Q000494:pharmacology*; D009496:Neurotensin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D018727:Muscarinic Antagonists; D009496:Neurotensin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D000255:Adenosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2