Leipziger J, Thomas J, Rubini-Illes P, Nitschke R, Greger R
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1996 Feb;353(3):295-301. doi: 10.1007/BF00168631.
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.
8 -（N，N - 二乙氨基）辛基 - 3,4,5 - 三甲氧基苯甲酸酯（TMB - 8）是一种广泛用于研究细胞内钙库参与细胞反应的药理学工具。在本研究中，我们研究了TMB - 8作为一种假定的“钙信号”抑制剂在单个用fura - 2负载的HT29结肠上皮细胞中的作用，这些细胞受到ATP、卡巴胆碱（CCH）和神经降压素（NT）的刺激。TMB - 8有效抑制了CCH诱导的（100μmol/L细胞内钙（[Ca2 +]i）瞬变，IC50为20μmol/L。然而，由其他磷脂酶C偶联激动剂ATP（10μmol/L，n = 4）和NT（10nmol/L，n = 4）诱导的[Ca2 +]i瞬变不受TMB - 8（50μmol/L）影响。当刺激浓度降低至ATP为0.5μmol/L（n = 4）或NT为1nmol/L（n = 4）时，激动剂诱导的[Ca2 +]i瞬变同样不受100μmol/L TMB - 8影响。通过在不存在和存在拮抗剂的情况下检查不同浓度的激动剂，证明了TMB - 8诱导的对CCH诱导的[Ca2 +]i瞬变抑制的竞争性性质。单独的高浓度TMB - 8（100μmol/L）诱导了小的[Ca2 +]i增加（Δ[Ca2 +]i：40±5nmol/L，n = 7）。我们假设这种增加是TMB - 8诱导的细胞内碱化（ΔpH：0.1±0.02，n = 7）与[Ca2 +]i增加同时发生的结果。从这些结果我们得出以下结论：（1）与大量其他研究形成鲜明对比，但与其他类型细胞的研究一致，这些结果极大地质疑了“工具”TMB - 8作为“细胞内钙拮抗剂”的价值；（2）TMB - 8在M3受体处作为毒蕈碱受体拮抗剂起作用；（3）当IP3信号转导由ATP或NT激活时，TMB - 8不影响细胞内钙库中钙的释放；（4）TMB - 8作为一种弱有机碱在高浓度下使细胞质碱化；（5）TMB - 8在较高浓度下诱导小的[Ca2 +]i瞬变。
