ISSN:
1432-1912
Keywords:
TMB-8
;
Fura-2
;
HT29
;
M3-receptor
;
ATP
;
Carbachol
;
Neurotensin
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract 8-(N, N-diethyl amino) 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 coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 μmol/l intracellular Ca2+ ([Ca2+]i) transient with an IC50 of 20 μmol/l. However, [Ca2+]i transients induced by other phospholipase C coupled agonists ATP (10 μmol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 μmol/l). The agonist-induced [Ca2+]i transients remained equally unaffected by 100 μmol/l TMB-8 when the stimulatory concentration was reduced to 0.5 μmol/I 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 μmol/l) alone induced a small [Ca2+]i increase (Δ[Ca2+]i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization (Δ pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca +]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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00168631
