Calcium channels activated by endothelin-1 in human trophoblast

Abstract

Ca2+ transfer across the syncytiotrophoblast (ST) of the human placenta is essential for normal fetal development. However, the nature of Ca2+ conductance in the ST and the mechanisms by which it is regulated are poorly understood. With the major signal transduction pathway of endothelin-1 (ET1) acting via phospholipase C (PLC) and Ca2+, we used ET1 to analyse the nature of Ca2+ channels on cultured trophoblastic cells by means of cytofluorimetric analysis using the ratiometric Ca2+ indicator Indo-1. Results indicate that ET1 (10(-7) M) stimulates a biphasic (transient and sustained) increase in [Ca2+]i in trophoblastic cells. This response is mediated by the endothelin receptor B (ETB) coupled to PLC, since treatment with BQ788 (10(-6) M) or U73122 (2 microM) totally abolished the response. Persistence of the rapid transient rise in [Ca2+]i in Ca2+-free extracellular medium confirms the release of Ca2+ from intracellular stores in response to ET1 stimulation. Furthermore, abolition of the sustained increase in [Ca2+]i in Ca2+-free extracellular medium argues in favour of the entry of Ca2+ during the plateau phase. Abolition of this plateau phase by Ni2+ (1 mM) in the presence of extracellular Ca2+ confirmed the existence of an ET1-induced Ca2+ entry. No evidence for the presence of voltage-operated channels was demonstrated during ET1 action since nifedipine (10(-6) M) did not reduce the Ca2+ response and depolarization with a hyper-potassium solution had no effect. Pharmacological studies using the imidazole derivatives SK&F96365 (30 microM) and LOE 908 (10 microM) partially inhibited the ET1-evoked Ca2+ response, thus providing evidence for the presence of both store-operated Ca2+ channels and non-selective cationic channels in the human ST.

Figure 1. Effect of endothelin-1 (ET1) on [Ca²⁺]_i in cultured syncytiotrophoblastic cells

A, Ca²⁺ change in response to exposure of a syncytiotrophoblast to ET1 (10⁻⁷ m). After a rapid rise to peak of the fluorescence ratio, a decrease in [Ca²⁺] to a level higher than that of the basal level was observed (plateau). Arrows indicate the time of measurement for calculation of the percentage change in ratio expressed in B. B, averaged data for the percentage increase in fluorescence ratio (F₄₀₅/F₄₈₅). Data are means ± s.e.m.; peak n = 49; plateau n = 20.

Figure 2. Analysis of ET1-induced Ca²⁺ response

A and B, the effects of BQ123 (A; 10⁻⁶ m) and BQ788 (B; 10⁻⁶ m) on the [Ca²⁺]_i increase induced by ET1 (10⁻⁷ m). C, recording of the Ca²⁺ response to ET1 (10⁻⁷ m) for a syncytiotrophoblast (ST) bathed in Ca²⁺-free extracellular medium. D, Ca²⁺ response to ET1 (10⁻⁷ m) after pre-incubation of the ST for 15 min in the presence of the phospholipase C (PLC) inhibitor, U73122 (2 μm). Arrows in A–D indicate the time of measurement used for the calculation of the percentage changes in ratios expressed in Fig. 4.

Figure 3. Effects of Ni²⁺, nifedipine and hyper-potassium solution on the ET1-induced Ca²⁺ response in a ST

Ca²⁺ response to ET1 (10⁻⁷ m) in the presence of 1 mm Ni²⁺ (A) or nifedipine (10⁻⁶ m; B). C, representative recording of the Ca²⁺ response to 100 mm K⁺ perfusion and subsequent response to ET1 perfusion (10⁻⁷ m). Arrows in A–C indicate the time of measurement used for the calculation of the percentage changes in ratios expressed in Fig. 4.

Figure 4. Average of percentage variation of fluorescence ratio during the spike obtained after various pharmacological treatments

Data represent means ± s.e.m.; ET1 alone (n = 49); ET1 + BQ123 (n = 24); ET1 + BQ788 (n = 13); ET1 +0 Ca²⁺ (n = 10); ET1 + U73122 (n = 12); ET1 + Ni²⁺ (n = 13); ET1 + nifedipine (n = 20); 100 mm K⁺ (n = 15). One-way analysis of variance revealed significant differences between treatments (F = 39.1; P < 0.0001; d.f. = 7). Dunnett's post hoc test showed a non-significant difference between ET1 alone and ET1 + BQ123, and that BQ788, 0 Ca²⁺, U73122 and Ni²⁺ significantly inhibited ET1-mediated effects on Ca²⁺ activity (***P < 0.01). On the other hand, nifedipine (Nif) slightly increased the ET1 response (**P < 0.05). Furthermore, hyper-potassium (HypK⁺) perfusion induced a slight decrease of the baseline Ca²⁺ level (two-tailed paired Student's t test; **P < 0.05).

Figure 5. Effects of SK&F96365 and LOE 908 on the ET1-induced calcium plateau phase

A and C, recordings of Ca²⁺ changes induced in response to ET1 (10⁻⁷ m) in the presence of 30 μm SK&F96365 (A) and 0.1, 1 and 10 μm LOE 908 (C) during the plateau phase. Arrows indicate the time of measurement used for the calculation of the percentage changes in ratios expressed in B and D. B, average percentage change in fluorescence ratio (F₄₀₅/F₄₈₅) for results of SK&F96365 treatment presented in A. Data represent means ± s.e.m.; ET1 plateau (n = 10); ET1 plateau + SK&F96365 (n = 10); (**P < 0.01; one-tailed paired Student's t test). D, average of percentage increases in fluorescence ratio (F₄₀₅/F₄₈₅) for results of LOE 908 treatment presented in A. Data represent means ± s.e.m.; ET1 plateau (n = 10); ET1 plateau + LOE 908 0.1 μm (n = 6); ET1 plateau + LOE 908 1 μm (n = 6); ET1 plateau + LOE 908 10 μm (n = 6); (*P < 0.05; one-tailed paired Student's t test).