Effects of methyl p-hydroxybenzoate (methyl paraben) on Ca2+ concentration and histamine release in rat peritoneal mast cells

Abstract

1 Mechanisms of methyl p-hydroxybenzoate (methyl paraben) action in allergic reactions were investigated by measuring the intracellular Ca(2+) concentration ([Ca(2+)](i)) and histamine release in rat peritoneal mast cells (RPMCs). 2 In the presence or absence of extracellular Ca(2+), methyl paraben (0.1-10 mM) increased [Ca(2+)](i), in a concentration-dependent manner. Under both the conditions, methyl paraben alone did not evoke histamine release. 3 In RPMCs pretreated with a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate (PMA) 3 and 10 nM), methyl paraben (0.3-3 mM) induced histamine release. However, a high concentration (10 mM) of the agent did not increase the histamine release. 4 U73122 (0.1 and 0.5 micro M), an inhibitor of phospholipase C (PLC), significantly inhibited the methyl paraben-induced histamine release in PMA-pretreated RPMCs. U73343 (0.5 micro M), an inactive analogue of U73122, did not inhibit the histamine release caused by methyl paraben. 5 In Ca(2+)-free solution, PLC inhibitors (U73122 0.1 and 0.5 micro M, D609 1-10 micro M) inhibited the methyl paraben-induced increase in [Ca(2+)](i), whereas U73343 (0.5 micro M) did not. 6 Xestospongin C (2-20 micro M) and 2 aminoethoxydiphenyl borate (30 and 100 micro M), blockers of the inositol 1,4,5-trisphosphate (IP(3)) receptor, inhibited the methyl paraben-induced increase in [Ca(2+)](i) in Ca(2+)-free solution. 7 In conclusion, methyl paraben causes an increase in [Ca(2+)](i), which may be due to release of Ca(2+) from storage sites by IP(3) via activation of PLC in RPMCs. In addition, methyl paraben possibly has some inhibitory effects on histamine release via unknown mechanisms.

Figure 1

Effects of methyl paraben on [Ca²⁺]_i in RPMCs. The fluo-3 AM-loaded RPMCs were stimulated with methyl paraben for 75 s at room temperature (22–25°C). (a) Methyl paraben (3 mM) increased the [Ca²⁺]_i in an RPMC incubated in PSS containing 2 mM Ca²⁺. (b) Methyl paraben was applied in Ca²⁺-free solution containing 0.5 mM EGTA after a 5 min removal of Ca²⁺. Also in Ca²⁺-free condition, methyl paraben increased the [Ca²⁺]_i in an RPMC. (c) The Concentration–response relation of methyl paraben-induced [Ca ²⁺]_i increase in Ca²⁺-containing (open circles) and Ca²⁺-free (filled circles) mediums. The ordinate shows the net maximum [Ca²⁺]_i of the response with baseline subtracted. The data points indicate mean±s.e.m. of 20 experiments. The asterisk shows a significant difference in Ca²⁺-containing and Ca²⁺-free solutions.

Figure 2

Effects of methyl paraben (3 and 10 mM) and various stimuli on histamine release from RPMCs. Cells were preincubated for 5 min at 37°C and subsequently incubated with methyl paraben (3 and 10 mM), compound 48/80 (0.3 μg ml⁻¹) or a combination of A23187 (0.1 μM) and PMA (10 nM) for 30 min in the external solution with (open columns) or without (closed columns) Ca²⁺. The released histamine in the 30 min period was calculated as a percentage of the total histamine content of the cells. Results are expressed as mean±s.e.m. of eight to 10 experiments.

Figure 3

Effects of combined application of methyl paraben and PMA on histamine release in PSS. Cells were preincubated for 5 min at 37°C in the external solution with Ca²⁺ and subsequently incubated without (open circles) or with PMA (3 nM, closed circles or 10 nM, closed squares) for 5 min. The cells were then stimulated with methyl paraben (3 mM) for 30 min in the continuous presence or absence of PMA. The spontaneous histamine release was used for normalization as a relative histamine release of 1.0. The symbols refer to the mean of four to 12 experiments and the error bars represent s.e.m.

Figure 4

Effects of U73122 and U73343 on histamine release induced by a combination of PMA and methyl paraben. After preincubation for 5 min at 37°C in PSS, cells were pretreated with PMA (10 nM) or a combination of PMA and U73122 (0.1 and 0.5 μM) or U73343 (0.5 μM) for 5 min. The cells were then stimulated with methyl paraben (closed columns) or not (hatched columns) and incubated for 30 min in PSS with continued treatment of PMA in the continuous presence or absence of U73122 or U73343. The spontaneous histamine release was used for normalization as a relative histamine release of 1.0 (open column). Results are expressed as mean±s.e.m. of five to 12 experiments. Asterisks indicate a significant difference from the combined application of PMA and methyl paraben in the absence of U73122 or U73343 with P<0.05.

Figure 5

Effects of U73122, U73343 and D609 on the increase in [Ca²⁺]_i induced by methyl paraben in Ca²⁺-free solution containing 0.5 mM EGTA. Methyl paraben (3 mM) was applied for 60–75 s following a 5 min incubation in the Ca²⁺-free solution. (a) Methyl paraben increased the [Ca²⁺]_i in a fluo-3 AM-loaded RPMC. (b) Pretreatment good for 5 min with U73122 (0.5 μM) markedly suppressed the increase, while U73343 (0.5 μM) had no effects. (c) Pretreatment for 5 min with D609 (10 μM) completely blocked the effect of methyl paraben. (d) Each column indicates the average of the peak increase above the base line (n=8–10). U73122 and D609 suppressed the [Ca²⁺]_i increase dose-dependently. The reduction by U73343 was not significant. Bars indicate s.e.m. Asterisks indicate significant difference from the control with P<0.05.

Figure 6

Effects of xestospongin C and 2APB on the increase in [Ca²⁺]_i induced by methyl paraben in Ca²⁺-free solution containing 0.5 mM EGTA. Methyl paraben (3 mM) was applied for 70–75 s in Ca²⁺-free solution after a 5 min removal of Ca²⁺. (a) Methyl paraben increased the [Ca²⁺]_i in a fluo-3 AM-loaded RPMC. (b) Pretreatment for 5 min with xestospongin C (6 or 20 μM) markedly suppressed the [Ca²⁺]_i increase. (c) Pretreatment for 5 min with 2APB (100 μM) greatly decreased the [Ca²⁺]_i increase. (d) Each column indicates the average of the peak increase above the base line (n=7–10). Xestospongin C and 2APB dose-dependently suppressed the [Ca²⁺]_i increase produced by methyl paraben. Bars indicate s.e.m. Asterisks show significant suppression from the control with P<0.05.