Rapid Disruption of Cellular Integrity of Zinc-treated Astroglia Is Regulated by p38 MAPK and Ca-dependent Mechanisms

Abstract

Cultured cortical primary astroglia treated with zinc died while rapidly detached from culture plates, a distinct part of zinc-treated astroglia. In the present study, we investigated the mechanism underlying the rapid change in the morphologic integrity of zinc-treated astroglia. Among the early cellular events occurring in zinc-treated astroglia, strong activation of p38 MAPK and JNK was evident. Although inhibitors of p38 (SB203580 and SB202190) or JNK (SP600125) did not protect zinc-insulted astroglia from cell death, the p38 inhibitors, but not the JNK inhibitor, suppressed actin filament and cell morphology disruption. The Ca(2+) ionophore, A23187, also suppressed actin filament and cell morphology disruption, but not cell death, of zinc-insulted astroglia. However, A23187 did not inhibit p38 MAPK activation in zinc-treated astroglia. Together these results suggest that zinc influx in astroglia results in rapid loss of the morphologic integrity via mechanisms regulated by p38 kinase and/or Ca(2+) signaling.

Keywords: actin filament; astroglia; morphology protection; p38 inhibitors; zinc.

Fig. 1

p38 and JNK MAPKs were activated in zinc-treated astroglia. (A, B) Astroglial cell death by zinc increased in a zinc-dose (A) and zinc-treated time (B) dependent manner. LDH assay was performed after 24 h-exposure of cultures to indicated concentrations of zinc. (C) Western blots showing zinc-dose dependent activation of p38 and JNK MAPKS in astroglia. Assay was performed 3 h after zinc treatment. Anti-phospho-p38 MAPK and anti-phospho-JNK were used to deduce the activation levels of these kinases. (D) Western blots showing time-dependent activation of p38 and JNK MAPKS in zinc (35 µM)-treated astroglia.

Fig. 2

The p38 MAPK inhibitor, SB203580, suppressed zinc-induced morphology disruption and detachment from the culture plate. (A~H) Phase contrast (A~D) or trypan-blue stained (E~H) astroglia with sham treatment (A, E) or astroglia treated with zinc (B, F), with zinc and SP600125 (C, G), or with zinc and SB203580 (D, H). Photomicrographs were taken 24 h after zinc (35 µM)-treatment in the presence of SP600125 (20 µM) or SB203580 (20 µM). Scale bar represents 100 µm. (I) LDH assay showing cell death levels of zinc (35 µM)-treated astroglia in the presence of PD98059 (PD: 20 µM), SP600125 (SP: 20 µM), or SB203580 (SB: 20 µM). LDH assay was performed 24 h after zinc treatment.

Fig. 3

The Ca²⁺ ionophore, A23187, suppressed cell morphology disruption of zinc-treated astroglia. (A) LDH assay showing cell death levels of astroglia treated with A23187 (0.1 µ M), zinc (35 µM), zinc (35 µM)+A23187 (0.1 µM), or zinc (35 µM)+SB203580 (20 µM). LDH assay was performed after 24 h of drug treatment. (A) A23187; SB, SB203580. (B~E) Trypan-blue stained astroglia treated with zinc (35 µM), zinc (35 µM)+SB203580 (20 µM), A23187 (0.1 µM) or zinc (35 µM)+A23187. Photomicrographs were taken after 24 h of zinc treatment in the presence of SB203580 or A23187. (F~K) Trypan-blue stained astroglia with sham treatment (F, G), with zinc (H, I), or with zinc and SB203580 (J, K) in the presence or absence of the Ca²⁺ chelator, BAPTA (10 µM). Photomicrographs were taken 24 h after treatment of zinc (35 µM) and SB203580 (20 µM). (L) LDH assay showing cell death levels of astroglia treated with zinc (35 µM) or zinc (35 µM)+SB203580 (20 µM) in the presence or absence of BAPTA (10 µM). LDH assay was performed after 24 h of drug treatment. Scale bar represents 100 µm. SB, SB203580. The data are presented as means±S.E.M. (n=6~12). ^**Denotes difference at p<0.05.

Fig. 4

SB203580 and A23187 suppressed actin-filament disruption in zinc-treated astroglia. (A~E) Photomicrographs showing actin-filament distributions in zinc-treated astroglia stained with TRITC-labeled phalloidin. Sham control (A) and astroglia treated with Zn (35 µM) alone (B), Zn (35 µM)+SB203580 (20 µM) (C), A23187 (0.1 µM) alone (D), or zinc (35 µM)+A23187 (0.1 µM) (E). Actin-filament disruptions were visualized after 10 h of zinc treatment. Scale bar represents 100 µm.

Fig. 5

A23187 did not suppress p38 MAPK activation in zinc-treated astroglia. Western blots showing p38 MAPK activation levels in zinc-treated astroglia. Western blot analysis was performed after 3 h of treatment with zinc (40 µM) alone or zinc (40 µM)+A23187 (A: 0.1 µM).