Elsevier

Brain Research

Volume 855, Issue 2, 14 February 2000, Pages 244-251
Brain Research

Research report
Possible involvement of cytosolic phospholipase A2 in cell death induced by 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in GH3 cells

https://doi.org/10.1016/S0006-8993(99)02340-9Get rights and content

Abstract

Previously we reported that 1-methyl-4-phenylpyridinium ion (MPP+), a dopaminergic neurotoxin, induced apoptosis of GH3 cells established from rat anterior pituitary. In the present study, the role of MPP+ along with that of other apoptotic factors such as Ca2+ and H2O2 in cell death was examined. Ionomycin induced DNA fragmentation and lactate dehydrogenase (LDH) leakage in GH3 cells. H2O2 also induced LDH leakage. Co-addition of MPP+, in conditions where MPP+ had no effect by itself, enhanced ionomycin- and H2O2-induced cell death. Because the stimulation of phospholipase A2 (PLA2) causing arachidonic acid (AA) release has been proposed to be involved in neuronal cell death, the effect of MPP+ on AA release in GH3 cells was investigated. MPP+ treatment for 8 h enhanced ionomycin- and H2O2-stimulated AA release mediated by activation of cytosolic PLA2 in a concentration-dependent manner, although MPP+ by itself had no effect on AA release. An inhibitor of cytosolic PLA2 inhibited MPP+-induced cell death. These findings suggest a synergistic effect of MPP+ on Ca2+- and H2O2-induced cell death, and the involvement of cytosolic PLA2 activation in MPP+-induced cell death in GH3 cells. Pretreatment with a caspase inhibitor or EGF did not modify the ionomycin- or H2O2-induced AA release, or enhancement by MPP+, but the pretreatment inhibited the cell death in the presence and absence of MPP+. The involvement of caspase(s) on activation of PLA2 by MPP+ was excluded, and EGF inhibited MPP+-induced cell death downstream of the AA release.

Introduction

The major anatomical abnormality characterizing Parkinson's disease is the death of dopaminergic neurons in the substantia nigra. The selective dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been used to create animal models of dopaminergic denervation and also produces Parkinson's syndrome in humans (for review, see Refs. 4, 25). MPTP is metabolized by monoamine oxidase B to 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite. MPP+ is thought to be incorporated by the dopamine transport system and induce cell death [25]. Recent studies show that programmed cell death known as apoptosis can be induced by MPP+ in various neuronal cells 7, 9, 10, 22. Previously we reported that GH3 cell line, a clonal strain established from rat anterior pituitary, is an appropriate model for dopaminergic neurons and MPP+ induced GH3 cell death [32]. MPP+-induced cell death of GH3 cells was shown to be apoptosis accompanied with release of cytochrome C from mitochondria, activation of caspase(s) and DNA fragmentation [33]. LDH leakage [32]and DNA fragmentation [33]by MPP+ was inhibited in GH3 cells treated with EGF. It has been shown that MPP+ causes marked inhibition of the mitochondrial respiratory chain and an energy crisis, but a number of mechanisms and/or other factors may also mediate MPP+ toxicity 4, 25. Recently, Sawada et al. [21]reported that MPP+ at nonlethal concentrations enhanced glutamate-induced cytotoxicity in dopaminergic neurons. Because chronic neurodegenerative disorders including death of dopaminergic neurons may be induced by a number of different pathways and/or factors, it is important to investigate the involvement of MPP+ with other factors which regulate cell death and/or apoptosis.

Numerous reports have shown that the disruption of Ca2+ homeostasis induces apoptosis of cells (for review, see Ref. [15]). The addition of Ca2+ to isolated nuclei can directly promote apoptotic nuclear changes by activation of caspase-3-like enzyme 11, 14. Activation of N-methyl-d-aspartate receptor, a type of glutamate receptor, stimulated Ca2+ influx and phospholipase A2 (PLA2) activity, and the subsequent release of arachidonic acid (AA) leads to the generation of reactive oxygen species (ROS) including H2O2[5]. ROS is another factor causing dopaminergic cell death, and neurotoxicity of MPP+ is at least partially mediated by ROS [4]. We have shown that addition of H2O2 induces DNA fragmentation of GH3 cells [31]. In neuronal cells, however, the interactive effect of MPP+ on Ca2+- and H2O2-induced cell death and/or apoptosis has not been extensively investigated.

The stimulation of PLA2 causing AA release is essential for cytotoxic action and/or apoptosis by stimulants including tumor necrosis factor 8, 12, 24, 27, 28, 30. Oxidative stress such as ischemia increases Ca2+-sensitive cytosolic PLA2 expression and free AA (and its metabolites) in brain (for review, see Ref. [2]). Bonventre et al. [3]have created “knockout” mice that lack cytosolic PLA2, and reported that these mice had smaller infarcts and developed less severe brain edema. These reports suggest the existence of a vicious cycle between Ca2+, ROS and AA in neuronal cells. However, MPP+ involvement in PLA2 activity and AA release has not been studied. In this study, we investigated the effects of MPP+ on Ca2+- and H2O2-induced cell death and AA release in GH3 cells. MPP+ enhanced cell death and AA release induced by ionomycin and H2O2 in GH3 cells. A possible role for PLA2 in MPP+-induced cell death and/or apoptosis is also discussed.

Section snippets

GH3 cells

GH3 cells obtained from the American Type Culture Collection (Rockville, MD, USA) were suspended and grown as described previously 31, 32, 33. A total of 48 h before the experiment, cells were plated in dishes and grown as monolayers in F10 medium with 15% horse serum (HS) and 5% fetal calf serum (FCS).

Materials

MPP+ iodide was purchased from Research Biochemicals (Natick, MA, USA). [5,6,8,9,11,12,14,15-3H]AA (215 Ci/mmol, 7.96 TBq/mmol) and [methyl-3H]thymidine (2 Ci/mmol, 74 GBq/mmol) were purchased

Ionomycin-induced apoptosis of GH3 cells and its enhancement by MPP+

A clonal strain, GH3 cells, is a model for studying the regulation of neuronal hormone secretion in anterior pituitary. Previously we reported that GH3 cell line is an appropriate model for dopaminergic neurons and that MPP+ induces cell death of GH3 cells [32]. Ca2+ is involved in several steps of cell death and apoptosis (for review, see Ref. [14]). First we investigated the effect of ionomycin on an apoptotic marker, DNA fragmentation in GH3 cells (Table 1). Ionomycin (1 μM) induced DNA

Synergistic effect of MPP+ on ionomycin- and H2O2-induced cell death of GH3 cells

MPP+ induced cell death [32]and apoptosis [33]in GH3 cells. In this study we examined the effect of MPP+ on other stimulants causing cell death. In GH3 cells, ionomycin caused DNA fragmentation (Table 1) and LDH leakage (Table 2). Ionomycin-induced DNA fragmentation was inhibited in GH3 cells treated with 50 ng/ml of EGF (Table 1), and with ZVAD-fmk as described in Section 3. Addition of H2O2 induced LDH leakage in a ZVAD-fmk-sensitive manner. These findings suggest that Ca2+ and H2O2

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