Protective effects of morphine in peroxynitrite-induced apoptosis of primary rat neonatal astrocytes: potential involvement of G protein and phosphatidylinositol 3-kinase (PI3 kinase)

Abstract

Opiates, such as morphine, have been used extensively in the clinical management of pain due to their potent analgesic effect. Astrocytes, representing a major non-neuronal cell population in the CNS, contain opioid receptors that are actively involved in several brain functions. This study was designed to evaluate the effects by which morphine, a preferential mu-opioid receptor agonist, contributes to cytotoxicity of nitric oxide (NO) species, including NO and peroxynitrite (ONOO⁻), in primary rat neonatal astrocytes. Primary astrocytes isolated from the cerebral cortex of 1- to 2-day-old Sprague–Dawley rats were treated with morphine, naloxone, and 3-morpholinosydnonimine (SIN-1), a donor of peroxynitrite. Morphine significantly protected primary rat astrocytes from apoptosis mediated by sodium nitroprusside, an NO donor, and SIN-1 in a dose-dependent manner, whereas it did not in other types of cells including C6 glioma, RAW 264.7, and HL-60 cells. Moreover, naloxone antagonized the protective effects of morphine on SIN-1-induced apoptosis. Morphine also inhibited the nuclear condensation and fragmentation of SIN-1-treated cells that was antagonized by naloxone pretreatment. The protective role of morphine in SIN-1-induced apoptosis was dependent on an intracellular antioxidant system such as GSH. Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were prohibited by pretreatment with the G_i protein inhibitor, pertussis toxin, and the phosphatidylinositol 3-kinase (PI3 kinase) inhibitors, wortmannin and LY294002. Taken together, these results suggest that morphine may protect primary rat astrocytes from apoptosis by NO species via the signaling cascades that involve both G protein and PI3 kinase.

Introduction

¹Morphine and endogenous opioid ligands are implicated in diverse functions, from development to immune modulation in the central and peripheral nervous system [1], [2]. These functions are mediated mostly via specific opioid receptors, uniquely localized in different regions and types of brain cells of which there are three major types, namely mu, delta, and kappa [3]. These receptors are coupled with Bordetella pertussis toxin-sensitive G proteins, which also modulate adenylyl cyclase, the voltage-gated Ca²⁺ channel, and K⁺ conductance [4]. Opiates also modulate the viability of neuronal and glial cells via an opioid receptor-mediated mechanism [5]. Mechanisms underlying an effect of opioid on CNS are known to be mediated via immune mediators, such as cytokines, beta-chemokines, reactive oxygen intermediates (ROI), and NO, which are produced by activated glial cells including microglia and astrocytes.

Astrocytes regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn function on neural or glial cells. These cells are ubiquitous in the brain and more resistant to oxidative stress than oligodendrocytes or neurons. However, neurons may undergo degenerative changes when astrocytes damaged by oxidative stress do not generate sufficient neuropeptides and nerve growth factors. Recently, it has been reported that longer exposure to peroxynitrite (ONOO⁻), a reactant of NO with superoxide anion (O₂⁻), increases astrocyte death [6]. Hydrogen peroxide (H₂O₂) also induces the apoptosis of cultured primary astrocytes [7]. The cytotoxic effect of NO remains elusive, although it has been postulated that NO cytotoxicity might be mediated by peroxynitrite [8], [9], [10]. Peroxynitrite is a strong oxidant that damages subcellular organelles, membranes, and enzymes through nitration of proteins, lipid peroxidation, and direct breakage of DNA. In our experimental model, primary astrocytes underwent nuclear shrinkage, chromatin condensation, and nuclear fragmentation in reactive nitrogen intermediates (RNI)-induced astrocyte death. It has recently been reported that NO is involved in the antinociceptive effects of multiple opioid receptor agonists, which represents a possible interaction between NO and opioid systems [11]. Singhal et al. [12] have demonstrated that morphine enhances apoptotic death of macrophages through NO generation. However, Meriney et al. [13] have reported the possibility that morphine delays the neuronal cell death of avian ciliary ganglion through an inhibition of neurotransmission. In this study, we examined the effects of morphine and opioids on the free radical-induced death of rat primary astrocytes.