μ-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase
Introduction
Endogenous opioid peptides and alkaloids such as morphine mediate their biological effects through membrane-bound heptahelical proteins called opioid receptors. The three main types of receptors for opioid drugs, μ (also known as OP3), δ (OP1) and κ (OP2) receptors (Dhawan et al., 1996) belong to the G protein-coupled receptor (GPCR) family (Connor and Christie, 1999). Agonist activation of GPCRs leads to a physical interaction between the receptor and the GDP-bound G protein heterodimer, which causes the dissociation of the guanine nucleotide and the incorporation of GTP in the G protein α subunit, thus releasing the βγ heterodimer. In turn, GTP-bound α subunits and βγ complexes initiate, independently, a variety of intracellular signaling pathways (Gilman, 1987).
Evidence has accumulated that, in addition to regulate classic effectors such as adenylate cyclase, phosphodiesterases, phospholipases and ion channels, some members of the GPCRs family are able to activate intracellular kinase cascades implicated in cell survival and translational control, such as the mitogen-activated protein kinases (MAPKs) and the phosphatidylinositol 3-kinase (PI 3-K)/protein kinase B (also known as Akt) signalling pathways (for a review see Katada et al., 1999, Lopez-Ilasaca, 1998, Luttrell et al., 1999). In this context, specific Gβγ heterodimers may directly activate PI 3-Ks (Kurosu et al., 1997, Leopoldt et al., 1998, Murga et al., 1998, Murga et al., 2000); whereas Gα subunits have no stimulatory effect (Bommakanti et al., 2000). Activated PI 3-Ks phosphorylate membrane inositol lipids at the D-3 position of the inositol ring, thus generating binding sites for the pleckstrin-homology (PH) domain of Akt. Once in the plasma membrane, Akt is fully activated by phosphorylation at specific residues (Alessi and Cohen, 1998).
It is well documented that when Akt is phosphorylated in response to a wide variety of growth factor stimuli, it plays a key role in promoting cell survival by opposing apoptotic pathways (Dudek et al., 1997, Lawlor and Alessi, 2001). In this context, morphine administration has been shown to reduce naturally occurring neuronal death (Meriney et al., 1985) and a role for endogenous opioid peptides acting as trophic factors regulating neuronal survival and cell proliferation during nervous system development has been proposed (Meriney et al., 1991, Pérez-Navarro et al., 1993, Schmahl et al., 1989). Moreover, the μ selective agonist [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) has synergistic effects with nerve growth factor (NGF) on neuronal survival in culture (Sakaguchi et al., 1999) and opioid agonists transiently prevent activation of apoptotic cell death following serum withdrawal in PC12 cells (Dermitzaki et al., 2000). However, no evidence has been reported for the implication of opioid-mediated PI 3-K/Akt activation on these cell survival-promoting effects.
The present work was designed first, to test whether activation of the μ-opioid receptor could have protective effects on neuronal survival after trophic factor deprivation and secondly, to study if these effects could be mediated through the PI 3-K/Akt signalling cascade. We first tested these hypotheses in the SH-SY5Y neuroblastoma cell line. This cell line is a subclone derived from the parent SK-N-SH that expresses predominantly μ-opioid receptors and, thus, has been widely employed as a model system for studying the molecular mechanisms of the function of these receptors (Elliott et al., 1997, Zadina et al., 1994). We have previously developed a protocol of differentiation for the SH-SY5Y cell line based on the sequential treatment with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) in serum-free medium. This procedure yields homogenous populations of non-proliferating cells exhibiting neuronal phenotype and depending on the continuous presence of BDNF for survival (Encinas et al., 2000). Removal of this neurotrophin or selective blockade of the PI 3-K/Akt pathway causes apoptotic cell death (Encinas et al., 1999, Encinas et al., 2000). Then, we tested the potential protective effects of μ-opioid receptor agonists on apoptotic cell death in a primary culture of mouse cortical neurons. It is well established that in these cells the PI 3-K/Akt pathway is necessary and sufficient for serum as well as BDNF-mediated survival (Hetman et al., 1999).
Section snippets
Materials
DAMGO and β-funaltrexamine (β-FNA) were purchased from Tocris Cookson Ltd (Bristol, UK). Morphine HCl was from Unión Química Farmacéutica, S.A.E. (Madrid, Spain). Human recombinant BDNF was from Alomone Laboratories (Jerusalem, Israel). The PI 3-K inhibitor LY294002 and the MAPK and extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD 98059 were from Calbiochem-Novabiochem (Läufelfingen, Switzerland). Naloxone HCl, pertussis toxin (PTX), culture media and other reagents were
Effects of μ-opioid agonists on SH-SY5Y survival following serum withdrawal
To study possible survival-promoting effects of μ-agonists, cell death was induced in SH-SY5Y cells by culturing them in serum-free medium. RA-differentiated SH-SY5Y cells in the presence of BDNF (50 ng/ml) were taken as 100% of survival (Encinas et al., 2000). Serum withdrawal for 24 h caused a significant decrease (48%) in the ability to metabolize MTT to formazan (Fig. 1(a)); indicating loss of mitochondrial membrane potential in dying cells (Ankarcrona et al., 1995). The presence of the
Discussion
Evidence has accumulated that, in addition to survival factors, agonists of the GPCR family can activate the PI 3-K/Akt pathway; some of these receptors include the β-adrenergic receptor (Moule et al., 1997), the m1 and m2 muscarinic receptors (Murga et al., 1998), the lysophosphatidic acid (LPA1/VZG-1) receptor (Weiner and Chun, 1999), the CB1 cannabinoid receptor (Gómez del Pulgar et al., 2000), the A2A adenosine receptor (Lee and Chao, 2001) and the μ-opioid receptor (Polakiewicz et al., 1998
Acknowledgements
This study was supported by Spanish Govern ‘Comisión Interministerial de Ciencia y Tecnología’ through the ‘Plan Nacional de Salud y Farmacia’ Grants SAF2000-0164-CO2-02 (G.O.) and SAF2000-0164-CO2-01 (J.X.C.). Research of the J.X.C. group is supported by ‘Generalitat de Catalunya (Distinció per a Joves Investigadors Universitaris and Suport a Grups de Recerca)’. M.F.S. was supported by a predoctoral fellowship from ‘Ministerio de Educación, Cultura y Deporte’ (Spain). We thank I. Sanchez, R.
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