μ‐Opioid Agonist Inhibition of κ‐Opioid Receptor‐Stimulated Extracellular Signal‐Regulated Kinase Phosphorylation Is Dynamin‐Dependent in C6 Glioma Cells

Opioid receptors (ORs) are members of the heptahelical, G protein-coupled receptor (GPCR) family that are targets of endogenous opioid peptides as well as opiate alkaloids, such as morphine. Opioid effects on neural development primarily entail suppression of neural cell proliferation as seen in studies performed in vivo (Johannesson et al., 1972; Vertes et al., 1982; Zagon and McLaughlin, 1983, 1991; Kornblum et al., 1987; Bartolome et al., 1991) and in primary cultures from embryonic brain (Barg et al., 1992, 1993a,b; for review, see Hammer and Hauser, 1992). The effects of opioids on glial cells have proven diverse but often also result in the inhibition of proliferation (Stiene-Martin and Hauser, 1991; Barg et al., 1993a; Gurwell et al., 1996; Hauser et al., 1996). The rat C6 glioma cell line expresses κ-ORs and low levels of a novel type of μ-OR that has been demonstrated by radioligand binding (Albouz et al., 1982; Reggiani et al., 1987; Barg et al., 1991; Bohn et al., 1998), RT-PCR (Bohn et al., 1998), and RNase protection assays (L.M.B., unpublished data). The C6 cells have been used as a model system to study opioid inhibition of mitogen-stimulated DNA synthesis (Barg et al., 1994). Endothelin-1 stimulates C6 cell proliferation that correlates with increases in DNA synthesis, phosphoinositide (PI) turnover, extracellular signal-regulated kinase (ERK) activity, and Ca2+ influx (MacCumber et al., 1990; Chuang et al., 1991; Couraud et al., 1991; Lin et al., 1992; Barg et al., 1994; Lazarini et al., 1996) similar to the extent of stimulation obtained with the κ-opioid agonist as reported in our preceding article (Bohn et al., 2000). The μ-OR agonists morphine, β-endorphin, and a morphine-mimicking, antiidiotypic antibody (Coscia et al., 1991) inhibit PI turnover, Ca2+ influx, and growth of endothelin-stimulated C6 cells (Barg et al., 1994). In our preceding article we reported that activation of endogenous κ-ORs stimulates C6 cell DNA synthesis by a mechanism that also involves activation of PI turnover and ERK1/2 phosphorylation (Bohn et al., 2000). This raises the question of whether the highly selective, endogenous μ-OR agonist endomorphin-1 (Zadina et al., 1997) attenuates κ-OR-mediated stimulation of DNA synthesis, PI turnover, and ERK1/2 phosphorylation in C6 cells. Internalization of GPCRs may represent a prerequisite event in initiating mitogenic signaling (for review, see Lefkowitz, 1998). Dynamin mediates clathrin-dependent internalization of many receptors by participating in the “pinching off” of endocytotic vesicles from the plasma membrane (Hinshaw and Schmid, 1995; Takei et al., 1995; Wang et al., 1995). A mutation in the dynamin molecule (K44A or K44E) rendering it GTPase-defective creates a dominant suppressor mutant that blocks epidermal growth factor receptor and GPCR endocytosis (Vieira et al., 1996; Zhang et al., 1996; Chu et al., 1997; Daaka et al., 1998). Such suppressors of receptor endocytosis can attenuate GPCR-mediated activation of ERK without interfering with classical second messenger [cyclic AMP, inositol phosphates (IPs), and Ca2+] production (Luttrell et al., 1997a; Daaka et al., 1998; Ignatova et al., 1999). In addition, ERK activation by β2-adrenergic receptors expressed in human embryonic kidney (HEK) 293 cells (Daaka et al., 1998), LPA receptors expressed in Rat-1 cells (Luttrell et al., 1997a), and μ-, κ-, and δ-ORs expressed in HEK293 or COS-7 cells (Ignatova et al., 1999) requires functional dynamin. In this article we show that μ-OR agonists inhibit not only endothelin-stimulated DNA synthesis, but also the κ-OR-mediated stimulation of DNA synthesis, PI turnover, and ERK phosphorylation. Moreover, κ-opioid agonist phosphorylation of ERK is unchanged in C6 cells on overexpression of dynamin K44A, whereas μ-opioid agonist inhibition of κ-opioid mitogenic signaling is attenuated.