Modulation of opioid signal transduction in SH-SY5Y neural cells by differentiating agents: Concurrent mu receptor upregulation and effector desensitization by phorbol ester

Abstract

Long-term exposure of SH-SY5Y human neural cells to retinoic acid (RA) increased the binding of [3H] DAMGO and [3H]DPDPE by up to 3-fold compared to membranes of untreated cells. In contrast, incubation of the cells with phorbol ester (TPA) selectively up-regulated the binding of [3H]DAMGO. RA enhanced the maximal inhibition of cAMP formation in SH-SY5Y cells by both DAMGO and DPDPE from 20% for both opioids in control cells to 75% and 50%, respectively. On the other hand, the effect of TPA was limited to a marginal increase in the inhibition of cAMP formation by DAMGO. Parameters of GTP3PsS binding as well as low-Km GTPase activity revealed no deterioration in the structure or function of total G protein in the TPA-treated cells, and initial Western blots showed no difference in the cell content of Go. Ongoing experiments are focusing on the covalent modification of specific G protein subtypes involved in # and ~ opioid signal transduction in SH-SY5Y cells differentiated by TPA. METHODS:Human neuroblastoma cells SH-SY5Y were cultured and differentiated with RA or TPA for 9-11 days as described (1). Ligand binding was resolved and quantified by computer fitting using the statistical program SYSTAT, and the activities of adenylyl cyclase and GTPase were determined by a radioligand binding assay and measurement of released 32p, respectively (2). Binding of GTP3Pss (3), Western blotting (4), and the determination of protein (3) were implemented as described previously.

Desensitization of the μ‐opioid activation of phospholipase C in SH‐SY5Y cells: the role of protein kinases C and A and Ca2+‐activated K+ currents

“…The phosphorylation of ion channels by effector protein kinases has previously been proposed as one of the mechanisms underlying the rapid desensitization of a number of responses (Lohse, 1993). It is worth noting here that the y-opioidmediated opening of the L-type VSCC in SH-SY5Y cells involves a pertussis toxin-sensitive G-protein 1995), and that PKC has been shown to phosphorylate, and thus inactivate, such G-proteins in SH-SY5Y cells (Lin et al, 1994). In addition, it has been reported that PKC uncouples 3-opioids from their G-proteins in guinea-pig striatal membranes .…”

“…However, all these mechanisms potentially involve increased protein kinase C (PKC) activity (Lohse, 1993). Indeed, PKC has been reported to uncouple G-proteins from opioid receptors in guinea-pig striatal membranes and SH-SY5Y cells (Lin et al, 1994). Furthermore, PKC is known to modulate the activity of various ion channels (Shearman et al, 1989;Petersen & Berridge, 1994;Tuominen et al, 1994), including the L-type VSCC (Schuhmann & Groschner, 1994).…”

Dissociation of Tolerance and Dependence for Opioid Peripheral Antinociception in Rats

“…Receptor phosphorylation and uncoupling of G-proteins from cell-surface receptors have been implicated in opioid tolerance and dependence (Fukagawa et al, 1992;Escriba et al, 1994;Sim et al, 1996). G-proteins can be coupled from opioid receptors by increased activity of the second messenger protein kinase C (PKC) (Fukushiama et al, 1994;Lin et al, 1994), and translocation and activation of PKC in spinal cord dorsal horn neurons have been implicated in the development of tolerance and dependence on the antinociceptive effects of morphine (Mayer et al, 1995a,b). The second messenger nitric oxide (NO) has also been suggested to be involved in morphine tolerance (Kolesnikov et al, 1993;Elliott et al, 1995;Herman et al, 1995;Pasternak et al, 1995;Vaupel et al, 1995;Dunbar and Yaksh, 1996); it was demonstrated that the NO synthase (NOS) inhibitor N G -methyl-Larginine (NMLA) prevents the development of antinociceptive tolerance and /or dependence on systemic morphine in the mouse (Kolesnikov et al, 1993;Majeed et al, 1994).…”