Down-regulation of μ-Opioid Receptor by Full but Not Partial Agonists Is Independent of G Protein Coupling

Abstract

SUMMARYIn C 6 glial cells stably expressing rat -opioid receptor, opioid agonist activation is negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. H]DAMGO binding in membranes with the rank order of etorphine Ͼ DAMGO ϭ ␤-endorphin Ͼ morphine Ͼ butorphanol, and the affinity of DAMGO in alkaloid-but not peptide-treated membranes was significantly lower in comparison with control. Pertussis toxin treatment of the cells before agonist treatment did not prevent the down-regulation by full agonists; DAMGO and etorphine exhibited ϳ80% internalization, whereas the ability of partial agonists was greatly impaired. In addition to establishing this cell line as a good model for further studies on the mechanisms of opioid tolerance, these results indicate important differences in the inactivation pathways of receptor triggered by full and partial agonists.Opioid receptors are activated by endogenous opioid peptides and alkaloids, which cause a multitude of important physiological functions. Recent cloning of -, ␦-, and -opioid receptors showed that these proteins contain seven transmembrane domains and belong to the family of GPCRs (1). The -opioid receptor is the molecular target for potent analgesics such as morphine and fentanyl, which are indispensable in the management of pain despite their abuse potential (2). The biochemical mechanisms of tolerance have been studied in many systems, including cell lines containing ␦-opioid receptors such as N4TG1 (3) and NG108 -15 (4) cells. Although studies conducted in the central nervous system often led to inconsistent results due to the heterogeneity of the system, experiments carried out in a single brain region, such as locus ceruleus, demonstrated the physiological relevance of the cellular model originally proposed by Sharma et al. (5) in NG108 -15 cells based on the alterations in the opioid/AC system. Subsequent studies using 7315c (6) and SH-SY5Y (7, 8) cells examined altered properties of -opioid receptor/effector components during tolerance; however, the exact mechanisms involved in this process are largely unknown. To study the molecular mechanisms of -opioid receptor selectively, we transfected C 6 glial cells that express many other receptors, but not opioid receptors (9), with the rat receptor cDNA. Transfected receptor in these cells is coupled to AC through PTX-sensitive G proteins (10). We characterized opioid agonist efficacies (11) and showed that this cell line exhibits sodium regulation of receptor in much the same fashion as SH-SY5Y cells (12).The major goal of the current study was to investigate the molecular changes involved in the development of tolerance by different agonists of varying efficacies. In the C 6 cell line stably expressing high levels of receptor (ϳ8 pmol/mg), tolerance to peptides and alkaloids was induced, and alterations were examined at every step of the signal transduction pathway (i.e., ligand/receptor interactions, G protein and effector functions). The diminished receptor activation of G protein, ...

Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking filamin A

“…In contrast, PTX treatment did not have any effect on morphine-induced down-regulation of MOP in A7 cells. Yabaluri et al (Yabaluri and Medzihradsky, 1997) reported that PTX treatment did not block agonist-induced down-regulation of MOP in C6 glioma cells. However, it should be noted that PTX treatment of N2A cells expressing MOP blocked etorphine-induced MOP down-regulation (Chakrabarti, 1997).…”

“…Extensive studies in many different cell lines demonstrated MOP down-regulation by different agonists (Baumhaker et al, 1993;Chakrabarti et al, 1995;Kato et al, 1998;Yabaluri and Medzihradsky, 1997). Chronic morphine-induced MOP down-regulation has been well investigated and characterized in a various cell culture systems, such as SH-SY5Y (Zadina et al, 1993), HEK (Onoprishvili et al, 1999), CHO (Kato et al, 1998), C6 (Yabaluri and Medzihradsky, 1997), Neuro2a (Chakrabarti et al, 1995), 7315c (only 20%) (Puttfarcken and Cox, 1989) and SK-N-SH (Baumhaker et al, 1993).…”

Butorphanol: Effects of a Prototypical Agonist-Antagonist Analgesic on κ-Opioid Receptors

“…Butorphanol pretreatment was associated with little or no tolerance development, with maximal DAMGOinduced binding of 325% above the control. Similarly, as a partial µ-opioid receptor agonist, 24-h treatment with butorphanol could reduce B max for [ by 97% from the control (45). The effects of opioid receptor binding in the whole organism are less clearly defined.…”

“…This agonist activity can be contrasted directly with the responses to butorphanol in C6 cells expressing the rat µ-opioid receptor (45). Treatment of such cells with the selective rat µ-opioid receptor agonist DAMGO stimulated […”

The Intracerebroventricular Administration of W-7, A Calmodulin Inhibitor, Attenuate the Development of Morphine Tolerance in Rats

“…Increased in cytosolic calcium concentration activate several intracellular enzymes including protein kinases (Wroblewski and Danysz, 1989). Protein kinases such as Ca 2+ /Calmodulin-dependent protein Kinases (CaMK) have been reported to phosphorylate opioid receptor, leading to receptor desensitization (Koch et al, 1997;Mestek et al, 1995); a phenomenon which plays a critical role in opioid tolerance (Breivogel et al, 1997;Yabaluri and Medzihradsky, 1997). If this were to be the case, an essential process for opioid receptor phosphorylation and desensitization could be the activation of the Ca 2+ /calmodulin complex.…”