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

Abstract

We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a sub clone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed, that after chronic morphine treatment (24 hr) of A7 cells, MOP binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was upregulated to 188% of control naïve cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced upregulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e. it decreased DAMGO-induced stimulation of GTPγS binding. In M2 cells DAMGO stimulation of GTPγS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture.

The Interaction Between the Mu Opioid Receptor and Filamin A

“…Even the mechanism of antagonist-induced uporegulation is not yet understood. Our results did provide preliminary evidence that neither increased protein synthesis nor decreased protein degradation appear to play a role, i.e., we saw no change in the density of the MOPr protein band in Western Blots [8].…”

“…A rather serendipitous finding was reported in a separate paper [8]. While investigating the downregulation of the MOPr in human melanoma cells, we found that in A7 cells chronic morphine induced a modest downregulation of [ 3 H]diprenorphine binding of ca.…”

“…Another interesting observation, summarized above, was the upregulation of MOPr by almost twofold in M2 cells, seen after long term treatment with morphine [8]. This is not seen after long term exposure to DAMGO, the ligand used in our earlier studies.…”

“…A similar result was obtained for the desensitization of forskolin-stimulated cAMP accumulation, which was absent in M2 cells. The activation of G protein, as measured by GTPcS binding, also failed to be desensitized in the absence of FLNA [8].…”

Filamin A Mutant Lacking Actin-Binding Domain Restores Mu Opioid Receptor Regulation in Melanoma Cells

“…Cell membranes from control and DAMGO-treated (5 lM for 24 h) M2, A7 and M2-ABD cells expressing myctagged MOPr were prepared as previously described [7] with some modifications. An aliquot of frozen membranes were diluted in a 50 mM Tris-HCl buffer, pH 7.4, containing 3 mM MgCl 2 , 0.2 mM EGTA, 100 mM NaCl, 10 lM GDP and 10 lg/ml Saponin.…”