Mu-opioid agonist inhibition of kappa-opioid receptor-stimulated extracellular signal-regulated kinase phosphorylation is dynamin-dependent in C6 glioma cells

Abstract

In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.

FIG. 1

μ-Opioids selectively inhibit endothelin-stimulated DNA synthesis. C6 cells were kept in “low MEM” for 48 h before treatment. For the final 20 h of this 48-h period, 5 μM DMI was included for some assays. Cells were treated with opioids for 1 h, and then endothelin-1 (Et-1) was added (in the presence of opioids). After 30 min, [³H]thymidine (0.5 μCi/ml) was included in the medium, and [³H]thymidine incorporation was determined after 23 h by scintillation counting. Data have been normalized to represent percent incorporation over unstimulated controls. A: Morphine (1 μM) inhibits ET-1 (30 nM) stimulated [³H]thymidine incorporation. *Significantly greater than control, ^#significantly less than ET-1, p < 0.01. Data are mean ± SEM (bars) values of three to six experiments performed in triplicate. B: Endomorphin-1 (Em-1; 1 μM) inhibits ET-1 (30 nM) stimulated [³H]thymidine incorporation. These effects are reversed by the opioid antagonist naloxone (Nalox; 1 μM, 1 h before Em-1). *Significantly greater than control, ^#significantly less than ET-1, p < 0.01. Data are mean ± SEM (bars) values of four to nine experiments performed in triplicate. C: The κ-OR-selective opioid U69,593 (U69; 1 μM) does not significantly affect ET-1-stimulated DNA synthesis. *Significantly greater than control, p < 0.01. Data are mean ± SEM (bars) values of five experiments performed in triplicate. Basal [³H]thymidine incorporation yielded an average of 15,970 ± 2,259 dpm per well for unstimulated controls and 8,982 ± 1,100 dpm per well for unstimulated, DMI-treated controls.

FIG. 2

Endomorphins inhibit κ-opioid-stimulated DNA synthesis. C6 cells were kept in “low MEM” for 48 h before treatment. In this same medium, endomorphin-1 (Em-1) or -2 (Em-2; 10 nM) was added 1 h before U69,593 (U69; 1 μM) in assays where both agonists are present. After 30 min, [³H]thymidine (0.5 μCi/ml) was added to the medium, and [³H]thymidine incorporation was assayed after 23 h by scintillation counting. CTAP (1 μM) was added 1 h before addition of either Em-1 or -2. Data are average ± SEM (bars) values of three experiments performed in triplicate. *Significantly greater than control, ^#significantly less than U69, φsignificantly greater than U69 + Em-1 and U69 + Em-2, p < 0.01. Basal [³H]thymidine incorporation yielded an average of 14,370 ± 1,985 dpm per well.

FIG. 3

Endomorphin-1 (Em-1) inhibits κ-opioid-stimulated PI turnover. C6 cells were kept in “low MEM” for 48 h and then labeled with 1.5 μCi of myo-[³H]inositol/ml per well in the same medium overnight. Medium was changed, and cells were incubated in “low MEM” plus 5 mM LiCl for 1 h before drug treatment. Em-1 (10 nM) was added 1 h before stimulation with U69,593 (U69; 1 μM). After a 10-min U69 treatment, cells were collected, and extracted ³H-IP_x fractions were eluted from anion exchange columns and assayed by scintillation counting. *Significantly greater than control, p < 0.01; ^#significantly less than U69, p < 0.01; ^φsignificantly greater than U69 + Em-1, p < 0.05. Data are mean ± SEM (bars) values from three to seven experiments performed in triplicate. Basal ³H-IP_x accumulation was measured as 35,920 ± 3,916 dpm per well.

FIG. 4

Endomorphin-1 (Em-1) inhibits κ-opioid phosphorylation of ERK1/2. C6 cells were kept in “low MEM” for 48 h before addition of opioids. Em-1 (10 nM) was added 1 h before a 10-min stimulation with U69,593 (U69; 10 nM). CTAP (1 μM) was added 1 h before Em-1 where indicated. In some cases, cells were treated for 10 min or 1 h with Em-1 alone. Data are mean ± SEM (bars) values of three experiments. *Significantly greater than control, p < 0.001; ^#significantly less than U69, p < 0.001; φsignificantly greater than U69 + Em-1, p < 0.01. Also shown is a representative membrane, blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 5

Time dependence for endomorphin-1 (Em-1) inhibition of U69,593 (U69)-stimulated ERK1/2 phosphorylation. C6 cells were kept in “low MEM” for 48 h before addition of opioids. Em-1 (10 nM) was added at the times indicated before a 10-min stimulation with U69 (10 nM). Data are mean ± SEM (bars) values of three or four experiments. *Significantly greater than control, p < 0.005; ^#significantly less than U69 ( p < 0.01) and U69 + Em-1 [0 ( p < 0.01), 10, and 30 min ( p < 0.05)]. Also shown is a representative membrane, blotted first with antiphospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).

FIG. 6

Dynamin K44A mutant has no effect on κ-OR-mediated phosphorylation of ERK1/2, yet blocks the inhibitory effects of endomorphin-1 (Em-1). C6 cells were transiently transfected with 1 μg of dynamin (Dyn; left panels) or dynamin K44A (K44A; right panels) cDNA and then maintained in “low MEM” for 48 h before treatment. Em-1 (10 nM) was added 1 h before the 10-min U69,593 (U69; 10 nM) stimulation. Data are mean ± SEM (bars) values of four to seven experiments. *Significantly greater than control, ^#significantly less than U69, p < 0.001. Also shown are representative membranes, blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).