Morphine enhances microglial migration through modulation of P2X4 receptor signaling

Abstract

Opioids, although fundamental to the treatment of pain, are limited in efficacy by side effects including tolerance and hyperalgesia. Using an in vitro culture system, we report that morphine increased microglial migration via a novel interaction between mu-opioid and P2X(4) receptors, which is dependent upon PI3K/Akt pathway activation. Morphine at 100 nm enhanced migration of primary microglial cells toward adenosine diphosphate by 257, 247, 301, 394, and 345% following 2, 6, 12, 24, and 48 h of stimulation, respectively. This opioid-dependent migration effect was inhibited by naloxone and confirmed to be mu-opioid receptor-dependent through the use of selective agonists and antagonists. PPADS [pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid)], a P2X(1-3,5-7) antagonist, had no effect on microglial migration; however, TNP-ATP [2',3'-O-(2,4,6-trinitrophenyl)-ATP], a P2X(1-7) antagonist, inhibited morphine-induced migration, suggesting a P2X(4) receptor-mediated effect. The PI3K inhibitors wortmannin and LY294002 decreased morphine-induced microglial migration. Iba1 protein, a microglial marker, and P2X(4) receptor expression were significantly increased after 6, 12, 24, and 48 h of morphine stimulation. Together, these results provide evidence for two phases of morphine effects on microglia. The initial phase takes place in minutes, involves PI3K/Akt pathway activation and leads to acutely enhanced migration. The longer-term phase occurs on the order of hours and involves increased expression of Iba1 and P2X(4) receptor protein, which imparts a promigratory phenotype and is correlated with even greater migration. These data provide the first necessary step in supporting microglial migration as an attractive target for the prevention or attenuation of morphine-induced side effects including tolerance and hyperalgesia.

Figure 1.

Morphine dose-dependently enhanced microglial migration which is inhibited by naloxone. Primary microglia were treated for 2 h with 0, 1, 10, or 100 nm morphine and then allowed to migrate toward 10 μm ADP. a, Images of stained microglia which migrated to the bottom of the transwell membrane. b, Quantification of microglial migration after morphine treatment, represented as mean ± SEM. Microglia were treated for 1 h with 0 or 100 pm or 1, 10, or 100 nm naloxone, then 0 or 100 nm morphine was added. Cells were allowed to migrate toward 10 μm ADP for 2 h; then the migrated cells were counted. c, Quantification of microglial migration after naloxone and morphine treatment, represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with media. ^##p < 0.01 compared with 1 nm morphine. Med, Media; mor, morphine.

Figure 2.

Morphine-induced microglial migration is μ-opioid receptor-dependent. Primary microglia were treated with opioid receptor agonists or antagonist, then allowed to migrate toward 10 μm ADP. Migrated cells were counted and represented as mean ± SEM. a, Quantification of microglial migration after treatment with 0, 1, 10, or 100 nm DAMGO, a selective μ-opioid receptor agonist. b, Quantification of microglial migration after treatment with 0, 1, 10, or 100 nm DPDPE, a selective δ-opioid receptor agonist. c, Quantification of microglial migration after treatment with 0, 1, 10, or 100 nm U-69593, a selective κ-opioid receptor agonist. d, Microglia were pretreated for 1 h with 0, 1, or 100 nm CTAP, a selective μ-opioid receptor antagonist, followed by 2 h of treatment with 0 or 100 nm morphine. **p < 0.01, ***p < 0.001. ^##p < 0.01 compared with media control. Med, Media; mor, morphine.

Figure 3.

P2X₄ inhibition reduced morphine-induced microglial migration. Primary microglia were treated with P2X inhibitors, then allowed to migrate toward 10 μm ADP. Migrated cells were counted and represented as mean ± SEM. a, Microglia were pretreated with 0 or 100 nm PPADS, a P2X_1–3,5–7 receptor antagonist, for 1 h, then treated with 0 or 100 nm morphine for 2 h. b, Microglia were pretreated with 0, 1, or 100 nm TNP-ATP, a P2X_1–7 receptor antagonist, for 1 h followed by 0 or 100 nm morphine. c, TNP-ATP dose–response inhibition of morphine-induced microglial migration. *p < 0.05, **p < 0.01, ***p < 0.001. ^###p < 0.001 compared with 100 nm morphine. ^††p < 0.01 compared with media control. ^‡‡p < 0.01 compared with 1 nm TNP-ATP. Med, Media; mor, morphine.

Figure 4.

PI3K inhibition reduced morphine-induced microglial migration. Primary microglial cells were treated with two PI3K inhibitors, wortmannin and LY294002, for 1 h then treated for 2 h with morphine and allowed to migrate toward 10 μm ADP. Migrated cells were counted and represented as mean ± SEM. a, Quantification of microglia pretreated with 0 or 100 nm wortmannin followed by 0 or 100 nm morphine. b, Quantification of microglia pretreated with 0 or 50 μm LY294002 followed by 0 or 100 nm morphine. ***p < 0.001. ^###p < 0.001 compared with media control. ^†††p < 0.001 compared with 100 nm morphine.

Figure 5.

Morphine enhanced Akt phosphorylation in a PI3K and μ-opioid receptor-dependent manner. Primary microglial cells were treated with 100 nm morphine for 0, 5, 15, 30, 60, and 120 min, subjected to Western blot analysis and probed with anti-pAkt then Akt antibody (a). b, Image of Western blot membrane of microglia pretreated for 15 min with 0 or 100 nm wortmannin, then treated for 15 min with 0 or 100 nm morphine. c, Image of Western blot membrane of microglia pretreated for 15 min with 0 or 50 μm LY294002, then treated for 15 min with 0 or 100 nm morphine. d, Image of Western blot membrane of microglia pretreated for 15 min with 0 or 100 nm of the μ-opioid receptor antagonist CTAP, then treated for 15 min with 0 or 100 nm morphine.

Figure 6.

Morphine enhances Iba1 expression in microglial cells. Primary microglia were treated with 0, 1, 10, or 100 nm morphine for 6 (a), 12 (b), 24 (c), or 48 (d, e) h, lysed, and subjected to Western blot analysis. a–d, Graphs indicate relative Iba1 expression normalized to β-actin loading control ± SEM. e, Image of membrane after 48 h treatment probed with anti-Iba1 and anti-β-actin antibodies. Graphs show data from at least four individual Western blots, n = 2 per blot. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Morphine enhances P2X₄ receptor expression in microglial cells. Primary microglia were treated with 0, 1, 10, or 100 nm morphine for 6 (a), 12 (b), 24 (c), or 48 (d, e) h, lysed, and subjected to Western blot analysis. a–d, Graphs indicate relative P2X₄ receptor expression normalized to β-actin loading control ± SEM. e, Image of membrane after 48 h treatment probed with anti-P2X₄ receptor and anti-β-actin antibodies. Graphs show data from at least four individual Western blots, n = 2 per blot. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8.

Morphine-induced Iba1 and P2X₄ receptor expression is μ-opioid receptor-dependent. Primary microglia were treated with 0 or 100 nm of the μ-opioid receptor-selective antagonist, CTAP, for 1 h, then treated with 0 or 100 nm morphine for 24 h. Cells were lysed and subjected to Western blot analysis. a, Image of Western blot membrane probed with anti-Iba1 and anti-β-actin antibodies. b, Graphical analysis of CTAP inhibition of morphine-induced Iba expression normalized to β-actin loading control ± SEM. c, Image of Western blot membrane probed with anti-P2X₄ receptor and anti-β-actin antibodies. d, Graphical analysis of CTAP inhibition of morphine-induced P2X₄ receptor expression normalized to β-actin loading control ± SEM. Graphs show data from at least six individual Western blots, n = 1 per blot. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9.

Six, twelve, twenty-four, and forty-eight hour morphine treatment dose-dependently enhanced microglial migration. Primary microglial were treated for 2, 6, 12, 24, or 48 h with 0, 1, 10, or 100 nm morphine and then allowed to migrate toward 10 μm ADP. Quantification of microglial migration after 2, 6, 12, 24, or 48 h morphine treatment, represented as mean ± SEM.