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Interleukin-1 beta-induced up-regulation of opioid receptors in the untreated and morphine-desensitized U87 MG human astrocytoma cells.

Byrne LS, Peng J, Sarkar S, Chang SL - J Neuroinflammation (2012)

Bottom Line: DOR expression was also elevated, although not significantly.Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.Our results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of NeuroImmune Pharmacology, Seton Hall University, 400 South Orange Ave, South Orange, NJ 07079, USA.

ABSTRACT

Background: Interleukin-1beta (IL-1β) is a pro-inflammatory cytokine that can be produced in the central nervous system during inflammatory conditions. We have previously shown that IL-1β expression is altered in the rat brain during a morphine tolerant state, indicating that this cytokine may serve as a convergent point between the immune challenge and opiate mediated biological pathways. We hypothesized that IL-1β up-regulates opioid receptors in human astrocytes in both untreated and morphine-desensitized states.

Methods: To test this hypothesis, we compared the basal expression of the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors in the human U87 MG astrocytic cell line to SH-SY5Y neuronal and HL-60 immune cells using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). To demonstrate that IL-1β induced up-regulation of the MOR, DOR and KOR, U87 MG cells (2 x 105 cells/well) were treated with IL-1β (20 ng/mL or 40 ng/mL), followed by co-treatment with interleukin-1 receptor antagonist protein (IL-1RAP) (400 ng/mL or 400 ng/mL). The above experiment was repeated in the cells desensitized with morphine, where U87 MG cells were pre-treated with 100 nM morphine. The functionality of the MOR in U87 MG cells was then demonstrated using morphine inhibition of forksolin-induced intracellular cAMP, as determined by radioimmunoassay.

Results: U87 MG cells treated with IL-1β for 12 h showed a significant up-regulation of MOR and KOR. DOR expression was also elevated, although not significantly. Treatment with IL-1β also showed a significant up-regulation of the MOR in U87 MG cells desensitized with morphine. Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.

Conclusion: Our results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

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The effects of morphine on MOR expression in U87 MG cells. U87 MG cells were treated with either vehicle (cell culture medium) or morphine (100 nM) for 0 (control), 45 minutes, 3, 6, 12, 24, or 48 hours. Real time RT-PCR was used to determine the copy number of the MOR and GAPDH. GAPDH levels were used to normalize the MOR levels. Each time-point was adjusted by the appropriate time-point control. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control.
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Figure 6: The effects of morphine on MOR expression in U87 MG cells. U87 MG cells were treated with either vehicle (cell culture medium) or morphine (100 nM) for 0 (control), 45 minutes, 3, 6, 12, 24, or 48 hours. Real time RT-PCR was used to determine the copy number of the MOR and GAPDH. GAPDH levels were used to normalize the MOR levels. Each time-point was adjusted by the appropriate time-point control. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control.

Mentions: Chronic exposure to morphine desensitizes the MOR [29]. To further examine the immune-opioid relationship, IL-1β’s ability to potentially up-regulate a desensitized MOR after chronic morphine treatment was examined. A desensitization time course indicated that U87 MG cells treated with 100 nM morphine for 45 minutes had a significant decrease in the copy number of the MOR (2.5 x 104 ± 3.7 x 103 copies of MOR/μg total RNA) compared to the control (9.4 x 103 ± 3.2 x 103 copies of MOR/μg total RNA). Interestingly, after 3 h of morphine treatment, the MOR was significantly increased (1.9 x 106 ± 2.9 x 104 copies of MOR/μg total RNA) compared to the control. However, MOR expression again decreased after 6 h (7.0 x 105 ± 2.9 x 104 copies of MOR/μg total RNA), 12 h (4.4 x 105 ± 1.4 x 104 copies of MOR/μg total RNA), 24 h (2.2 x 105 ± 2.3 x 104 copies of MOR/μg total RNA), and 48 h (2.9 x 105 ± 9.3 x 104 copies of MOR/μg total RNA) of morphine treatment compared to control (Figure 6).


Interleukin-1 beta-induced up-regulation of opioid receptors in the untreated and morphine-desensitized U87 MG human astrocytoma cells.

Byrne LS, Peng J, Sarkar S, Chang SL - J Neuroinflammation (2012)

The effects of morphine on MOR expression in U87 MG cells. U87 MG cells were treated with either vehicle (cell culture medium) or morphine (100 nM) for 0 (control), 45 minutes, 3, 6, 12, 24, or 48 hours. Real time RT-PCR was used to determine the copy number of the MOR and GAPDH. GAPDH levels were used to normalize the MOR levels. Each time-point was adjusted by the appropriate time-point control. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526549&req=5

Figure 6: The effects of morphine on MOR expression in U87 MG cells. U87 MG cells were treated with either vehicle (cell culture medium) or morphine (100 nM) for 0 (control), 45 minutes, 3, 6, 12, 24, or 48 hours. Real time RT-PCR was used to determine the copy number of the MOR and GAPDH. GAPDH levels were used to normalize the MOR levels. Each time-point was adjusted by the appropriate time-point control. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control.
Mentions: Chronic exposure to morphine desensitizes the MOR [29]. To further examine the immune-opioid relationship, IL-1β’s ability to potentially up-regulate a desensitized MOR after chronic morphine treatment was examined. A desensitization time course indicated that U87 MG cells treated with 100 nM morphine for 45 minutes had a significant decrease in the copy number of the MOR (2.5 x 104 ± 3.7 x 103 copies of MOR/μg total RNA) compared to the control (9.4 x 103 ± 3.2 x 103 copies of MOR/μg total RNA). Interestingly, after 3 h of morphine treatment, the MOR was significantly increased (1.9 x 106 ± 2.9 x 104 copies of MOR/μg total RNA) compared to the control. However, MOR expression again decreased after 6 h (7.0 x 105 ± 2.9 x 104 copies of MOR/μg total RNA), 12 h (4.4 x 105 ± 1.4 x 104 copies of MOR/μg total RNA), 24 h (2.2 x 105 ± 2.3 x 104 copies of MOR/μg total RNA), and 48 h (2.9 x 105 ± 9.3 x 104 copies of MOR/μg total RNA) of morphine treatment compared to control (Figure 6).

Bottom Line: DOR expression was also elevated, although not significantly.Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.Our results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of NeuroImmune Pharmacology, Seton Hall University, 400 South Orange Ave, South Orange, NJ 07079, USA.

ABSTRACT

Background: Interleukin-1beta (IL-1β) is a pro-inflammatory cytokine that can be produced in the central nervous system during inflammatory conditions. We have previously shown that IL-1β expression is altered in the rat brain during a morphine tolerant state, indicating that this cytokine may serve as a convergent point between the immune challenge and opiate mediated biological pathways. We hypothesized that IL-1β up-regulates opioid receptors in human astrocytes in both untreated and morphine-desensitized states.

Methods: To test this hypothesis, we compared the basal expression of the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors in the human U87 MG astrocytic cell line to SH-SY5Y neuronal and HL-60 immune cells using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). To demonstrate that IL-1β induced up-regulation of the MOR, DOR and KOR, U87 MG cells (2 x 105 cells/well) were treated with IL-1β (20 ng/mL or 40 ng/mL), followed by co-treatment with interleukin-1 receptor antagonist protein (IL-1RAP) (400 ng/mL or 400 ng/mL). The above experiment was repeated in the cells desensitized with morphine, where U87 MG cells were pre-treated with 100 nM morphine. The functionality of the MOR in U87 MG cells was then demonstrated using morphine inhibition of forksolin-induced intracellular cAMP, as determined by radioimmunoassay.

Results: U87 MG cells treated with IL-1β for 12 h showed a significant up-regulation of MOR and KOR. DOR expression was also elevated, although not significantly. Treatment with IL-1β also showed a significant up-regulation of the MOR in U87 MG cells desensitized with morphine. Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.

Conclusion: Our results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

Show MeSH
Related in: MedlinePlus