Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain

Abstract

1. This study was designed to assess the influence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. 2. The acute treatment of rats with the mu-opioid receptor agonist morphine (3-30 mg x kg(-1), i.p., 2 h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100 mg x kg(-1), i.p., 2 h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain. 3. Chronic morphine (10-100 mg x kg(-1), 5 days, and 10 mg x kg(-1), 13 days) induced marked increases (47-123%) in the immunodensity of Fas receptor in the cerebral cortex. In contrast, chronic morphine (5 and 13 days) decreased the immunodensity of Bcl-2 protein (15-30%) in brain cortex. Chronic naloxone (10 mg x kg(-1), 13 days) did not alter the immunodensities of Fas and Bcl-2 proteins in the cerebral cortex. 4. The concurrent chronic treatment (13 days) of naloxone (10 mg x kg(-1)) and morphine (10 mg x kg(-1)) completely prevented the morphine-induced increase in Fas receptor and decrease in Bcl-2 protein immunoreactivities in the cerebral cortex. 5. The results indicate that morphine, through the sustained activation of opioid receptors, can promote abnormal programmed cell death by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein.

Figure 1

Representative autoradiographs of Western blots depicting labelling of immunodetectable pro-apoptotic Fas receptor (A) and anti-apoptotic Bcl-2 oncoprotein (B) in rat brain membranes. Samples from the cerebral cortex were subjected to SDS – PAGE, transferred to nitrocellulose membranes (immunoblotting), incubated with the specific primary and secondary antibodies, and visualized by the Enhanced Chemiluminiscence (ECL or ECL-Plus) method. The apparent molecular masses of Fas (48/49 kDa) and Bcl-2 (25/26 kDa) proteins were determined by calibrating the blots with prestained molecular weight markers as shown on the left hand side. For Fas the amounts of total protein loaded per gel well were: 31.4; 62.7; 94.1; 125.4 (μg) and the corresponding IOD: 0.37; 0.75; 1.08; 1.21 (arbitrary units) (standard curve; μg protein vs IOD, r=0.98). For Bcl-2 the amounts of total protein loaded per gel well were: 31.4; 62.7; 94.1; 125.4 (μg) and the corresponding IOD: 0.56; 1.29; 2.48; 2.90 (arbitrary units) (standard curve; μg protein vs IOD, r=0.99). (A) The specificity of the antibody anti-Fas was assessed by preincubating the antibody with the antigenic peptide (preabsorbed antibody), which resulted in the blockade of the immunoreaction for the specific protein (48/49 kDa) and other unknown related peptides (≈35 – 40 kDa). (B) For Bcl-2 immunodetection, omission of the primary antibody was used as a negative control and the immunoreactivity was absent under this condition.

Figure 2

(A) Representative immunoblots using antisera against Fas, Bcl-2 and NF-L proteins in the cerebral cortex of saline-treated rats (two animals, S1 and S2) and morphine-treated rats (3 and 30 mg kg⁻¹, 2 h) (two animals for each acute treatment, M1 and M2). The amount of total protein loaded per gel well ranged from 87 to 89 μg for the different treatments. Note that the immunodensities of Fas, Bcl-2 and NF-L did not change significantly in morphine-treated rats (IOD, percentage of saline-treated rats, range 95 – 106%). (B) Columns are means±s.e.mean of 6 – 8 experiments per group performed in duplicate with an animal per experiment, and expressed as percentage of saline-treated rats. One-way ANOVA did not detect significant differences for Fas (F=0.35, P=0.71), Bcl-2 (F=0.96, P=0.39) and NF-L (F=1.09, P=0.34) immunodensities after the acute treatments with morphine. (C) Representative immunoblots using antisera against Fas, Bcl-2 and NF-L proteins in the cerebral cortex of saline-treated rats (two animals, S1 and S2) and naloxone-treated rats (1 and 100 mg kg⁻¹, 2 h) (two animals for each acute treatment, N1 and N2). The amount of total protein loaded per well ranged from 88 to 91 μg for the different treatments. Note that the immunodensities of Fas, Bcl-2 and NF-L did not change significantly in naloxone-treated rats (IOD, percentage of saline-treated rats, range 98 – 102%). (D) Columns are means±s.e.mean of 6 – 8 experiments per group performed in duplicate with an animal per experiment, and expressed as percentage of saline-treated rats. One-way ANOVA did not detect significant differences for Fas (F=1.04, P=0.36), Bcl-2 (F=1.58, P=0.21) and NF-L (F=3.06, P=0.05) immunodensities after the acute treatments with naloxone.

Figure 3

(A). Representative immunoblots using antisera against Fas, Bcl-2 and NF-L proteins in the cerebral cortex of saline-treated rats (two animals, S1 and S2) and chronic morphine-treated rats (10 to 100 mg kg⁻¹ for 5 days or 10 mg kg⁻¹ every 12 h for 13 days) (two animals for each chronic treatment, M1 and M2). The amount of total protein loaded per gel well ranged from 92 to 96 μg for the different treatments. Note that the immunodensity of Fas increased (IOD, percentage of saline-treated rats: 155 – 238%) and that of Bcl-2 (96 – 80%) or NF-L (58 – 45%) decreased in chronic morphine-treated rats. (B) Columns are means±s.e.mean of 6 – 8 experiments per group performed in duplicate with an animal per experiment, and expressed as percentage of saline-treated rats. One-way ANOVA detected significant differences between groups with respect to protein immunodensities after the chronic treatments with morphine: Fas (F=27.56, P<0.0001), Bcl-2 (F=51.52, P<0.0001) and NF-L (F=54.81, P<0.0001). ^* P<0.05; ^** P<0.001 when compared with the saline group (ANOVA followed by Scheffé's test). (C) Representative immunoblots using antisera against Fas, Bcl-2 and NF-L proteins in the cerebral cortex of saline-treated (S), naloxone-treated (N, 10 mg kg⁻¹ for 13 days), morphine-treated (M, 10 mg kg⁻¹ for 13 days) and naloxone plus morphine-treated (N – M) rats. The amount of total protein loaded per gel well ranged from 90 to 91 μg for the different treatments. Note that naloxone completely prevented the effects of morphine (group N – M) on Fas, Bcl-2 and NF-L immunoreactivity. (D) Columns are means±s.e.mean of 6 – 8 experiments per group performed in duplicate with an animal per experiment, and expressed as percentage of saline-treated rats. One-way ANOVA detected significant differences between groups: Fas (F=53.77, P<0.0001), Bcl-2 (F=36.66, P<0.0001) and NF-L (F=25.82, P<0.0001). ^** P<0.001 when compared with the saline group (ANOVA followed by Scheffé's test).