Involvement of Dopamine Receptors in Binge Methamphetamine-Induced Activation of Endoplasmic Reticulum and Mitochondrial Stress Pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D1 receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D1 and D2 receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D1 or D2 receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58IPK, in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D2-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors

Raclopride did not block METH-induced ATF4 and ATF3 expression.

<p>(A) Representative immunoblots of ATF4, ATF3, CHOP and caspase-12. Quantification of ATF4 (B), ATF3 (C), CHOP (D) and caspase-12 (E) are shown. Key to statistics: * = p<0.05; ** = p<0.01; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the Rac group. !! = p<0.01; !!! = p<0.001, in comparison of METH group to the Rac+METH group.</p

Effects of raclopride on METH-induced HSP40 and HSP70.

<p>(A) Representative immunoblots of the effects of the drugs. (B, C) Quantitative analysis of the proteins. Protein expression was normalized to α-Tubulin. Key to statistics: * = p<0.05; ** = p<0.01; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the Rac group. !! = p<0.01; !!! = p<0.001, in comparison of METH group to the Rac+METH group.</p

Effects of SCH23390 and raclopride on METH-induced hyperthermia.

<p>Animals in the control group received an injection of saline followed 30 min later by another injection of saline, this pattern of injections was repeated four times at 2-hr intervals. The METH treatment group of rats received four injections of saline at 2-hr intervals, each saline injection being followed by an injection of METH (10 mg/kg). (A) Two other groups of animals were pretreated with SCH23390 30 min before each of four saline or METH injections given according to the intervals described above. (B) Two other groups were pretreated with raclopride and treated with either saline or METH injections as above. Temperature was recorded 1 hr prior to the first injection (−1 hr), 30 min after each combined set of injections (shown in arrows), and 2 hr after the final injection. Statistical differences in temperature were considered significant at p values less than 0.05. Key to statistics: *** = p<0.001, in comparison to the control group. ! = p<0.05, in comparison to the METH group (post-hoc test).</p

Effects of METH and SCH23390 administration on the expression of cytosolic chaperones HSPs.

<p>(A) Representative western blot bands (1 band for Saline or SCH representing each time-point, 3 bands for METH and SCH+METH). METH administration caused rapid and stable induction of the chaperones HSP40 (B) and HSP70 (C). Pretreatment with SCH23390 prevented these increases. Protein expression was normalized to α-Tubulin. Key to statistics: * = p<0.05; ** = p<0.01; *** = p<0.001, in comparison to the Saline group. ## = p<0.01; ### = p<0.001, in comparison to the SCH group. !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p

The effects of METH and raclopride on the expression of the Bcl-2 family of proteins and cytochrome c.

<p>(A) Representative immunoblots. (B) Pretreatment with raclopride attenuated METH-induced decreases in Bcl-2 protein levels, and (C, D) increases in Bax and Bad expression. In contrast, raclopride was ineffective to block cytochrome c induction (E). Protein expression was normalized to α-Tubulin. Key to statistics: ** = p<0.01; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the Rac group. ! = p<0.05; !! = p<0.01; !!! = p<0.001, in comparison of METH group to the Rac+METH group.</p

Effects of METH on the transcript levels of pro-death genes.

<p>(A) METH caused rapid induction in Chop/Gadd153 mRNA levels. (B) Gadd34 was up-regulated at late time-points. Key to statistics: ** = p<0.01, in comparison to the Saline group. # = p<0.05; ## = p<0.01; ### = p<0.001, in comparison to the SCH group. ! = p<0.05; !! = p<0.01; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p

Effects of METH injections and SCH23390 treatment on the expression of stress response regulated proteins.

<p>(A) Representative immunoblots showing the effects of METH and SCH23390. (A–E) Pretreatment with SCH23390 blocked the METH-induced changes on ATF3 (B), ATF4 (C), CHOP (D) and caspase-12 (E). Protein expression was normalized to α-Tubulin. Key to statistics: * = p<0.05; *** = p<0.001, in comparison to the Saline group. ## = p<0.01; ### = p<0.001, in comparison to the SCH group. !! = p<0.01; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p

Binge METH injections caused time-dependent increases in the expression of the ER chaperone, BiP/GRP-78, and of the co-chaperone, P58^IPK.

<p>Levels of (A) BiP/GRP-78 and (B) P58^IPK transcripts were rapidly increased at 30 min after METH injections. RT-PCR was performed on total RNA isolated from the striatal tissue. Data were obtained from RNA isolated from six animals per group and determined individually. The levels of mRNA were normalized to clathrin mRNA levels. Values obtained for the treatment groups were compared by analysis of variance (ANOVA) followed by post-hoc analyses when ANOVA revealed significant changes. Key to statistics: * = p<0.05; *** = p<0.001, in comparison to the Saline group. # = p<0.05; ### = p<0.001, in comparison to the SCH group. ! = p<0.05; !!! = p<0.001, in comparison of METH group to the SCH+METH group.</p