Hydrogen sulfide attenuates opioid dependence by suppression of adenylate cyclase/cAMP pathway

Abstract

Aims: The best-established mechanism of opioid dependence is the up-regulation of adenylate cyclase (AC)/cAMP pathway, which was reported to be negatively regulated by hydrogen sulfide (H2S), a novel endogenous neuromodulator. The present study was, therefore, designed to determine whether H2S is able to attenuate the development of opioid dependence via down-regulating AC/cAMP pathway.

Results: We demonstrated that application of sodium hydrosulphide (NaHS) and GYY4137, two donors of H2S, significantly alleviated naloxone-induced robust withdrawal jumping (the most sensitive and reliable index of opioid physical dependence) in morphine-treated mice. Repeated treatment with NaHS inhibited the up-regulated protein expression of AC in the striatum of morphine-dependent mice. Furthermore, NaHS also attenuated morphine/naloxone-elevated mRNA levels of AC isoform 1 and 8, production of cAMP, and phosphorylation of cAMP response element-binding protein (CREB) in mice striatum. These effects were mimicked by the application of exogenous H2S or over-expression of cystathione-β-synthase, an H2S -producing enzyme, in SH-SY5Y neuronal cells on treatment with [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin, a selective μ-opioid receptor agonist. Blockade of extracellular-regulated protein kinase 1/2 (ERK1/2) with its specific inhibitor attenuated naloxone-induced CREB phosphorylation. Pretreatment with NaHS or stimulation of endogenous H2S production also significantly suppressed opioid withdrawal-induced ERK1/2 activation in mice striatum or SH-SY5Y cells.

Innovation: H2S treatment is important in prevention of the development of opioid dependence via suppression of cAMP pathway in both animal and cellular models.

Conclusion: Our data suggest a potential role of H2S in attenuating the development of opioid dependence, and the underlying mechanism is closely related to the inhibition of AC/cAMP pathway.

FIG. 1.

Effect of NaHS and GYY4137 on naloxone-induced withdrawal behaviors in morphine-dependent mice. (A, D) jumping; (B, E) weight loss; (C, F) rearing. Mean±S.E.M, n=6–11. **p<0.01, ***p<0.001, versus Con; ^#p<0.05, ^##p<0.01, versus Mor. Con, control; Mor, morphine; S, NaHS; G, GYY4137; NaHS, sodium hydrosulphide.

FIG. 2.

Effect of NaHS pretreatment on AC protein expression in the brain regions of morphine-treated mice. (A) striatum; (B) prefrontal cortex; (C) hippocampus. Mean±S.E.M, n=3–4. *p<0.05, ***p<0.001, versus Con; ^#p<0.05, versus Mor. Con, control; Mor, morphine; S, NaHS; AC, adenylate cyclase.

FIG. 3.

Effect of NaHS pretreatment on AC isoforms mRNA expression in the striatum of morphine-treated mice. (A) Representative gel images. (B) Quantifications by densitometric measurement. Mean±S.E.M, n=4. **p<0.01, versus Con; ^#p<0.05, ^###p<0.001, versus Mor. Con, control; Mor, morphine; S, NaHS.

FIG. 4.

NaHS treatment attenuated morphine-induced elevation of cAMP level and CREB phosphorylation in the striatum of mice. (A) Effect of NaHS on cAMP production in the striatum of morphine-treated mice. Mean±S.E.M, n=9. (B, C) Effect of NaHS pretreatment on the up-regulation of CREB phosphorylation induced by naloxone-precipitated withdrawal in mice striatum. Mean±S.E.M, n=4. *p<0.05, **p<0.01, versus Con; ^#p<0.05, versus Mor. Con, control; Mor, morphine; S, NaHS; CREB, cAMP response element-binding protein.

FIG. 5.

Effect of NaHS on cAMP rebound, AC mRNA levels in SH-SY5Y cells treated with DAMGO. (A) NaHS pretreatment attenuated cAMP rebound induced by opioid withdrawal in SH-SY5Y cells. Cells were treated with DAMGO (10 μM) for 24 h followed by addition of naloxone (100 μM) (D+Nal) or washing the cell with low-serum medium (D+W) to induce opioid withdrawal. NaHS (10 μM) was given 10 min before the addition of DAMGO. Mean±S.E.M., n=6–12. **p<0.01, ***p<0.001, versus the corresponding values without NaHS treatment in the same group; ^##p<0.01, ^###p<0.001, versus D (DAMGO alone without NaHS treatment). (B) Naloxone-induced cAMP rebound was abolished by NaHS (10 μM) given 10 min before the addition of DAMGO (S+D+Nal), but not by NaHS given 30 min before the addition of naloxone (D+S+Nal). Mean±S.E.M, n=6. ***p<0.001, versus Con; ^###p<0.001, versus D;+++p<0.001, versus D+Nal. (C, D) NaHS pretreatment abolished DAMGO-induced up-regulation of mRNA level of AC1 and AC8 in SH-SY5Y cells. Mean±S.E.M, n=4–6. **p<0.01, versus Con; ^##p<0.01, versus D. Con, control; FSK, forskolin; S, NaHS; D, DAMGO; Nal, naloxone; Veh, vehicle; DAMGO, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-Enkephalin.

FIG. 6.

Effect of endogenous H₂S on cAMP rebound, AC mRNA levels in SH-SY5Y cells treated with DAMGO. (A) Western blotting analysis showing the protein expression of CBS protein in SH-SY5Y cells transfected with CBS-PME185-HA vector or its empty vector. Mean±S.E.M., n=6. *p<0.05, versus Con; ^#p<0.05, versus CBS. (B) Over-expression of CBS, but not its empty vector, reduced cAMP production in either forskolin-stimulated, chronic DAMGO-treated, or naloxone-precipitated cells. Mean±S.E.M., n=5–14. ***p<0.001, versus Con; ^#p<0.05, ^##p<0.01, ^###p<0.001, versus the corresponding values without NaHS treatment in the same group. (C, D) Effects of CBS over-expression on DAMGO-induced AC1 expression in SH-SY5Y cells. Mean±S.E.M, n=5. ***p<0.001, versus Con; ^#p<0.05, versus D. Con, control; FSK, forskolin; S, NaHS; D, DAMGO; Nal, naloxone; Veh, vehicle; Vec, vector; CBS, cystathione-β-synthase; H₂S, hydrogen sulfide.

FIG. 7.

Western blotting analysis showing the effect of NaHS on the up-regulation of CREB phosphorylation induced by opioid withdrawal in SH-SY5Y cells. (A) The up-regulation of CREB phosphorylation caused by DAMGO withdrawal was attenuated by NaHS treatment. Mean±S.E.M, n=7. (B) DAMGO-induced CREB phosphorylation was suppressed by over-expression of CBS but not its empty vector. Mean±S.E.M., n=4. *p<0.05, ***p<0.001 versus Con; ^#p<0.05, ^###p<0.001, versus D+Nal; ⁺p<0.05, versus CBS+D+Nal. Con, control; S, NaHS; D, DAMGO; Nal, naloxone; Vec, vector.

FIG. 8.

Role of ERK1/2 in CREB phosphorylation in DAMGO-treated SH-SY5Y cells. (A, B) Western blots showing the effects of NaHS and inhibition of ERK1/2 on naloxone-induced CREB phosphorylation in SH-SY5Y cells. Mean±S.E.M, n=4. ***p<0.001, versus Con; ^###p<0.001, versus D+Nal. (C, D) Western blots showing the effect of CBS over-expression on the up-regulated p-ERK1/2 induced by naloxone-precipitated withdrawal in SH-SY5Y cells. Mean±S.E.M, n=4. **p<0.01, versus Con; ^##p<0.01, versus D+Nal. Con, control; S, NaHS; D, DAMGO; Nal, naloxone; PD, PD98059; t-ERK, total ERK; ERK1/2, extracellular regulated protein kinase 1/2.

FIG. 9.

Effect of NaHS on ERK1/2 phosphorylation in the striatum of morphine-dependent mice. (A, B) Western blots showing the effects of NaHS pretreatment on naloxone-induced ERK1/2 phosphorylation in mice striatum. Mean±S.E.M, n=8. *p<0.05, versus Con; ^##p<0.01, versus Mor. (C) Immunostaining showing the effect of NaHS administration on the expression of p-ERK1/2 in mice striatum. Green: immunostaining of p-ERK1/2; Blue: the nuclei of neurons stained with Hoechst 33342. Con, control group receiving saline; Mor, morphine-treated group; S+Mor, NaHS-pretreated morphine group. Scale bar=20 μm. Con, control; S, NaHS; Mor, morphine. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars