Hydrogen sulphide induces micro opioid receptor-dependent analgesia in a rodent model of visceral pain

Abstract

Background: Hydrogen sulphide (H2S) is a gaseous neuro-mediator that exerts analgesic effects in rodent models of visceral pain by activating KATP channels. A body of evidence support the notion that KATP channels interact with endogenous opioids. Whether H2S-induced analgesia involves opioid receptors is unknown.

Methods: The perception of painful sensation induced by colorectal distension (CRD) in conscious rats was measured by assessing the abdominal withdrawal reflex. The contribution of opioid receptors to H2S-induced analgesia was investigated by administering rats with selective mu, kappa and delta opioid receptor antagonists and antisenses. To investigate whether H2S causes mu opioid receptor (MOR) transactivation, the neuronal like cells SKNMCs were challenged with H2S in the presence of MOR agonist (DAMGO) or antagonist (CTAP). MOR activation and phosphorylation, its association to beta arrestin and internalization were measured.

Results: H2S exerted a potent analgesic effects on CRD-induced pain. H2S-induced analgesia required the activation of the opioid system. By pharmacological and molecular analyses, a robust inhibition of H2S-induced analgesia was observed in response to central administration of CTAP and MOR antisense, while kappa and delta receptors were less involved. H2S caused MOR transactivation and internalization in SKNMCs by a mechanism that required AKT phosphorylation. MOR transactivation was inhibited by LY294002, a PI3K inhibitor, and glibenclamide, a KATP channels blocker.

Conclusions: This study provides pharmacological and molecular evidence that antinociception exerted by H2S in a rodent model of visceral pain is modulated by the transactivation of MOR. This observation provides support for development of new pharmacological approaches to visceral pain.

Figure 1

Na₂S induces antinociception. CRD induces a volume-dependent increase of the AWR score in both fasting and fed rats (panels A and B respectively) and Na₂S (100 μMol/kg i.p.) causes a significant reduction of visceral sensitivity and pain (panel C). Data are mean ± SEM of 5 rats. *p < 0.05 versus CRD. CRD induces the increase of spinal cFos expression that is downregulated by Na₂S (panel D). Data are mean ± SEM of 5 rats. *p < 0.05 versus control.

Figure 2

CTAP reverses the Na₂S-induced antinociception. Pre-treating rats with the selective μ opioid receptor antagonist CTAP (0.09 mg/kg i.c.v. thirty minutes before Na₂S; panel C) abrogates the antinociceptive effect of Na₂S (100 μMol/kg i.p.). In contrast, the selective δ opioid receptor antagonist NTI (4 μg/kg i.c.v. five minutes before Na₂S, panel A) and the selective κ opioid receptor antagonist GNTI (0.08 μmg/kg i.c.v. three days before Na₂S, panel B) do not inhibit the analgesic effect of Na₂S, indicating that δ and κ opioid receptors have no effects on Na₂S-induced antinociception. Data are mean ± SEM of 5 rats. *p < 0.05 versus CRD.

Figure 3

The selective antisense oligodeoxynucleotide probes against DOR and MOR reverse the Na₂S-induced antinociception. Pre-treating rats with both the mismatched antisense oligodeoxynucleotides (panel A) and the κ opioid receptor antisense oligodeoxynucleotides (panel C) does not modify the H₂S-induced decrease of the AWR score, confirming that KOR does not cause any change on the Na₂S-induced analgesia. In contrast, oligodeoxynucleotide probes against DOR and MOR reverse the antinociception caused by Na₂S (panel B and D respectively). Data are mean ± SEM of 5 rats. *p < 0.05 versus CRD.

Figure 4

Glibenclamide reverses the Na₂S-induced antinociception. In a different experiment we have analyzed the role of the K_ATP channels on the H₂S-induced analgesia (panel A). Pre-treating rats with the K_ATP channels selective blocker glibenclamide (2.8 μmol/kg i.v.) completely reverses the Na₂S-induced analgesia (panel B) without any effects on the change of the colonic compliance induced by Na₂S. Data are mean ± SEM of 5 rats. *p < 0.05 versus CRD.

Figure 5

Na₂S induces MOR activation and phosphorylation, the recruitment of β arrestin and MOR internalization. Both DAMGO (1 μM) and Na₂S (50 μM) induce MOR activation (panel A and B respectively). Treating SKNMCs with both DAMGO and Na₂S results in MOR phosphorylation that is time-dependent. DAMGO induces MOR phosphorylation at Ser(377) that is maximal at 30 minutes and, similarly, H₂S induces MOR phosphorylation that peaks at 3-6 minutes and persists until 30 minutes (panel C). The total DAMGO-induced and H₂S-induced MOR phosphorylation is unchanged within the duration of the experiment (panel D). Co-immunoprecipitation experiments demonstrate that DAMGO induces the rapid complex between MOR and β arrestin with the peak at 5-15 minutes and, similarly, H₂S induces the co-immunoprecipitation of MOR and β arrestin that peaked at 30 minutes (panel E), indicating that H₂S induces the interaction between β arrestin and MOR. At the cell membrane fractioning experiments, DAMGO (1 μM) causes the disappearance of MOR from the plasma membrane fraction at 5 minutes and this effect is maximal at 60 minutes. At the same time there is a progressive increment of MOR presence in the cytoplasmatic fraction (panel F). After Na₂S (50 μM), MOR disappears from the plasma membrane fraction at 30 minutes with the maximal effect at 60 minutes and, in contemporary, it passes into the cytoplasmatic fraction (panel G). At the confocal microscopy SKNMCs exhibit MOR immunoreactivity predominantly localized at the cell surface in nonstimulated condition (panel H) and it translocates to cytoplasm after activation with DAMGO (panel I), which is known to induce MOR internalization. Na₂S induces a massive translocation of MOR from plasma membrane into the cytoplasm in most neurons (panel L). Data are representative of at least 3 experiments. *p < 0.05 vs control.

Figure 6

CTAP only partially inhibits the Na₂S-induced MOR internalization. SKNMCs are stimulated with DAMGO (1 μM) or Na₂S (50 μM) in presence or in absence of CTAP (1 μM) and the effects on MOR activation and internalization are detected. CTAP inhibits the MOR activation induced by DAMGO while it has no effect on that induced by Na₂S (panel A). CTAP blocks the DAMGO-induced MOR internalization but, in contrast, it only partially inhibits the Na₂S-induced MOR internalization (panel B). Data are representative of at least 3 experiments. *p < 0.05 vs control, **p < 0.05 vs DAMGO alone.

Figure 7

Na₂S induces AKT phosphorylation. Exposure to both DAMGO (1 μM) and Na₂S (50 μM) causes AKT phosphorylation on Threonine 308 as detected by Western blot analysis (panel A). Moreover, Na₂S induces AKT phosphorylation on Ser(473) as detected by phospho AKT assay (panel B). CTAP inhibits the DAMGO-induced AKT phosphorylation on Thre(308) (panel C), while it does not prevent that induced by Na₂S on Thre(308) and Ser(473) (panels C and D). Data are representative of at least 3 experiments. Data on AKT phosphorylation are mean ± SE of 5 experiments. *p < 0.05 vs control.

Figure 8

LY294002 inhibits the Na₂S-induced MOR internalization and AKT phosphorylation. The selective PI3K inhibitor LY294002 has no effects on DAMGO-induced MOR internalization (panel A), while it blocks that induced by Na₂S (panel B). Furthermore, LY294002 inhibits the AKT phosphorylation induced by Na₂S on Ser(473) (panel C). Data are representative of at least 3 experiments. Data on AKT phosphorylation are mean ± SE of 5 experiments. *p < 0.05 vs control.

Figure 9

Glibenclamide inhibits Na₂S-induced MOR activation and internalization and AKT phosphorylation. Qualitative PCR (panel A) and Quantitative Real-Time PCR (panel B) showing the expression of Kir6.2 and SUR1 in HepG2 (positive control) demonstrate that SKNMCs express both the K_ATP channels subunits Kir6.2 and SUR1. SKNMCs are stimulated with Na₂S (50 μM) in presence or in absence of glibenclamide (1 μM) for 60 minutes. Glibenclamide prevents the Na₂S-induced MOR activation (panel C) and internalization (panel D), while it has no effect on MOR internalization induced by DAMGO (panel D). Finally, glibenclamide inhibits AKT phosphorylation induced by Na₂S, as assessed by phospho-immunoassay (panel F). Data are representative of at least 3 experiments. Data on AKT phosphorylation are mean ± SE of 5 experiments. *p < 0.05 vs control; **p < 0.05 vs DAMGO or Na₂S alone.

Figure 10

Schematic representation of H₂S and opioid receptor interaction. The selective μ opioid receptor enkephalin analog DAMGO acts as a direct agonist of MOR leading to its activation, phosphorylation on Ser(377), co-immunoprecipitation with β arrestin and internalization (panel A and B). The selective MOR antagonist CTAP blocks the effects induced by DAMGO, while it only partially inhibits those induces by H₂S. In contrast, H₂S opens the K_ATP channels that activate the PI3K/AKT pathway leading to MOR activation, phosphorylation, co-immunoprecipitation with β arrestin and internalization, as the selective K_ATP channels blocker glibenclamide and the selective PI3K inhibitor LY294002 inhibit these effects (panel C and D).