Hydrogen sulfide acts as a mediator of inflammation in acute pancreatitis: in vitro studies using isolated mouse pancreatic acinar cells

Ramasamy Tamizhselvi, Philip K Moore, Madhav Bhatia, Ramasamy Tamizhselvi, Philip K Moore, Madhav Bhatia

Abstract

Hydrogen sulphide (H(2)S) is synthesized from L-cysteine via the action of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS). We have earlier shown that H(2)S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H(2)S and the expression of H(2)S synthesizing enzymes, CSE and CBS, in isolated mouse pancreatic acini. Pancreatic acinar cells from mice were incubated with or without caerulein (10(-7) M for 30 and 60 min). Caerulein increased the levels of H(2)S and CSE mRNA expression while CBS mRNA expression was decreased. In addition, cells pre-treated with DL-propargylglycine (PAG, 3 mM), a CSE inhibitor, reduced the formation of H(2)S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H(2)S formation in mouse acinar cells. Furthermore, substance P (SP) concentration in the acini and expression of SP gene (preprotachykinin-A, PPT-A) and neurokinin-1 receptor (NK-1R), the primary receptor for SP, are increased in secretagogue caerulein-treated acinar cells. Inhibition of endogenous production of H(2)S by PAG significantly suppressed SP concentration, PPT-A expression and NK1-R expression in the acini. To determine whether H(2)S itself provoked inflammation in acinar cells, the cells were treated with H(2)S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 muM), that resulted in a significant increase in SP concentration and expression of PPT-A and NK1-R in acinar cells. These results suggest that the pro-inflammatory effect of H(2)S may be mediated by SP-NK-1R related pathway in mouse pancreatic acinar cells.

Figures

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Hydrogen sulphide (H2S) concentration in caerulein-induced mouse pancreatic acinar cells. Freshly prepared pancreatic acinar cells were incubated with or without caerulein (10−7 M for 30 and 60 min). H2S concentration was measured as described in Section ‘Materials and methods’. Results shown are the mean ± S.E.M. of at least six separate determinations. ***P < 0.001 when caerulein treated cells (60 min) were compared with control cells (60 min).
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Cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) mRNA expression in control and caerulein-treated mouse pancreatic acinar cells. (A) RT-PCR of CSE in control and caerulein (10−7 M for 30 and 60 min) treated acinar cells. The graphs represent the optical density of the bands of CSE generated from six independent experiments that were normalized with the expression of 18S. ***P < 0.001 when caerulein treated cells (60 min) were compared with control cells (60 min). (B) RT-PCR of CBS in control and caerulein treated acinar cells. The graphs represent the optical density of the bands of CBS generated from six independent experiments that were normalized with the expression of 18S. ***P < 0.001 when caerulein treated cells (60 min) were compared with control cells (0 hr).
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Effect of DL-propargylglycine (PAG) on H2S synthesizing activity in caerulein-treated mouse pancreatic acinar cells. (A) Freshly prepared pancreatic acinar cells were incubated for 30 and 60 min at 37°C with different concentration of PAG (2–5 mM). After which the pellet was used to detect the H2S synthesizing activity. Results shown are the mean ± S.E.M. of at least six separate determinations. ***P < 0.001 when 3 mM PAG treated cells (60 min) were compared with control cells (60 min). (B) Freshly prepared pancreatic acinar cells were pretreated with PAG (3 mM) 60 min before caerulein (10−7 M for 30 and 60 min) treatment. Pancreatic acinar cell homogenates were assessed for ability to synthesize H2S from exogenous L-cysteine. Results shown are the mean ± S.E.M. of at least six separate determinations. ***P < 0.001 when caerulein treated cells (60 min) were compared with control cells (60 min). ***P < 0.001 when caerulein treated cells (60 min) were compared with PAG pretreated cells (60 min).
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PAG treatment attenuated caerulein-induced increase in substance P (SP) level in mouse pan-creatic acinar cells.Freshly prepared pancreatic acinar cells were pre-treated with PAG (3 mM) 60 min before caerulein (10−7 M for 60 min) treatment. Substance P level was measured as described in Section ‘Materials and methods’. Results shown are the mean ± S.E.M. of at least six separate determinations. ***P < 0.001 when caerulein treated cells (60 min) were compared with control cells (60 min). *P < 0.05 when caerulein treated cells (60 min) were compared with PAG pretreated cells (60 min).
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PAG treatment suppressed caerulein-induced up regulation of preprotachykinin-A (PPT-A) and neurokonin-1 receptor (NK-1R) mRNA expression in mouse pancreatic acinar cells. (A) PPT-A mRNA expression in control, caerulein-treated (10−7 M for 60 min) and PAG pretreated (3 mM) (30 min before caerulein treatment) cells. The graphs represent the optical density of the bands of PPT-A generated from six independent experiments that were normalized with the expression of 18S. ***P < 0.001 between control and caerulein treated cells. (B) NK-1 receptor mRNA expression in control, caerulein-treated (10−7 M for 60 min) and PAG pretreated (3 mM) (60 min before caerulein treatment) cells. The graphs represent the optical density of the bands of NK-1 receptor generated from six independent experiments that were normalized with the expression of 18S. ***P < 0.001 between caerulein and PAG pretreated cells.
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Effect of H2S donor, sodium hydro-sulphide (NaHS), on SP level in pancreatic acinar cells. Freshly prepared pancreatic acinar cells were treated with NaHS (10, 50 and 100 μM) for 30 min. Substance P level was measured as described in Materials and Methods. Results shown are the mean ± S.E.M. of at least six separate determinations. **P < 0.01 between control and NaHS-induced cells.
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H2S donor, sodium hydrosulphide (NaHS), induces PPT-A and NK-1R mRNA expression in mouse pancreatic acinar cells. (A) PPT-A mRNA expression in control and NaHS-treated (10, 50 and 100 μM) cells. The graphs represent the optical density of the bands of PPT-A generated from six independent experiments that were normalized with the expression of β-actin . *P < 0.05 between control and NaHS-treated cells. (B) NK-1R mRNA expression in control and NaHS-treated (10, 50 and 100 μM) cells. The graphs represent the optical density of the bands of NK-1R generated from six independent experiments that were normalized with the expression of β-actin. ***P < 0.001 between control and NaHS-induced cells.

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