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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 25, 2005 as doi:10.1096/fj.04-3023fje. |
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* Department of Pharmacology, National University of Singapore, Singapore; and
Centre for Biomedical Sciences, Defense Medical and Environmental Research Institute, Singapore
1Correspondence: Department of Pharmacology, National University of Singapore, Faculty of Medicine, Bldg. MD2, 18 Medical Dr., Singapore 117597, Singapore. E-mail: mbhatia{at}nus.edu.sg
SPECIFIC AIMS
Hydrogen sulfide (H2S) is a naturally occurring gas that has been shown to be a potent vasodilator. This study aims to investigate the presence of the H2S synthesizing enzyme cystathionine-
-lyase (CSE) in the pancreas. We report the effect of treatment with the CSE inhibitor DL-propargylglycine (PAG) on the severity of acute pancreatitis and associated lung injury.
PRINCIPAL FINDINGS
1. Induction of acute pancreatitis in mice by caerulein hyperstimulation resulted in an increase in plasma H2S levels
In control mice given 10 hourly injections of saline, plasma levels of H2S were 22.5 ± 1.9 µM. In mice given multiple i.p. injections of caerulein (50 µg/kg hourly for 10 h), levels of H2S were found to be 31.1 ± 3.3 µM (P<0.05; n=20). These results point to a role of H2S in the pathogenesis of acute pancreatitis.
2. Mouse pancreas expresses the gene CSE and exhibits H2S synthesizing activity
Mouse pancreas was found to express the gene for CSE, as evidenced by RT-PCR of the RNA extracted from the pancreas (Fig. 1
A). Incubation of mouse pancreas homogenates with exogenous cysteine resulted in the formation of H2S. To determine the effect of PAG treatment on pancreatic H2S formation, mice were administered PAG (100 mg/kg, i.p.) and killed 2 h later. Treatment of animals with PAG significantly inhibited pancreatic H2S synthesis (Fig. 1B
). These results pointed to the potential usefulness of PAG as a H2S synthesis inhibitor to investigate the role of H2S in acute pancreatitis and associated lung injury.
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3. Prophylactic, as well as therapeutic, treatment with PAG reduces the severity of pancreatic injury in acute pancreatitis
Evidence of pancreatic injury in acute pancreatitis induced by i.p. administration of caerulein (50 µg/kg hourly for 10 h) was indicated by increased plasma amylase and pancreatic MPO as a measure of neutrophil infiltration in mice treated with caerulein (saline-injected mice). Administration of PAG (100 mg/kg i.p.) 1 h before (prophylactic) or 1 h after (therapeutic) the first caerulein injection resulted in reduced plasma amylase levels (animals in which pancreatitis was induced by caerulein (plasma amylase (U/L) (control, 1204±59)); prophylactic treatment: placebo, 10635 ± 305; PAG, 7904 ± 495; therapeutic treatment: placebo, 10427 ± 470; PAG, 7811 ± 428. MPO activity in the pancreas was significantly reduced in both groups of caerulein-injected animals treated with PAG (pancreatic MPO activity, fold increase over control). Prophylactic treatment: placebo, 5.78 ± 0.63; PAG, 2.97 ± 0.39. Therapeutic treatment: placebo, 5.48 ± 0.52; PAG, 3.03 ± 0.47). Morphological changes in acute pancreatitis were significantly attenuated by PAG treatment (Fig. 2
). Figure 2A
shows a normal mouse pancreas; Fig. 2B
shows the pancreas of a mouse with caerulein-induced pancreatitis, clear evidence of edema, acinar cell injury/necrosis, and infiltration of inflammatory cells into the necrotic areas. Figure 2C, D
show a protective action of prophylactic, as well therapeutic, administration of PAG on these changes in acute pancreatitis.
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4. Prophylactic, as well as therapeutic treatment with PAG reduces the severity of lung injury associated with acute pancreatitis
Lungs from mice treated with caerulein exhibited increased MPO activity compared with control, vehicle-injected animals, implying lung neutrophil infiltration that was secondary to pancreatitis. Administration of PAG 1 h before or 1 h after the first caerulein injection significantly reduced lung MPO levels in caerulein-pretreated animals (lung MPO activity, fold increase over control); prophylactic treatment: placebo, 1.99 ± 0.16; PAG, 1.34 ± 0.14; therapeutic treatment: placebo, 2.03 ± 0.12; PAG, 1.41±0.97. The morphological changes in the lung after induction of acute pancreatitis consist principally of alveolar thickening and infiltration by inflammatory cells, the vast majority of which are neutrophils. After prophylactic or therapeutic treatment with PAG, alveolar thickening and inflammation were both consistently reduced.
CONCLUSIONS AND SIGNIFICANCE
Gases such as nitric oxide (NO) and carbon monoxide (CO) play important roles both in normal physiology and in disease. In recent years, interest has been directed toward other naturally occurring gases such as hydrogen sulfide (H2S). Two pyridoxal-5'-phosphate-dependent enzymes, cystothionine ß-synthase (CBS) and CSE, are responsible for the majority of the endogenous production of H2S in mammalian tissues. Both enzymes utilize L-cysteine as substrate. The transcriptional expression of CBS in rat brain has previously been confirmed using Northern blot analysis, but in that study no CSE mRNA was detected in the brain. CSE, on the other hand, appears to be the primary H2S-producing enzyme in peripheral tissues. In the vascular tissues studied so far, CSE is the only H2S-generating enzyme that has been identified. Although H2S has been shown to relax vascular smooth muscle in vitro and in vivo, and has been implicated in different conditions, such as septic shock, endotoxin shock, and pulmonary hypertension, its possible role as a mediator of inflammation has not yet been investigated.
To the best of our knowledge, this is the first report of a potential proinflammatory action of endogenously produced H2S. We show here that mRNA for CSE is expressed in mouse pancreas and that pancreas homogenates convert L-cysteine to H2S ex vivo. Plasma levels of H2S are increased in mice upon induction of acute pancreatitis. It has previously been shown that complete and irreversible inactivation of CSE activity is achieved in vitro with PAG. When administered to rodents, PAG produces an almost complete inhibition of liver CSE enzyme activity, is well absorbed, and readily crosses biological membranes. In our experiments, the conversion of L-cysteine to H2S in pancreas homogenates was significantly reduced in mice pretreated with PAG.
We show that treatment of animals with PAG (prophylactic or therapeutic) reduces the severity of pancreatitis as evidenced by a significant attenuation of hyperamylasemia and pancreatic MPO activity and by histological evidence of diminished pancreatic injury. Severe, but not mild, acute pancreatitis is associated with lung injury, which is characterized by sequestration of neutrophils within the lung (i.e., increased lung MPO activity) and histological evidence of lung injury. In the present experiments, we demonstrated that prophylactic/therapeutic administration of PAG protected mice against acute pancreatitis-associated lung injury as evidenced by a significant attenuation of lung MPO activity and by histological evidence of diminished lung injury (alveolar thickening and leukocyte infiltration).
Our results suggest that H2S is likely to play an important role in determining the severity of pancreatitis. The mechanism(s) by which H2S acts to amplify the severity of pancreatitis remain to be elucidated. From studies probing the role of H2S in other conditions, it seems reasonable to suggest that H2S acts primarily to increase vascular permeability and promote edema as a result of its vasodilator activity. Although this may account for the finding that PAG treatment lessens the increased pancreatic and lung edema, it would not account for some of the other effects of PAG treatment such as diminished acinar cell injury/necrosis, since this injury is believed to be caused by intra-acinar cell activation of digestive enzymes, with trypsinogen activation being an important early step in this cascade. It is thus possible that H2S may be produced within pancreatic acinar cells or other cells in close proximity to the acinar cells and may stimulate intra-acinar activation of zymogens.
That H2S is a potent vasodilator in large blood vessels such as the aorta and portal vein of rats is clear. In addition, a relaxant effect of H2S on resistance arterioles has recently been reported. However, to the best of our knowledge, no reports in the literature have attempted to assess the effect of H2S on inflammation.
Although the precise role of H2S in inflammation has yet to be determined, the possibility of an interaction with NO, which is known to play an important role in inflammation, should be considered. An interaction of H2S with NO has already been postulated. NO is a reactive nitrogen species and acts by impairing the reduced glutathione/oxidized glutathione balance and/or by inhibiting enzymes and ion channels through S-nitrosylation processes. H2S may be involved in the reduction of thiols. Numerous studies have pointed to a role of NO in acute pancreatitis. The possibility that NO and H2S may work together to promote inflammation warrants further investigation.
Another possibility is a contribution of H2S in neurogenic inflammation in acute pancreatitis. We previously showed a role of substance P and neurogenic inflammation in the pathogenesis of acute pancreatitis and associated lung injury by the use of knockout mice deficient in the gene for substance P and neurokinin-A (preprotachykinin-A gene), for neurokinin-1 (NK1) receptors, and by the use of the specific NK1 receptor antagonist CP 96,345. In a recent study, it was shown that H2S stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder, pointing to a role of H2S in neurogenic inflammation. Therefore, it is possible that H2S may be acting as an inducer of neurogenic inflammation in acute pancreatitis and other inflammatory conditions. The precise molecular mechanism of the role of H2S as an inflammatory mediator will be the subject of future studies.
Although the mechanisms by which H2S acts as a proinflammatory mediator in acute pancreatitis remain to be understood, results presented in this paper strongly suggest that H2S synthesis blockers may be of potential use to treat acute pancreatitis and its systemic complications. Whether such inhibitors may be applicable to treatment of other inflammatory diseases warrants further investigation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3023fje;
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