Hydrogen Sulfide and Substance P in Acute Pancreatitis

Lara Jupp, Jack R. Rivers*, and Madhav Bhatia

Dept of Pathology, University of Otago Christchurch

In Adaptation Biology and Medicine. Vol. 7. A.R. Popescu and P.K. Singal, editors. Narosa Publishing House, New Delhi.

*Corresponding author: Jack Rivers-Auty, Ph.D. Faculty of Life Sciences University of Manchester, Manchester, UK Email. jack.rivers-auty@manchester.ac.uk Web site: www.jackauty.com

Abstract

The pancreas produces hormones that are secreted into the blood which regulate

metabolism and it produced digestive enzymes that are secreted into the duodenum. During

pancreatitis these digestive enzymes are activated and released into the pancreas, which results in

the digestion of the organ itself causing extensive tissue damage. This leads to severe local

inflammation that can develop into a pathological systemic inflammatory state. Hydrogen sulfide

(H2S) and substance P (SP) have recently been discovered to play a significant role in the

pathological development of acute pancreatitis (AP). H2S is a gaseous molecule previously

considered to be an inconsequential byproduct of reactions in the body. However, recently it has

been discovered that the enzymes involved in the endogenous production of H2S are upregulated

during inflammatory conditions and that inhibition of these enzymes reduces inflammation. As a

result of these and other studies, H2S is now thought of as gaseous signalling molecule involved

in mediating inflammation and plays a crucial role in the pathophysiology of AP. SP is a

tachykinin that is produced from primary nerve terminals and has been found to be a key

signalling molecule in the neurogenic inflammatory response. SP activates neurokinin receptors,

particularly neurokinin-1 receptor (NK1R). Activation of NK1R causes inflammatory symptoms

such as vasodilation, cytokine release and leukocyte activation. Animal studies have shown that

SP levels increase 24-fold during the first 12 hours of AP. Furthermore, mice lacking a gene

necessary for SP production had significantly less severe pancreatitis symptoms. Interestingly,

several studies have shown that H2S levels significantly effect SP production and NK1R

expression. This research suggests that H2S and SP have significant roles in the signalling of

inflammation during AP and these signalling pathways are interdependent. Therefore, both

pathways are excellent candidates for pharmacological intervention as a treatment for AP.

Key Words

Pancreatitis, inflammation, substance P, Hydrogen Sulfide, cytokines, immune cells, signalling

pathways, neurokinin receptor, tachykinin, gaseous signalling molecule, acini, digestion,

leukocyte, L-cysteine, cystathionine-γ-lyase, CSE, cystathionine-β-synthase, CBS, vanilloid,

preprotachykinin-A.

Introduction

The pancreas is a vital organ responsible for regulating energy homeostasis. It is

composed of two different cellular groups, endocrine and exocrine, with quite differing

functionalities. The endocrine pancreatic islet cells produce and secrete hormones required for

energy and glucose homeostasis including insulin, glucagon, somatostatin, and pancreatic

polypeptide [1]. The exocrine acinar cells are responsible for the synthesis and secretion of

hydrolytic enzymes that act in the small intestine to aid in food digestion. The three main

categories of digestive enzymes secreted by the acini are α-amylase; to digest carbohydrates,

lipases; to hydrolyse fatty acids, and proteases; to digest protein in the diet. The digestive

enzymes are stored in secretory granules near the apical portion of the acini and in response to

food ingestion they are transported through a series of ducts of increasing size to the duodenum.

The proteases are stored in the acini as inactive zymogen precursors and once in the duodenum

they are then activated by the enterocyte-associated enzyme enterokinase [2]. This activation

mechanism prevents pathological autodigestion of the gland and related gastrointestinal

structures. Other mechanisms to prevent premature activation of these zymogens include low

intracellular Ca2+, low pH and the presence of protease inhibitors within the zymogen storage

granules [2]. However, in some disease states, such as acute pancreatitis (AP), these zymogens

are activated within the pancreas and this results in the digestion of functional cellular proteins

and ultimately widespread cellular death and tissue damage.

AP is an inflammatory disease of the pancreas characterised by pancreatic tissue oedema,

acinar cell necrosis, haemorrhage and inflammation of the damaged pancreas [1]. In developed

countries, the main causes are alcohol abuse (36%) and obstruction of the bile duct by gallstones

(38%) [3]. Other rare and more controversial causes include pancreas divisum; a congenital

morphological aberration of the pancreatic duct system, an intraduct papillary mucinous tumour,

and hypercalcaemia [3]. The pathophysiology of AP begins with the premature intra-acinar

activation of zymogens and the autodigestion of the proteins within the organ [4,5]. The resulting

pancreatic damage leads secretion of inflammatory signalling molecules and a local

inflammatory response, which can then be followed by a systemic inflammatory condition

referred to as the systemic inflammatory response syndrome (SIRS) [6]. SIRS is a systemic

response to a local inflammatory assault and can include fever, tachycardia, respiratory

insufficiency and multiple organ dysfunction syndrome (MODS) [6,7]. The overall mortality of

AP is approximately 5%, and this is higher in patients with necrotizing pancreatitis (17%). This

increase in mortality is due to the heightened risk of organ failure, as opposed to those with

intersitital pancreatitis (3%) where little necrosis occurs [5].

The severity of AP can differ widely, however, the cellular immune response and

processes are very similar regardless of etiology and involves both the local and systemic

overproduction of inflammatory mediators. One of the primary focuses of research into AP has

been inflammatory cytokines. Increased concentrations of the inflammatory cytokines

interleukin-1 (IL-1), IL-6, IL-8 and tumour necrosis factor-α (TNF-α) have been associated with

greater disease severity [7]. Intervention studies have shown that these inflammatory signalling

molecules are crucial to the development of pancreatitis [8,9]. The damaged acinar cells release

chemotactic factors facilitating the recruitment of inflammatory cells, leading to the release of

these inflammatory cytokines and other key inflammatory mediators including platelet-activating

factor (PAF) and bradykinin [10]. Recently, substance P (SP) and hydrogen sulfide (H2S) have

been identified as signalling molecules in novel pathways of inflammation and the pathogenesis

of AP. This chapter will discuss the roles of these compounds in AP outlining the key research

that elucidated the involvement of SP and H2S in inflammation signalling [10].

Hydrogen Sulfide

H2S is a foul smelling gas associated with swamps and rotten eggs where it is formed as a

byproduct of bacterial anaerobic digestion [11]. Historically it has been viewed as a toxicant

responsible for many industrial deaths, as it is also generated as a byproduct in petrol refineries,

paper mills and tanneries [11]. However, this ubiquitous molecule has more recently been found

to be produced by the body and is involved in the regulation of many endogenous processes. The

physiological concentrations of H2S have been a contentious issue with reported plasma

concentrations ranging from sub-micromolar levels to 300 µM [12]. Despite these issues, the

majority of groups in the field agree that H2S is produced by the body and this production is

upregulated during several pathologies [13-16]. Furthermore, inhibiting this production has been

shown to be beneficial during some of these pathologies [17]. Exogenous H2S exhibits a steep

dose-response curve, with obvious odour at 3-10 ppm, respiratory tract irritation at 50-100 ppm

and dizziness, unconsciousness and death occurring at levels of 500 ppm and above [18]. As a

broad-spectrum toxicant it affects many organ systems including lung, brain, kidney and

gastrointestinal tract. H2S is weakly acidic and exists in plasma as the un-dissociated gas or in

one of two dissociation states, the hydrosulfide anion (HS-) and the sulfide anion (S2-). At

physiological pH of 7.4, the total sulfide pool is comprised of 18.5% un-dissociated H2S and

81.5% as HS- [19]. Due to the current limitations in H2S measurement, it is difficult to confirm

whether the physiological actions of H2S are mediated by H2S directly or by the dissociated

form.

H2S Synthesis

The amino acid L-cysteine is the predominate sulfide donor molecule in the endogenous

production of H2S. L-cysteine can be obtained from the diet, synthesised from L-methionine or

‘liberated’ from endogenous proteins [20]. In mammalian tissues, H2S is produced from the

enzymatic desulfhydration of cysteine by at least three separate endogenous pathways [11]. The

key enzymes in the two primary pathways are cystathionine-γ-lyase (CSE, EC 4.4.1.1) or

cystathionine-β-synthase (CBS, EC 4.2.1.22) (Fig.1) [21]. A third minor pathway involves the

enzymes 3-mercapto-sulfurtransferase (3-MST, EC 2.8.1.2) and cysteine aminotransferase

(CAT, EC 2.6.1.3) (Fig.1) [21]. Both CSE and CBS are pyridoxal phosphate (PLP)-dependent

enzymes and govern H2S synthesis in quite separate organ systems.

CBS is the largest contributor to endogenous H2S in the central nervous system with roles

in both neuroinflammation and cognition [22,23]. It also has a minor role in H2S production in

the periphery including the liver, kidney and ileum [24]. CBS activity is enhanced by the

allosteric activator S-adenosyl-L-methionine (SAM) as well as by binding of Ca2+/Calmodulin,

L-glutamate and also electrical stimulation [23]. At this time, two H2S-generating pathways have

been described for CBS. The first and best characterised involves the condensation of

homocysteine and serine to form cystathione, with the other pathway involving hydrolysation of

L-cysteine as the substrate to form L-serine and H2S (Fig.1) [24].

CSE is the enzyme primarily involved in peripheral H2S synthesis, including muscle

tissues, vascular systems, digestive tract, liver, pancreas and kidneys [24]. Hydrolysis of L-

cysteine by CSE produces H2S, pyruvate and ammonia (NH3), and CSE can also catalyse the β-

disulfide elimination reaction of cystine to thiocysteine, which is then hydrolysed by CSE to

cysteine and H2S (Fig. 1) [21]. CSE activity is irreversibly inhibited by D,L-propargylglycine

(PAG) and reversibly inhibited by β-cyano-L-alanine (BCA) [25].

3-MST and CAT are non-PLP dependent enzymes located both in the mitochondria and

cytosol of the vascular endothelium and brain. CAT catalyses the reaction of L-cysteine with

keto-acids forming 3-mercaptopyruvate which is then desulfurated in a zinc-dependent reaction

by 3-MST, yielding H2S and pyruvate (Fig. 1) [26]. This pathway is of particular interest in the

brain where it produces H2S more efficiently than the pathway catalysed by CBS [27]. H2S can

also be formed non-enzymatically via reduction of thiols and thiol-containing molecules as a

way of liberating H2S from intracellular sulfur stores [19].

H2S is rapidly oxidised in the mitochondria yielding thiosulfate (S2O32-), which is then converted

to sulfite (SO32-) by rhodanese (EC 2.8.1.1), followed by oxidation by sulfate oxidase to sulfate

(SO42-) and renal excretion [20]. It is interesting to note that the oxidation of sulfide takes

preference over the oxidation of other carbon-based substrates in the mitochondria, ensuring that

the intracellular levels of H2S remain constitutively low [12].

Figure 1: Potential pathways for H2S production. CAT, cysteine aminotransferase; CBS, cystathionine-β-synthase; CSE, cystathionine-γ-lyase; DHLA, Dihydro lipoid acid; 3-MP, 3-mercaptopyruvate; 3-MST, 3-mercapto-sulfurtransferase; R-SH, thiol; Trx, thioredoxin.

Pathological Effects of H2S

The main pathological effect of H2S is by direct inhibition of mitochondrial cytochrome c

oxidase, hindering oxidative phosphorylation [28]. This is thought to be the primary mechanism

of H2S-related toxicity and deaths. It has been found that H2S is a more potent inhibitor of

mitochondrial activity than cyanide [18].

H2S is also a potent mediator of inflammation in many pathological conditions. H2S

levels were shown to be markedly increased in rat septic and endotoxic shock models, and to

correlate negatively with blood pressure and cardiac function [29]. H2S was also found to be a

crucial inflammatory mediator in a lipopolysaccharide-induced mouse model of septic shock

[30]. The deleterious haemodynamic effects of H2S are due to the interaction of H2S with KATP

channels of vascular smooth muscle cells, causing hyperpolarisation of the cell membrane [31].

This effect also increases vascular permeability and promotes oedema by allowing neutrophil

and leukocyte infiltration [32]. H2S contributes to inflammation by a variety of other routes. H2S

promotes transient receptor potential vanilloid-1 (TRPV1)-mediated neurogenic inflammation in

sepsis [33]. Activation of TRPV1 by H2S in sepsis evoked lung injury causes the upregulation of

COX-2 and PGE-2 metabolites, which are implicated in the augmentation of inflammation in

both sepsis and respiratory disease [14]. In polymicrobial sepsis H2S activation of TRPV1

resulted in release of the inflammatory tachykinin SP culminating in an increase in ERK1/2 and

IkB-α phosphorylation [13]. H2S acts as an inflammatory mediator in cecal ligation and

puncture-induced sepsis in the mouse by increasing levels of inflammatory cytokines including

IL-1β, IL-6, TNF-α and monocyte chemotactic protein-1 (MCP-1) via nuclear factor-κB (NFκB)

activation [34]. Furthermore, H2S inhibition decreases severity of both local and systemic

inflammation [34]. Therefore, not only does H2S cause deleterious hypotensive haemodynamic

effects due to its vasorelaxation properties, it also promotes the development and progression of

inflammation and multi-organ damage that is associated with sepsis and other inflammatory

conditions.

H2S in Acute Pancreatitis

The use of high doses of the peptide secretagogue cholecystokinin (CCK) or its analogue

caerulein has been well validated as an experimental model of AP [35]. This model has identified

H2S as an important mediator in the pathogenesis of AP. Pancreatic acinar cells express both the

genes for CSE and CBS, however, only CSE mRNA expression is upregulated in response to

administration of caerulein. This suggests that CSE is responsible for the increase in H2S

concentrations during AP and therefore, is the most suitable target for pharmacological

intervention in AP [36]. Furthermore, the upregulation of CSE corresponds to a significant

increase in H2S production by isolated acinar cells, an effect that was abated with the pre-

administration of 3 mM of the CSE inhibitor DL-proplyargylglycine (PAG) [36]. Mice with

caerulein-induced AP (50 µg/kg i.p. per hour for 10 hours) showed a 27% increase in plasma

H2S levels compared to saline controls, as well as increased myeloperoxidase (MPO) levels

indicating neutrophil infiltration [37]. Both prophylactic and therapeutic treatment with PAG

significantly decreased these levels as well as decreasing amylase secretion and the degree of

pancreatic injury. Furthermore, there is evidence that H2S is involved in AP-related pain and

hyperalgesia through interaction with T-type Ca2+ channels [16].

A predominant mechanism of H2S-induced inflammation in AP is through the activation

of pro-inflammatory CC type chemokines. In vitro, caerulein hyperstimulation augmented the

pancreatic production and secretion of the CC chemokines MCP-1, MIP-2 and MIP-1α [17]. This

was also shown to occur in vivo, with both the pancreas and the lungs showing increased mRNA

expression of these chemokines, implicating them in the development of both local and systemic

inflammation in AP. As PAG pre-treatment decreased this chemokine expression, it is thought

that the pro-inflammatory effects of H2S in AP are at least partly mediated by these CC

chemokines [17]. Brady et al. [38], found in an in vivo model of AP that CXC type chemokines

may also be involved with increased concentrations of the CXC chemokine CINC, in caerulein-

hyperstimulated mice. Furthermore, Tamizhelvi et al. [39], found that H2S causes neutrophil

attraction and attachment through intra-cellular adhesion molecule-1 (ICAM-1) activation,

mediated via NFκB and SFKs. Therefore, H2S seems to mediate the activation of CC and CXC

chemokines as well as other inflammatory cytokines in AP.

Substance P

SP belongs to the tachykinin family of neuropeptides. Tachykinins are one of the largest families

of neuropeptides and are characterised by a conserved C-terminal sequence of Phe-X-Gly-Leu-

Met-NH2, in which X encodes an aromatic (Phe, Tyr) or aliphatic (Val, Ile) amino acid [40]. The

integrity of this C-terminal sequence is important for mediating their biological effects. SP is

encoded by the preprotachykinin-A (PPT-A) gene, which also encodes another tachykinin,

Neurokinin A (NKA). Alternative splicing of the PPTA genes results in three primary mRNAs:

α-, β-, and γ-PPT-A. The α-PPT-A encodes SP solely in the CNS, while β-, and γ-PPT-A code

for the synthesis of both NKA and SP [40]. SP is released primarily from neuronal cells in

peripheral endings of widely dispersed capsaicin-sensitive primary afferent neurons as well as

enteric neurons of the submucosal and myenteric plexuses in the gut [41]. SP synthesis and

release from primary nerve terminals is increased remarkably in both the CNS and PNS during

inflammation, in a phenomenon known as ‘neurogenic inflammation’ [41,42]. SP has been

shown to be a major mediator of neurogenic inflammation in a range of tissues including skin,

cardiovascular tissue, and cephalic structures, as well as in the respiratory and gastrointestinal

tract [10].

The biological actions of SP are mediated primarily through the NK1R, though at high

concentrations SP can also activate NK2 and NK3 receptors in a number of tissues [42].

Neurokinin receptors are G-protein coupled receptors (GPCRs), and activation of NK1R leads to

phospholipase activation, inositol-1,4,5-triphosphate (IP3) turnover and increased levels of

intracellular calcium [42]. In neurogenic inflammation, the binding of SP to NK1Rs leads to

activation of a variety of immune and non-immune cells [41]. Activation of NK1R on endothelial

cells of blood vessels results in increased vascular permeability, vasodilation and plasma

extravasation of neutrophils and monocytes [41]. Macrophage NK1R activation results in release

of inflammatory cytokines and additional SP, which causes release of histamine and bradykinin

from mast cells and contributes to vascular permeability and oedema [41].

Substance P in Acute Pancreatitis

The first indication of the role of SP in AP was from a study by Bhatia et al. [43] that

induced AP in mice using supramaximally-stimulating doses of caerulein, and found a time-

dependent increase in pancreatic SP levels [43]. This culminated in a 24-fold increase in SP

levels over control after 12 hours and was correlated with an increase in NK1R density in the

pancreas. Genetic deletion of NK1Rs (NK1R-/- mice) markedly reduced the extent of pancreatic

injury as well as lung injury in response to caerulein compared to the wild type controls. This

study also showed no effect of either NK1R or PPT-A gene deletion on acinar amylase secretion,

indicating that acinar cell responsiveness to caerulein was not altered and that SP mediates more

downstream inflammatory effects [43]. This indicates SP, through the NK1R, plays a significant

role in the pathophysiology of AP and ensuing lung injury. The use of the specific NK1R

inhibitor CP-96345 also reduced the extent of caerulein-induced AP and lung injury in mice [44].

Neutral Endopeptidase (NEP) is an enzyme that hydrolyses tachykinins, including SP thereby

terminating their effects. NEP knock out mice (NEP-/-) were found to have more severe

pancreatitis symptoms (increased serum amylase, neutrophil sequestration and acinar cell

necrosis) and a greater degree of lung injury after 4 and 8 hours of caerulein administration

compared to wild type [45]. However after 12 hours the level of pancreatic injury was similar

between NEP-/- and NEP+/+ mice, indicating NEP only plays a role in the early stages of AP.

Several in vitro studies have probed the mechanisms by which SP contributes to AP.

Hyperstimulation with caerulein increased SP levels, as well as those of its receptor, NK1R, in

isolated pancreatic acini [36]. This inflammatory upregulation of SP is mediated by the kinases

extracellular signal-regulated kinase (ERK1/2), and c- Jun N-terminal kinase (JNK) as well as

the transcription factors NFκB and activator protein-1 (AP-1) [46]. SP caused a dose-dependent

upregulation of the pro-inflammatory chemokines MCP-1, MIP-1α and MIP-2 in pancreatic

acinar cells via an NFκB-dependent pathway [47]. Further studies found that SP-induced NFκB

activation of these inflammatory chemokines is mediated through several distinct pathways all

involving kinase activation. The first pathway involves the activation of the Ca2+-independent

kinase protein kinase-δ (PKC-δ) (Fig. 2). SP phosphorylation of PKC-δ resulted in increased

activation of MAPKKK, MEKK1, and MAPK (ERK and JNK), leading to transcription factor

NFκB and AP-1 activation and chemokine synthesis (Fig. 2) [48]. SP also activates

phospholipase C (PLC) resulting in increased intracellular Ca2+ levels and PKC-α

phosphorylation, also culminating in ERK, JNK, NFκB and AP-1 activation (Fig. 2) [49]. Src

family kinase (SFK) phosphorylation (Tyr416) is also increased in response to SP-NK1R

interaction as well as ERK and JNK and activation of the STAT3, NF-κB and AP-1 transcription

factors (Fig. 2) [49].

Figure 2: Proposed pathways of Substance P-induced chemokine synthesis.

H2S and SP interplay in AP

Both H2S and SP have been shown to mediate inflammation in AP independently, but

there is increasing evidence that some of the inflammatory effects described above may be due to

direct H2S and SP interaction. Treatment of pancreatic acinar cells with the H2S donor NaHS

caused a significant increase in SP levels along with upregulation of PPT-A and NK1R

expression [36]. Furthermore, administration of the CSE inhibitor PAG to caerulein-treated

acinar cells, decreased the levels of SP, PPT-A and NK1R expression compared to the control

acinar cells. This was also seen in the in vivo model with PAG administration significantly

attenuating the upregulation of SP and NK1R expression in the plasma, pancreas and lung tissue

in response to caerulein administration [50]. This indicates that the elevated concentration of H2S

in AP stimulates expression of the PPT-A and NK1R genes in the pancreas and lungs leading to

increased SP production and activity in these tissues. The inflammation and pancreatic injury

attributed to the action of H2S may be partially, or completely, attributed to SP mediated

mechanisms, though further elucidation of the mechanism(s) of this interplay is required to fully

understand the relationship between SP and H2S in AP.

Summary

During AP the digestive enzymes produced by the pancreas are activated and cause

autodigestion of the organ. The subsequent inflammatory response causes further damage to the

pancreas and can develop into a systemic inflammatory condition. H2S and SP have recently

been discovered to be integral signalling molecules in the inflammatory response of AP.

Inhibition of either of these pathways has been shown to reduce the severity of the pathology.

Interestingly, these pathways appear to be interdependent. More research is need to completely

elucidate the mechanisms by which H2S and SP contribute to inflammation during AP.

Furthermore, research into the development of novel and specific compounds that inhibit the

production of H2S and SP is needed as it may lead to new treatment strategies for AP in the

clinic.

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