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Hydrogen sulfide as a novel mediator for pancreatic pain in rodents
  1. S Nishimura1,
  2. O Fukushima1,
  3. H Ishikura2,
  4. T Takahashi1,
  5. M Matsunami1,
  6. T Tsujiuchi3,
  7. F Sekiguchi1,
  8. M Naruse2,
  9. Y Kamanaka4,
  10. A Kawabata1
  1. 1
    Division of Pharmacology and Pathophysiology, Kinki University School of Pharmacy, Higashi-Osaka, Japan
  2. 2
    Division of Emergency and Critical Care Medicine, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan
  3. 3
    Department of Life Science, Kinki University School of Science and Engineering, Higashi-Osaka, Japan
  4. 4
    Ono Pharmaceutical Co Ltd, Mishima-gunn, Osaka, Japan
  1. Professor A Kawabata, Division of Pharmacology and Pathophysiology, Kinki University School of Pharmacy, 3-4-1 Kowakae, Higashi-Osaka, 577-8502, Japan; kawabata{at}phar.kindai.ac.jp

Abstract

Objective: Hydrogen sulfide (H2S) is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H2S in the processing of somatic pain was identified. Here, the involvement of H2S in pancreatic pain is examined.

Methods: Anaesthetised rats or mice received an injection of NaHS, a donor for H2S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively.

Results: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca2+ channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice.

Conclusions: The data suggest that pancreatic NaHS/H2S most probably targets T-type Ca2+ channels, leading to nociception, and that endogenous H2S produced by CSE and possibly T-type Ca2+ channels are involved in pancreatitis-related pain.

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Hydrogen sulfide (H2S) is a colourless and flammable gas with a characteristic smell, and has long been regarded as a toxic environmental agent with little physiological significance. However, H2S is now considered a gaseous messenger like nitric oxide (NO) or carbon monoxide (CO), and is indeed produced endogenously from l-cysteine mainly by two pyridoxal 5′-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), in the mammalian body.13 In addition, H2S can be generated by sulfate-reducing bacteria in the intestinal lumen.46 Recent studies have shown that H2S plays several physiological and/or pathological roles in many organs or tissues, including the gastrointestinal tract.13

Among the functions of H2S in the alimentary systems, an interesting finding is that H2S protects against gastric mucosal injury induced by non-steroidal anti-inflammatory drugs (NSAIDs)7 or ischaemia–reperfusion.8 In contrast, proinflammatory roles for H2S have been described in various organs including the pancreas.9 10 In the colon, H2S is generally believed to be involved in inflammatory bowel diseases and colorectal cancer,46 while anti-inflammatory roles for colonic H2S have also been described.11 Furthermore, there is evidence that H2S regulates smooth muscle tone in the gastrointestinal tract12 as well as in the vascular and respiratory systems.1317 According to a recent study, H2S is also considered a prosecretory neuromodulator in the guinea-pig and human colon.18 Mechanisms for some of the above-described biological actions of H2S involve the activation of ATP-sensitive K+ (KATP) channels13 or mitogen-activated protein (MAP) kinase pathways,8 stimulation of capsaicin-sensitive sensory neurons16 1820 and an interaction with NO and/or NO synthase isoforms.13 14 21 Most recently, we reported electrophysiological and behavioural evidence that H2S sensitises T-type Ca2+ channels possibly through redox modulation and that H2S mediates hyperalgesia in rat hindpaw in a manner dependent on the activation of T-type Ca2+ channels.22 This is consistent with evidence for a critical role for T-type Ca2+ channels in the processing of pain perception.23 In addition, multiple independent studies have shown that H2S stimulates capsaicin-sensitive sensory nerves in distinct tissues including the colonic mucosa.16 1820 Nonetheless, roles for H2S in visceral nociceptive processing are complex—that is, luminal H2S appears to be pronociceptive in the mouse colon,24 while systemic (intraperitoneal) administration of a donor for H2S suppresses responses to colorectal distension in rats.25

Clinically, pancreatic pain is a serious problem for patients with pancreatitis or pancreatic cancer. In humans, pain elicited from the pancreas is often referred to the upper abdominal area and radiates to the back, and these skin areas are usually tender to touch (ie, referred hyperalgesia/allodynia). In animal models for pancreatitis, pancreatic pain is characterised as an increased response to mechanical stimulation in upper abdominal areas.26 27 Apart from the possible involvement of H2S in pancreatitis,9 it is likely that H2S may play direct roles in the processing of pancreatic pain. In this context, the present study investigated the effects of exogenously applied H2S or inhibition of CSE, a major H2S-forming enzyme in the pancreas,9 on pancreatic nociception in rodents, and examined the possible involvement of the T-type Ca2+ channel, a target for H2S, in the underlying mechanism.

MATERIALS AND METHODS

Experimental animals

Male Wistar rats weighing 240–300 g and male ddY mice weighing 16–23 g were purchased from Japan SLC (Shizuoka, Japan). All experimental protocols were approved by the Committee for the Care and Use of Laboratory Animals at Kinki University and were in accordance with the Guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Injection of NaHS or capsaicin into the pancreatic duct in rats or mice

NaHS, a donor for H2S, or capsaicin was infused into the pancreatic duct, as described by Hoogerwerf et al28 with minor modifications.29 Briefly, rats or mice were anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneally, Dainippon Sumitomo Pharmaceutical, Osaka, Japan), the peritoneum was incised and the duodenal loop was exposed. The pancreatic/common bile duct entering the duodenum was identified under a dissecting microscope, and ligated at the hepatic end. After a small incision was made into the duct at a position close to the duodenal end, a polyethylene cannula (0.61 mm diameter for rats, Becton Dickinson, Franklin Lakes, New Jersey, USA; 0.50 mm diameter for mice, Nazme, Tokyo, Japan) was inserted into the duct and tied with a thread. NaHS at 50–1500 nmol or capsaicin at 490 nmol in a volume of 0.5 ml was infused into the pancreatic duct through the cannula in the rat, while NaHS at 100 nmol in a volume of 25 μl was infused into the pancreatic duct of the mouse. Mibefradil, a T-type Ca2+ channel inhibitor, at 9 mg/kg was administered intraperitoneally 30 min before challenge with NaHS or capsaicin in rats or mice.

Analysis of neurons expressing immunoreactive Fos protein in the spinal dorsal horn of rats or mice

As described previously,29 2 h after infusion of NaHS or capsaicin into the pancreatic duct, the animals were perfused transcardially with physiological saline and subsequently with 2% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for fixation. Dorsal root ganglia (DRGs) at T8 and T9 levels were identified by counting up from the T13 DRG that was adjacent to the last rib, and were marked for the later identification of the T8 and T9 spinal cord segments. The spinal cord was removed, postfixed in the same fixative overnight at 4°C and then cryoprotected in a phosphate-buffered 30% sucrose solution overnight at 4°C. The T8 and T9 spinal cord segments (also C5–C8 and T5 spinal segments in some experiments) were serially sectioned at 30 μm thickness using a freezing microtome. The sections were collected in 0.1 M phosphate-buffered saline (PBS) and processed for the immunolocalisation of Fos. The consecutive sections were incubated in 1% normal goat serum for 30 min, and then with the primary antibody, a rabbit antiserum against a peptide mapping at the N-terminus of human Fos p62 (Santa Cruz Biotechnology, Santa Cruz, California, USA), at a dilution of 1:7000 for 16 h at 4°C. The sections were then incubated with the biotinylated goat antiserum against rabbit immunoglobulin G (IgG) for 1 h and subsequently treated with a peroxidase-conjugated avidin–biotin complex (Vectastain ABC kit, Vector Laboratories, Burlingame, California, USA) for 30 min at 4°C. To develop the ABC reaction, the sections were incubated in 0.05 M Tris–HCl buffer (pH 7.6) containing 0.05% 3,3′-diaminobenzidine-tetra HCl, 0.4% nickel ammonium sulfate and 0.035% hydrogen peroxide for 10 min at 24°C. The sections were then rinsed three times with 0.1 M PBS for 10 min. In five sections chosen from each animal, the number of Fos-positive cells was determined bilaterally in laminae I–II and III–IV of the T8 and T9 spinal sections. The mean of the number of Fos-positive cells per number of slices for each animal was first calculated and the data are calculated and statistically analysed from the number of animals.

Creation of a mouse model for caerulein-evoked pancreatitis accompanied by referred allodynia/hyperalgesia in the upper abdomen

As described previously,26 pancreatitis was created in mice by six intraperitoneal administrations of caerulein at 50 μg/kg at 1 h intervals. Referred allodynia/hyperalgesia in the upper abdomen was evaluated, as mentioned below, at 30 min after the final dose of caerulein. In the time course experiments, referred allodynia/hyperalgesia was also assessed at 0.5, 1.5, 2.5, 3.5 and 24 h after the final caerulein treatment. For determination of the severity of the evoked pancreatitis, blood samples were collected from the abdominal aorta in the mice under urethane (1.5 g/kg, intraperitoneally) anaesthesia, and the pancreas was excised and weighed. Plasma amylase activity was determined using an automatic analyser (Dri-Chem 3500i) with its exclusive colorimetric assay kit (AMYL-P) (Fujifilm, Tokyo, Japan). For histological observation, the pancreata were fixed, embedded in paraffin, sectioned and stained with H&E. dl-Propargylglycine (PPG), a CSE inhibitor, was administered at 100 mg/kg intraperitoneally 1 h before the first dose of caerulein, as described previously.9 To examine its effect on the established pancreatitis-related allodynia/hyperalgesia, mibefradil, a T-type Ca2+ channel inhibitor, was administered at 9 mg/kg intraperitoneally 30 10 min after the last dose of caerulein (20 min before the nociceptive testing). To examine its preventive effect, mibefradil at the same dose was administered intraperitoneally 20 min before the first dose of caerulein (for prophylactic effect).

Determination of sensitivity to mechanical stimulation in the upper abdomen of mice

The sensitivity of each mouse to mechanical stimuli to the upper abdomen was determined using von Frey filaments, as described previously.26 Each mouse was placed on a raised wire mesh floor under a clear plastic box (23.5×16.6×12.4 cm), and acclimated to the experimental environment for 20–30 min. The upper abdomen of each mouse was stimulated using three distinct filaments with strengths of 0.02, 0.16 and 1.0 g, respectively, in ascending order of strength. The mechanical stimulation with each filament was applied five times at intervals of 5–10 s, and, after a 1 min resting period, another five times in the same manner, for a total of 10 times. Two successive applications of stimulation to the same point was avoided, taking into account “wind-up” effects or desensitisation. Scoring of nociceptive behaviour was defined as follows: score 0 = no response; score 1 = immediate escape or licking/scratching of the site stimulated by application of von Frey hairs; score 2 = strong retraction of the abdomen or jumping. The data are expressed as the total score of responses from 10 challenges with each hair.

The activity of CSE, an H2S-forming enzyme, in mouse pancreatic tissues

CSE activity in pancreatic tissue homogenates was determined, essentially as reported previously.10 Briefly, the pancreas was perfused transcardially with saline under anaesthesia with intraperitoneal urethane at 1.5 g/kg, and then excised and homogenised in 20 volumes of an ice-cold 100 mM potassium phosphate buffer (pH 7.4). The reaction mixture for assessment of CSE activity contained the tissue homogenate, 10 mM l-cysteine and 2 mM pyridoxal 5′-phosphate. The mixture was incubated at 37°C for 90 min in the presence or absence of 2.5 mM PPG, and the reaction was then stopped by addition of 1% (w/v) zinc acetate and 10% trichloroacetic acid (final concentration). After the addition of N,N-dimethyl-p-phenylenediamine sulfate (2.35 mM) and FeCl3 (3.15 mM), the mixture was incubated in an ice-cold bath for 20 min, and the absorbance of the supernatant was measured at a wavelength of 670 nm. All samples were assayed in duplicate. The concentration of H2S in each sample was calculated against a calibration curve of Na2S solution (15–960 μM), and CSE activity was defined as the difference in the formation of H2S in the presence or absence of PPG.

Western blot analysis of CSE in mouse pancreatic tissues

CSE protein was analysed by western blotting, as described previously.22 The mice were anaesthetised with intraperitoneal urethane at 1.5 g/kg, and the pancreas was perfused transcardially with saline. The pancreas was isolated, weighed, and homogenised in a radioimmunoprecipitation assay (RIPA) buffer (PBS, 1% Igepal CA-630 (Sigma-Aldrich, St Louis, Missouri, USA), 0.5% sodium deoxycholate and 0.1% SDS) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.15 U/ml aprotinin and 1 mM sodium orthovanadate. After addition of 2-mercaptoethanol and bromophenol blue, the supernatant was denatured at 95–100°C for 5 min, and the proteins were separated by electrophoresis on a 12.5% sodium dodecylsulfate (SDS)–polyacrylamide gel (Wako Pure Chemicals, Osaka, Japan) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Billerica, Massachusetts, USA). The membrane was blocked with a blocking solution containing 5% skim milk, 137 mM NaCl, 0.1% Tween 20 and 20 mM Tris–HCl (pH 7.6). After washing, the membrane was incubated overnight at 4°C with the rabbit anti-CSE polyclonal antibody (dilution 1:1000) (Sigma-Genosys/Sigma-Aldrich) against a peptide corresponding to the amino acid sequence, (C)80GGTNRYFRR89V, in rat CSE,31 and with the rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (dilution 1:1000) (Santa Cruz Biotechnology, Santa Cruz, California, USA). After washing the primary antibodies, the membrane was then incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (dilution 1:2000) (Cell Signaling Technology, Beverly, Massachusetts, USA). Immunolabelled proteins (CSE, 44 kDa; GAPDH, 37 kDa) were visualised by the enhanced chemiluminescence (ECL) detection reagent (Amersham Biosciences, Little Chalfont, UK). Resulting films were scanned and quantified using densitometric software (Scion Image downloaded from www.microsoft.com/DirectX/). The specificity of the positive band for CSE was confirmed by checking the inhibitory effect of a blocking peptide, GGTNRYFRRV, at 2 mg/ml.

Major chemicals

NaHS was purchased from Kishida Chemical (Osaka, Japan) or Sigma-Aldrich. Capsaicin, mibefradil and PPG were obtained from Sigma-Aldrich, and caerulein was from Bachem (Bubendorf, Switzerland). Capsaicin was dissolved in a solution containing 10% ethanol, 10% Tween 80 and 80% saline, and all other chemicals were dissolved in saline.

Statistical analysis

Data are represented as means with the SEM. Statistical significance was analysed by Student t test or Wilcoxon test for two-group comparisons and by analysis of variance followed by Tukey test for multiple comparisons, and was set at a level of p<0.05.

RESULTS

Infusion of an H2S donor into the pancreatic duct evokes expression of Fos in the spinal dorsal horn of the rat

In anaesthetised rats, infusion of NaHS at 500 nmol per rat as well as capsaicin at 490 nmol per rat into the pancreatic duct caused the expression of Fos protein, a marker for neuronal activation, in the superficial layers of the T8 and T9 spinal cord segments, 2 h after administration (fig 1A,B), while no expression of Fos was detected in the spinal segments at T5 levels (fig 1C) or cervical levels (data not shown) after administration of capsaicin. Spinal Fos expression following either NaHS or capsaicin infusion was observed bilaterally, and to a similar extent (data not shown). NaHS, administered into the pancreatic duct at 500 nmol per rat, as did ductal capsaicin at 490 nmol per rat, significantly increased the number of Fos-positive cells in laminae I and II, but not clearly in laminae III and IV, of bilateral T8 and T9 spinal dorsal horn segments in a dose-dependent manner, although NaHS at the highest dose, 1500 nmol per rat, failed to produce an additional effect (fig 2). Few Fos-positive cells were observed in the deeper layers, including laminae V and VI of the spinal segments, in any rats (data not shown).

Figure 1

Typical microphotographs for Fos immunostaining in spinal cord sections following injection of NaHS (A) or capsaicin (B, C) into the pancreatic duct in rats. The spinal cord segments at T8 and T9 levels (A, B) and at T5 and T9 levels(C) were perfused, fixed and excised 2 h after injection of NaHS or capsaicin. Magnifications, ×40; scale bars, 100 μm.

Figure 2

Ductal NaHS- or capsaicin-evoked expression of Fos in laminae I–II and III–IV of the T8 and T9 dorsal horns in rats. NaHS at 50, 500 and 1500 nmol per rat or capsaicin at 490 nmol per rat was infused into the pancreatic duct of rats. The spinal cord was perfused, fixed and excised 2 h after injection of NaHS or capsaicin. Data show the mean (SEM) from 4–5 rats for capsaicin and 5–9 rats for NaHS.

Blockade of T-type Ca2+ channels abolishes expression of spinal Fos caused by intraductal injection of NaHS, but not capsaicin, in rats or mice

Mibefradil, a T-type Ca2+ channel blocker, administered intraperitoneally at 9 mg/kg, completely inhibited the ductal NaHS-evoked Fos expression in laminae I and II of the T8 and T9 spinal cord (figs 3A and 4A), although mibefradil alone had no effect on Fos expression (fig 4B). In contrast, the same dose of mibefradil did not reduce the ductal capsaicin-evoked Fos expression in the same spinal regions (figs 3B and 4B). Similar to the results from experiments performed in rats, NaHS at 100 nmol per mouse administered into the mouse pancreatic duct significantly increased the number of Fos-positive cells in laminae I and II (fig 4C), although NaHS at 20 nmol per mouse produced no significant effect (data not shown). Further, the NaHS-evoked Fos expression in mice was significantly attenuated by intraperitoneal administration of mibefradil at 9 mg/kg (fig 4C).

Figure 3

Typical microphotographs for Fos expression caused by ductal NaHS (A) or capsaicin (B) in T9 spinal cord sections in rats pretreated with the T-type Ca2+ channel blocker mibefradil or vehicle. Mibefradil at 9 mg/kg was administered intraperitoneally 30 min before infusion of NaHS at 500 nmol per rat or capsaicin at 490 nmol per rat into the pancreatic duct of rats. The spinal cord was perfused, fixed and excised 2 h after injection of NaHS or capsaicin. Magnifications, ×40; scale bars, 100 μm.

Figure 4

Effect of mibefradil on expression of Fos in laminae I–II of T8 and T9 dorsal horns following ductal NaHS or capsaicin in rats (A, B) or mice (C). Mibefradil at 9 mg/kg was administered intraperitoneally 30 min before infusion of NaHS at 500 nmol per rat (A) or 100 nmol per mouse (C) and capsaicin at 490 nmol per rat (B) into the pancreatic duct of rats (A, B) or mice (C). Data show the mean (SEM) from 6–7 (A) or 4–7 (B) rats and 6–9 mice (C). NS, not significant.

Inhibition of CSE prevents referred allodynia/hyperalgesia, but not altered objective measures of pancreatic inflammation, in mice with caerulein-induced pancreatitis

Caerulein, administered six times at 1 h intervals, caused significant increases in nociceptive scores in response to mechanical stimuli of three different strengths applied to the upper abdomen in mice (fig 5A). These increases in nociceptive scores were accompanied by increases in pancreatic wet tissue weight and plasma amylase activity, and by histological signs of pancreatitis—that is, leucocyte infiltration and acinar cell vacuoles in the tissues (fig 5B–D). The pancreatitis-related referred allodynia/hyperalgesia persisted at least for 1.5–2.5 h after the final dose of caerulein (fig 6). A single intraperitoneal preadministration of PPG, an inhibitor of CSE, at 100 mg/kg prevented the caerulein-evoked abdominal allodynia/hyperalgesia (fig 5A), indicating involvement of endogenous H2S. Nonetheless, PPG failed to attenuate the elevated pancreatic weight and plasma amylase activity, or the histological feature of pancreatitis (fig 5B–D).

Figure 5

Effect of pretreatment with dl-propargylglycine (PPG), a cystathionine-γ-lyase inhibitor, on caerulein-evoked abdominal allodynia/hyperalgesia and pancreatitis in mice. Caerulein at 50 μg/kg was administered intraperitoneally to mice at 1 h intervals, six times in total. PPG at 100 mg/kg or saline was administered intraperitoneally to mice 1 h before the first administration of caerulein. The nociception test (A) was performed 30 min after the final (sixth) dose of caerulein. Then, blood samples were collected, and the mice were killed for determination of pancreatitis-related parameters including pancreatic weight (B) and plasma amylase activity (C), and for histological analysis of pancreatic tissues (D). Data show the mean (SEM) from 12–15 mice (A, B, C). NS, not significant (A). Arrows, leucocyte infiltration; arrowheads, acinar cell vacuole; magnifications, ×200; scale bars, 50 μm (D).

Figure 6

The time course of maintenance and disappearance of the referred allodynia/hyperalgesia after repeated treatment with caerulein. Caerulein at 50 μg/kg or saline was administered intraperitoneally to mice at 1 h intervals, six times in total. The nociception test was performed 0.5, 1.5, 2.5, 3.5 and 24 h after the final dose of caerulein. Data show the mean (SEM) from five mice.

A blocker of T-type Ca2+ channels suppresses established pancreatitis-related abdominal allodynia/hyperalgesia in mice

To determine the possible involvement of T-type Ca2+ channels targeted by H2S in the maintenance of pancreatitis-related pain, we examined the effect of a single dose of mibefradil, a blocker of T-type Ca2+ channels, on the established abdominal allodynia/hyperalgesia in caerulein-treated mice. The enhanced sensitivity to mechanical stimuli in the upper abdomen was completely reversed by a single intraperitoneal administration of mibefradil at 9 mg/kg (fig 7A). In contrast, a single intraperitoneal preadministration of mibefradil did not prevent the development of abdominal allodynia/hyperalgesia in caerulein-treated mice (fig 7B). It is to be noted that mibefradil, administered either before or after the administration of caerulein in the same manner, had no effect on pancreatitis itself, as evaluated by parameters including pancreatic weight and plasma amylase activity and by histological observation (data not shown).

Figure 7

Effect of mibefradil on the pancreatitis-related allodynia/hyperalgesia in mice. Caerulein at 50 μg/kg was administered intraperitoneally to mice at 1 h intervals, six times in total. Mibefradil at 9 mg/kg or saline was administered intraperitoneally to mice 10 min after the final (sixth) administration of caerulein (A), or 20 min before the first administration of caerulein (B). The nociception test was performed 30 min after the final dose of caerulein. Data show the mean (SEM) from 4–9 mice. NS, not significant.

Activity and protein levels of CSE in the pancreatic tissues of mice with caerulein-evoked pancreatitis

The activity of CSE in pancreatic tissues of mice significantly increased following the repeated administration of caerulein (fig 8A). Similarly, the expression of pancreatic CSE protein was enhanced significantly after the development of caerulein-induced pancreatitis in mice as assessed by western blotting (fig 8B). The specificity of the band corresponding to CSE was confirmed using a blocking peptide (data not shown).

Figure 8

Activity (A) and protein levels (B) of pancreatic cystathionine-γ-lyase (CSE) in the mice treated with caerulein. Caerulein at 50 μg/kg was administered intraperitoneally to mice at 1 h intervals, six times in total. Western blotting shows that a band for CSE protein was detected at around 44 kDa (B, top panel) and the protein levels were quantified by densitometry (B, bottom panel). Data show the mean (SEM) from four mice.

DISCUSSION

The mibefradil-reversible expression of Fos in the spinal superficial layers following application of NaHS into the pancreatic tissues through the duct in rats and mice suggests that pancreatic NaHS/H2S elicits the excitation of spinal nociceptive neurons by targeting T-type Ca2+ channels. Our data from experiments employing mice with caerulein-evoked acute pancreatitis imply that endogenous H2S formed by CSE in the pancreas plays an emerging role in the manifestation of pancreatitis-related pain, but not in the development of pancreatic inflammation, and that T-type Ca2+ channels mediate the pronociceptive effect of pancreatic endogenous H2S. H2S is thus considered a pronociceptive gasotransmitter targeting T-type Ca2+ channels in the pancreas.

Our present results showing a pronociceptive role for pancreatic NaHS/H2S through T-type Ca2+ channels are consistent with our recent evidence for NaHS facilitation of T-type Ca2+ channel-dependent membrane currents in NG108-15 cells and for the mibefradil-reversible hyperalgesia following intraplantar administration of NaHS.22 The finding that NaHS-evoked Fos expression was not clearly dose dependent (see fig 2) is consistent with our previous studies that indicated a bell-shaped dose–response curve for NaHS-evoked somatic and visceral hyperalgesia.22 24 Such an unusual profile for the effects of NaHS in vivo might be characteristic of the action of NaHS on T-type Ca2+ channels, because the dose–response curve for NaHS facilitation of T-type Ca2+ channel-dependent membrane currents in NG108-15 cells and DRG neurons in vitro is also bell-shaped.22 24 The observation that mibefradil blocked the expression of spinal Fos caused by the ductal application of NaHS, but not capsaicin, indicates distinct mechanisms for nociceptive processing by those two agents in terms of the involvement of T-type Ca2+ channels. It is noteworthy that capsazepine, an inhibitor of transient receptor potential vanilloid 1 (TRPV1), had no significant effect on the hyperalgesia provoked by the intraplantar administration of NaHS in the previous study.22 Although H2S may activate KATP channels, which play a role in suppression of nociception,25 such an action of H2S, if any, would be overcome by its pronociceptive action through T-type Ca2+ channels at least in the present model.

It was a little surprising that pretreatment with PPG, a CSE inhibitor, at 100 mg/kg did not improve pancreatitis-related inflammatory parameters in mice given six injections of caerulein at 1 h intervals. Bahtia et al9 have reported that PPG at the same dose slightly attenuated the increase in plasma amylase levels and partially inhibited the elevation of pancreatic myeroperoxidase activity after 10 injections of caerulein at 1 h intervals in mice. This discrepancy might be explained by the difference in severity of pancreatitis (ie, 6 vs 10 injections of caerulein). Thus, the inhibitory effect of PPG on the pancreatitis-related allodynia/hyperalgesia is not secondary to an anti-inflammatory effect, if any, suggesting a pronociceptive role for H2S formed by CSE in the pancreas, in addition to its proinflammatory role.9 32 Similar antiallodynia/antihyperalgesia effects of PPG have been observed in somatic inflammatory pain models induced by intraplantar administration of lipopolysaccharide22 and of formalin,33 although the effect of PPG in the latter model appears to depend on the nociceptive intensity level. Interestingly, the finding that a single administration of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia strongly suggested that persistent activation/sensitisation of T-type Ca2+ channels targeted by endogenous H2S is involved in the maintenance of pancreatic pain. The importance of tonic activation of T-type Ca2+ channels in the maintenance of rather than the developing process of pancreatitis-related allodynia/hyperalgesia is also suggested by the lack of effect of a single pretreatment with mibefradil, although more confirmatory and mechanistic data are necessary to demonstrate our hypothesis. It should be taken into consideration that the blood concentration of mibefradil, preadministered only once before six administrations of caerulein at 1 h intervals, would not remain at effective levels, after 6 h or later, according to the pharmacokinetic profile of mibefradil.34 Mibefradil is the most selective for T-type Ca2+ channels,35 whereas it is also capable of inhibiting Na+ channels and L-type Ca2+ channels at relatively high concentrations.36 Since T-type Ca2+ channel inhibitors with selectivity higher than mibefradil are not currently available, further studies using gene silencing by RNA interference are now in progress to verify our conclusion in the present study. Nevertheless, considering our most recent study showing the involvement of Cav3.2 T-type Ca2+ channels in NaHS-evoked somatic hyperalgesia using the antisense method,37 the present study on pancreatic pain further supports the critical roles of T-type Ca2+ channels in sensory neurons, particularly in the processing of pathological pain including neuropathic or inflammatory pain/hyperalgesia.23 3840

The enhancement of CSE activity and the upregulation of CSE protein in pancreatic tissues during caerulein-evoked pancreatitis are in agreement with the results of an independent report,9 and are consistent with our present results that the CSE inhibitor PPG abolished the pancreatitis-related allodynia/hyperalgesia. CSE is abundant in the liver and kidney, but relatively poor in the brain of rodents.41 It is noteworthy that CSE inhibitors do not inhibit H2S production in the brain tissue.42 To the best of our knowledge, there is no clear information about the blood–brain permeability of PPG, while mibefradil has been shown to penetrate poorly into the brain.35

In conclusion, we propose that endogenous H2S produced from l-cysteine by CSE targets T-type Ca2+ channels most probably expressed on the peripheral ending of the sensory nerve, and contributes to acute pancreatitis-related pain, although involvement of such pathways needs to be assessed further when more selective tools become available. Our study thus implies a therapeutic benefit of pharmacological intervention in the l-cysteine/H2S/T-type Ca2+ channel pathway for future clinical treatment of pancreatitis-related pain.

REFERENCE

Footnotes

  • Competing interests: None.

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