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Am J Physiol Cell Physiol 291: C503-C510, 2006. First published April 5, 2006; doi:10.1152/ajpcell.00547.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulfide

Yang Cao,* Sharmila Adhikari,* Abel Damien Ang, Philip K. Moore, and Madhav Bhatia

Department of Pharmacology, National University of Singapore, Singapore

Submitted 27 October 2005 ; accepted in final form 30 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study investigated the mechanism of mouse pancreatic acinar cell apoptosis induced by H2S in an in vitro system, using isolated pancreatic acini. Treatment of pancreatic acini with 10 µM NaHS (a donor of H2S) for 3 h caused phosphatidylserine externalization as shown by annexin V binding, an indicator of early stages of apoptosis. This treatment also resulted in the activation of the caspase cascade and major changes at the mitochondrial level. Caspase-3, -8, and -9 activities were stimulated by H2S treatment. Treatment with inhibitors of caspase-3, -8, and -9 significantly inhibited H2S-induced phosphatidylserine externalization as shown by reduced annexin V staining. The mitochondrial membrane potential was collapsed in H2S-treated acini as evidenced by fluorescence microscopy and quantitative analysis. Furthermore, the treatment of acini with H2S caused the release of cytochrome c by the mitochondria. To investigate the mechanism underlying pancreatic acinar cell apoptosis, we also characterized the protein expression of a range of molecules that are each known to influence the apoptotic pathway. Among proapoptotic proteins, Bax expression was activated in H2S-treated cells but not Bid, and the antiapoptotic proteins Bcl-XL and Bcl-2 did not show any activation in pancreatic acinar cell apoptosis. The death effector domain-containing protein Flip is downregulated in H2S-treated acini. These results demonstrate the induction of pancreatic acinar cell apoptosis in vitro by H2S and the involvement of both mitochondrial and death receptor pathways in the process of apoptosis.

caspase; H2S; mitochondria; Bcl-2; Flip

HYDROGEN SULFIDE (H2S), a colorless and flammable gas with the characteristic smell of rotten eggs, has long been regarded as a toxic environmental agent of little physiological significance (21, 26, 28, 38). However, recent studies showed that H2S is indeed produced naturally by mammalian tissues (26). H2S may be synthesized by a number of pathways, but two pyridoxal-5'-phosphate-dependent enzymes, cystathionine beta-synthase (CBS, EC 4.2.1.22 [EC] ) and cystathionine {gamma}-lyase (CSE, EC 4.4.1.1 [EC] ) appear to be most abundant in mammalian cells. The enzymes utilize L-cysteine as substrate for the production of H2S. Interestingly, a difference in distribution of the two enzymes has been noted. In the brain CBS transcript levels are relatively high, with little or no CSE (1), whereas in vascular smooth muscle both CSE and CBS are expressed (24). In the brain, H2S increases responses to glutamate mediated by NMDA receptors and thereby facilitates the induction of long-term potentiation in the hippocampus (1, 15, 25, 29). In smooth muscle, H2S causes relaxation, enhancing the outward flux of potassium ions by opening ATP-sensitive potassium channels, thereby causing hyperpolarization of the membrane and leading to smooth muscle relaxation (15, 25, 48, 58). Importantly, H2S also plays an important role in determining the severity of pancreatitis in the mouse induced by caerulein hyperstimulation (7) and in the inflammation associated with lipopolysaccharide-induced endotoxic shock (34) and the carrageenan-induced hindpaw edema model (6).

It has also been reported that H2S-induced hypoxia triggers the proliferative phases of cell cycle entry in nontransformed rat intestinal epithelial cells (13). However, it was noted that a higher concentration of H2S induced late apoptotic events in these cells (13). Indeed, the proapoptotic effect of H2S in human vascular smooth muscle cells via activation of mitogen-activated protein kinase pathways has been suggested to be an important endogenous modulator of cellular apoptosis (55).

Apoptosis or programmed cell death is an essential physiological process that is required for the normal development and maintenance of tissue homeostasis (52). When misregulated, apoptosis can contribute to various diseases including cancer and autoimmune and neurodegenerative diseases (52). Caspases are synthesized as dormant proenzymes that on proteolytic activation acquire the ability to cleave key intracellular substrates, resulting in the morphological and biochemical changes associated with apoptosis (52). Two separable pathways leading to caspase activation have been characterized (3, 9). In death receptor-mediated apoptosis, a death-inducing signaling complex is formed by the death receptors, adapter proteins, and caspases like caspase-8 and caspase-10 (3, 11, 50). In contrast, in mitochondrion-dependent apoptosis, major changes occur like outer mitochondrial membrane permeabilization, decrease in mitochondrial membrane potential ({Delta}{Psi}m), and release of cytochrome c (10). On the other hand, members of the Bcl-2 family play important roles and interact as homodimers and heterodimers either promoting or inhibiting apoptosis (10, 27, 49). Bcl-2 classically inhibits apoptosis (10, 22, 27, 49), and overexpression of Bcl-2 occurs in a range of neoplasms, including follicular lymphoma (32). Bax, another member of the Bcl-2 family, also promotes apoptosis (16).

As yet, there have been no reports of the possible effect of H2S to induce apoptosis in isolated pancreatic acinar cells. We therefore considered it of value to investigate the proapoptotic effect of the H2S donor sodium hydrosulfide (NaHS) in isolated mouse pancreatic acini by investigating the involvement of apoptotic pathways and characterizing the protein expression of a range of molecules that are each known to influence the apoptotic pathway.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996) and approved by the animal ethics committee of National University of Singapore. Swiss mice (male, 25–30 g) were housed in a controlled environment with an ambient temperature of ~22–26°C and a 12:12-h light-dark cycle. They were fed with standard laboratory chow and given water ad libitum.

Preparation of pancreatic acini. Pancreatic acini were obtained from mouse pancreas by collagenase treatment. Briefly, pancreata from mice were infused with buffer A (mM: 140 NaCl, 4.7 KCl, 1.13 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.2) containing 200 IU/ml collagenase and incubated in a shaking water bath for 10 min at 37°C. The digested tissue was passed through a solution of 50 mg/ml BSA and then washed twice with buffer A before further experiments. Cell viability was determined by Trypan blue exclusion.

Induction of pancreatic acinar cell apoptosis. The prepared acini were distributed into microfuge tubes containing 500 µl of buffer A. NaHS was added into these tubes with a working concentration of 1, 5, 10, 20, 50, or 100 µM. Acini were incubated with or without NaHS at 37°C in a shaking water bath for 1, 2, 3, 4.5, and 6 h, respectively. In some experiments, caspase inhibitors were used together with NaHS treatment.

Annexin V-FITC and propidium iodide staining detection. The extent of apoptosis and necrosis of pancreatic acini was determined by annexin V-FITC and propidium iodide (PI) staining (35) with a BD ApoAlert Annexin V-FITC apoptosis kit. After treatment with NaHS (10 µM), acini were incubated with 5 µl of annexin V (20 µg/ml in Tris-NaCl) and 5 µl of PI (50 µg/ml in 1x binding buffer) for 15 min in the dark at room temperature. Cells were observed by fluorescence microscopy (Zeiss) with a "dual band-pass" filter designed to detect simultaneously fluorescein (excitation 490 nm, emission 520 nm) and rhodamine (excitation 540 nm, emission 570 nm). Cells showing visible annexin V staining (with no PI staining) were categorized as apoptotic cells. Samples were also quantified with a Gemini EM Microplate spectrofluorometer measuring red fluorescence (excitation 535 nm, emission 617 nm) and green fluorescence (excitation 488 nm, emission 530 nm). Data are expressed as relative fluorescent units per microgram of DNA per microliter. In some experiments, caspase inhibitors were used together with NaHS treatment.

Caspase assay. Caspase-3, -8, and -9 enzyme activities were quantified with a fluorometric assay in which the extent of cleavage of a enzyme-specific fluorometric peptide substrate was determined. After treatment, cells were incubated with a fluorometric substrate for caspase-3 (Ac-DEVD-AFC; BD PharMingen), caspase-8 (Ac-IETD-AFC; BD PharMingen), or caspase-9 (Ac-LEHD-AFC; Calbiochem). The acini were centrifuged, and 100 µl of lysis buffer was added. Freeze and thaw steps were carried out three times by transferring from liquid nitrogen to a 37°C water bath. The lysed cells were centrifuged at 4°C for 30 min at 13,000 rpm. The supernatant was used for the assay. Fluorescence was measured at respective wavelengths (excitation 394 nm, emission 535 nm for caspase-3; excitation 400 nm, emission 505 nm for caspase-8 and -9) with a fluorescence plate reader (Tecan) at 5-min intervals for 75 min. Caspase activity was expressed as relative fluorescent units per hour per microgram of DNA per microliter as calculated with the linear range of the assay. The caspase activity in untreated cells was considered to be 100%. For caspase inhibitor assays, caspase-3-specific inhibitor Z-DEVD-FMK, caspase-8-specific inhibitor Z-IETD-FMK, and caspase-9-specific inhibitor Z-LEHD-FMK (R&D Systems) were used at a working concentration of 100 µM according to the manufacturer’s instructions.

Mitochondrial {Delta}{Psi}m detection. After treatment with NaHS, the mitochondrial {Delta}{Psi}m in acinar cells were detected by staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) at 37°C for 15 min in a shaking water bath. Changes in {Delta}{Psi}m were assessed by fluorescence microscopy (Carl Zeiss) with a dual-band pass filter designed to detect simultaneously fluorescein (excitation 490 nm, emission 520 nm) and rhodamine (excitation 540 nm, emission 570 nm). The results were also quantified with a fluorescence plate reader (Tecan) measuring green fluorescence (excitation 485 nm, emission 535 nm) and red fluorescence (excitation 550 nm, emission 600 nm). Data were expressed as cell retention of JC-1 (relative ratio of red to green fluorescence). The level of JC-1 retained by untreated cells at 3 h was considered to be 100%. In some experiments, caspase inhibitors were used together with NaHS treatment.

Measurement of cytochrome c release from mitochondria. After treatment with 10 µM NaHS for 3 h, acinar cells were washed in fresh isolation buffer (mM: 20 HEPES, 10 KCl, 1.5 MgCl2, 1 EDTA, 1 EGTA, 1 DTT, and 250 sucrose pH 7.5) and homogenized in a 7-ml glass tissue Dounce with up and down strokes of the pestle. The homogenate was centrifuged at 800 g for 10 min at 4°C, and then the supernatant was retained and centrifuged at 10,000 g for 10 min at 4°C. Pellet was washed twice and resuspended in wash buffer (mM: 210 mannitol, 70 sucrose, and 5 HEPES, pH 7.4) to obtain the mitochondria suspension. Cytochrome c release from isolated mitochondria in acinar cells was then measured with a cytochrome c DuoSet IC ELISA kit (R&D Systems). Absorbance was measured at 450 nm, with the reference wavelength at 540 nm (Tecan). Results were adjusted by total cytochrome c in mitochondria by addition of 0.1% Triton X-100. The level of cytochrome c determined in untreated cells at 3 h was considered to be 100%.

Preparation of cell lysates for Western blot analysis. After treatment, pancreatic acinar cells were homogenized on ice in RIPA buffer supplemented with 1 mM PMSF and a protease inhibitor cocktail containing pepstatin, leupeptin, chymostatin, antipain, and aprotinin (5 µg/ml of each) and centrifuged at 4°C for 15 min at 13,000 rpm. The supernatants were collected and stored at –80°C. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

Western blot analysis. Cell lysates (100 µg) were separated on 12% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked by 1-h incubation of the membranes in 5% nonfat dry milk in 0.05% Tween 20 in PBS (PBST). The blots were then incubated overnight with primary antibodies at 1:1,000 dilution in a buffer containing 2.5% nonfat dry milk in PBST, washed four times with PBST, and finally incubated for 1 h with a secondary antibody at 1:3,000 dilution in a buffer containing 2.5% nonfat dry milk in PBST. The blots were developed for visualization with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL).

Antibodies. Primary antibodies rabbit anti-Flip polyclonal antibody, rabbit anti-bax polyclonal antibody, and rabbit anti-bcl2 polyclonal antibody were purchased from Chemicon International (Temecula, CA); rabbit anti-bid polyclonal antibody, rabbit anti-Bcl-XL, and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Statistical analysis. All experiments were repeated at least three times. Results are expressed as means ± SE. Treatment effects were compared with Student’s t-test or one-way ANOVA followed by a post hoc analysis (Tukey’s test). A P value <0.05 was regarded as a significant difference.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Effect of NaHS treatment on annexin V-FITC binding and PI staining in pancreatic acinar cells. To determine whether NaHS treatment resulted in translocation of phosphatidylserine to the outer leaf of the plasma membrane, we examined the cellular binding of annexin V as an index of perturbation of the membrane bilayer. As is well known, PI is excluded by early apoptotic cells but labels necrotic cells and those in the final stages of apoptosis (36, 56). Therefore, we used PI as an index of total cell death (necrosis and final stages of apoptosis). After dose and time course analysis by fluorescent plate reader, only 3-h treatment with NaHS at the dose of 10 µM showed a significant increase in annexin V-FITC binding (Fig. 1, Aa and Ba) compared with control cells. In terms of PI staining (Fig. 1, Ab and Bb), during the early time points or low doses no significant difference was found between NaHS-treated and untreated cells. However, NaHS was shown to increase PI staining significantly in the 6-h treated group at a dose of 10 µM as well as in the 3-h treated cells at a higher dose (≥20 µM). Consistently, annexin V-FITC binding was observed by microscopy in pancreatic acinar cells exposed to 3-h NaHS (10 µM) but not in untreated cells (Fig. 1C). As our H2S analysis data showed that the level of H2S in normal pancreatic acini is ~11–14 µM, these results indicated that treatment of pancreatic acinar cells with a physiological dose of NaHS (10 µM) for 3 h induces apoptosis but not necrosis.


Figure 1
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  		Fig. 1. Detection of annexin V-FITC and propidium iodide (PI) staining in pancreatic acinar cells. Samples were quantified by fluorescent plate reader with dual filter set for FITC (excitation 488 nm, emission 530 nm; a) and PI (excitation 535 nm, emission 617 nm; b) after 3-h treatment with different doses of NaHS (A) or after 1-, 2-, 3-, 4.5-, and 6-h treatment with 10 µM NaHS (B) (n = 6; *P < 0.01 compared with untreated samples). Data are expressed as relative fluorescent units (RFU) per µg of DNA per µl. C: acinar cell staining by fluorescence microscopy using a "dual-band pass" filter designed to simultaneously detect fluorescein (excitation 490 nm, emission 520 nm) and rhodamine (excitation 540 nm, emission 570 nm); x400 magnification. Cells with bound annexin V show punctuated green staining in the plasma membrane.

 
Evaluation of caspase-3, -8, and -9 activity in pancreatic acinar cells. Caspase-3 activity was significantly elevated in cells exposed to NaHS for 3 h, which was significantly inhibited when cells were cotreated with the caspase-3 inhibitor Z-DEVD-FMK (Fig. 2A). Moreover, the caspase-3 inhibitor treatment was observed to be able to significantly attenuate the annexin V binding induced by NaHS (Fig. 2B) with no significant increase of PI staining (Fig. 2C). These results suggest that caspase-3 in pancreatic acini was activated by NaHS treatment, which was involved in the process of apoptosis induced by NaHS.


Figure 2
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  		Fig. 2. Detection of caspase-3 activity in pancreatic acinar cells. Isolated pancreatic acini were incubated with or without 10 µM NaHS in a 37°C water bath for 3 h. A: effect of NaHS with or without Z-DEVD-FMK on caspase-3 activation. Caspase-3 activity in untreated cells at 3 h was considered to be 100%. B: effect of NaHS with or without Z-DEVD-FMK on annexin V-FITC binding. C: effect of NaHS with or without Z-DEVD-FMK on PI staining. n = 6; {ddagger}P < 0.002 compared with 3-h untreated samples; *P < 0.001 compared with 3-h NaHS-treated cells.

 
The activity of caspase-8 and -9 was also elevated in pancreatic acini by NaHS, and such activation could be blocked by the corresponding inhibitors (Z-IETD-FMK and Z-LEHD-FMK, respectively) as well. (Fig. 3). These results suggest that both caspase-8 and -9 were activated in NaHS-treated pancreatic acini. To further determine the role of caspase-8 and -9 activation during the process of apoptosis induced by H2S, both inhibitors were used in the assessment of NaHS treatment on caspase-3 activity. Figure 4A shows that the caspase-9 inhibitor caused significantly more blocking effect on caspase-3 than the caspase-8 inhibitor. However, in the annexin V binding assay, both inhibitors were shown to significantly decrease annexin V-FITC binding by NaHS to a similar extent (Fig. 4B) but were not shown to significantly increase PI staining (Fig. 4C). These results indicated that both caspase-8 and -9 were involved in H2S-induced apoptosis.


Figure 3
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  		Fig. 3. Detection of caspase-8 and -9 activity in pancreatic acinar cells. Isolated pancreatic acini were incubated with or without 10 µM NaHS in a 37°C water bath for 3 h. A: effect of NaHS with or without Z-IETD-FMK on caspase-8 activation. B: effect of NaHS with or without Z-LEHD-FMK on caspase-9 activation. Caspase activity in untreated cells at 3 h was considered to be 100%. n = 6; {ddagger}P < 0.002 compared with 3-h untreated samples; *P < 0.001 compared with 3-h NaHS-treated cells.

 

Figure 4
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  		Fig. 4. Effect of caspase-8 and -9 inhibitors in pancreatic acinar cell apoptosis. Isolated pancreatic acini were incubated with or without 10 µM NaHS in a 37°C water bath for 3 h. A: effect of Z-IETD-FMK or Z-LEHD-FMK on caspase-3 activation induced by NaHS. Caspase activity in untreated cells at 3 h was considered to be 100%. B: effect of Z-IETD-FMK or Z-LEHD-FMK on annexin V-FITC binding induced by NaHS. C: effect of Z-IETD-FMK or Z-LEHD-FMK on PI staining induced by NaHS. n = 6; {ddagger}P < 0.002 compared with 3-h untreated samples; *P < 0.001 compared with 3-h NaHS-treated cells; {dagger}P < 0.002 compared with 3-h NaHS-treated cells in the presence of Z-IETD-FMK.

 
Assessment of H2S treatment on mitochondrial {Delta}{Psi}m in pancreatic acinar cells. To investigate the contribution of the mitochondrial pathway in NaHS-induced cell death, changes in mitochondrial inner {Delta}{Psi}m were assessed in pancreatic acini treated with NaHS. As shown by Fig. 5A, a–f, control cells displayed the phenomenon of polarized mitochondria, whereas NaHS-treated cells showed depolarized mitochondrial signal, which indicated that in H2S-treated pancreatic acinar cells the mitochondrial {Delta}{Psi}m was already collapsed.


Figure 5
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  		Fig. 5. Detection of mitochondrial membrane potential ({Delta}{Psi}m) in pancreatic acinar cells. Isolated pancreatic acini were incubated with 10 µM NaHS for 3 h, followed by treatment with JC-1 reagent for 15 min. A: changes in {Delta}{Psi}m were assessed by fluorescence microscopy [a–c: 3-h control; d–f: 3-h NaHS; a and d, green staining filter set for fluorescein (excitation 490 nm, emission 520 nm); b and e, red staining filter set for rhodamine (excitation 540 nm, emission 570 nm); c and f, combined images]; x400 magnification. Polarized mitochondria are visualized by punctuated red staining, whereas depolarized mitochondria are visualized by their diffuse green staining. B: effect of Z-IETD-FMK or Z-LEHD-FMK on mitochondrial {Delta}{Psi}m induced by NaHS. Samples were quantified with a fluorescent plate reader with dual filter set for green fluorescence (excitation 485 nm, emission 535 nm) and red fluorescence (excitation 550 nm, emission 600 nm). Data are expressed as cell retention of JC-1 (relative ratio of red to green fluorescence). The level of JC-1 retained by untreated cells at 3-h incubation time was considered to be 100%. n = 6; *P < 0.001 compared with incubation control.

 
We also quantified the mitochondria data by using a fluorescence plate reader (Fig. 5B). Consistently, the relative ratio of red to green fluorescence was obviously decreased in H2S-treated cells compared with the untreated group. However, pretreatment with either Z-LEHD-FMK or Z-IETD-FMK in both NaHS-treated and untreated cells showed no alteration of mitochondrial {Delta}{Psi}m.

Assessment of H2S treatment on cytochrome c release from mitochondria. The release of mitochondrial cytochrome c from mitochondria in pancreatic acinar cells after treatment with NaHS was determined with a semiquantitative sandwich ELISA kit. After isolation of mitochondria from NaHS-treated cells, released cytochrome c was measured, whereas the retained cytochrome c in the mitochondria was removed during washing. As shown in Fig. 6, the release of mitochondrial cytochrome c in treated pancreatic acinar cells was significantly increased compared with untreated controls. These results indicated that treatment of acini with NaHS over this time period induces the release of cytochrome c by mitochondria in pancreatic acinar cells.


Figure 6
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  		Fig. 6. Detection of the release of cytochrome c from mitochondria in pancreatic acinar cells. Isolated pancreatic acini were incubated with 10 µM NaHS for 3 h, followed by isolation of the mitochondria. The released cytochrome c from mitochondria was determined with a semiquantitative sandwich ELISA kit. Absorbance was measured at 450 nm, with the reference wavelength at 540 nm. n = 6; *P < 0.001 compared with 3-h untreated samples. OD, optical density.

 
Western blot analysis of pro- and antiapoptotic proteins in H2S treatment of pancreatic acini. To investigate the processes underlying our observations of pancreatic acinar cell apoptosis, we examined protein levels by Western blot analysis of apoptosis-associated genes (Bid, Bax, Bcl-2, Bcl-XL, and Flip).

The proapoptotic protein bax recognized two isoforms, Bax-{alpha}, a 21-kDa protein, as well as a longer 24-kDa Bax-beta isoform, in both control and treated acinar cells. The level of Bax expression in treated cells showed an increase compared with the control cells. Another proapoptotic protein, Bid (22 kDa), did not show any change in the level of expression between control and treated cells (Fig. 7). Antiapoptotic proteins Bcl-XL (32 kDa) and Bcl-2 (25 kDa) did not show any difference in their expression between control and H2S-treated cells. We also evaluated the expression of FLIP (also known as Casper, I-Flice, Flame-1, Cash, Clarp), a cytosolic protein with homology to caspase-8 that acts as a dominant-negative inhibitor of caspase-8 activation. We found that FLIP was markedly downregulated in H2S-treated acinar cells (Fig. 7).


Figure 7
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  		Fig. 7. A: Western blot analysis of pancreatic Bax, Bid, Bcl-XL, Bcl-2, and Flip protein levels. Isolated pancreatic acini were incubated with or without 10 µM NaHS in a 37°C water bath for 3 h. B: densitometric measurements of Bax, Bid, Bcl-XL, Bcl-2, and Flip protein levels. The band intensity in untreated (Cont) cells at 3 h was considered to be 100%. Results shown are representative of 3 independent experiments. *P < 0.001 compared with 3-h untreated samples.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recently, H2S has attracted a lot of interest as a novel inorganic gaseous mediator involved in physiological and pathophysiological events. However, little is known about the effect of H2S on the modulation of cell proliferation and apoptosis.

In the present study, we investigated the effect of H2S on apoptosis of pancreatic acinar cells. We noted that treatment of isolated mouse pancreatic acini with a low concentration of NaHS caused phosphatidylserine externalization, stimulation of caspase-3, -8, and -9 activities, decrease in mitochondrial {Delta}{Psi}m, and release of cytochrome c.

Among the surface changes, the most common and best characterized in apoptotic cells is the loss of phospholipid asymmetry in the plasma membrane and the translocation of phosphatidylserine from the inner to the outer leaflet of the lipid bilayer (17–19, 23, 46). The exposure of phosphatidylserine occurs very early during the apoptotic process and is almost universally required for engulfment (17–19, 23, 46). Early apoptosis results in membrane exposure of phosphatidylserine that is recognized by the phospholipid-binding protein annexin V. In the present study, there was a significant increase (P < 0.05) in annexin V binding with NaHS treatment over that in untreated acini. However, propidium iodide staining showed no significant difference between treated and untreated acini. This observation clearly indicates that NaHS treatment induced apoptosis but not necrosis.

In apoptosis, caspases function both in cell disassembly (effectors) and in initiating this disassembly in response to proapoptotic signals (initiators) (50). Caspase-3 has been implicated in the effector phase of apoptosis. Both death receptor and mitochondrial pathways converge at the level of caspase-3 activation to amplify initiation signals into commitment to apoptosis (39, 42, 45, 50). Caspase-8 and -9 are known as the two major upstream activators of caspase-3. Caspase-8- and -9-mutant mice exhibit severe defects in developmental apoptosis, suggesting that they are critical for activation of apoptosis and caspase-3 during development (5, 31). In addition, situated at pivotal junctions, caspase-8 and -9 initiate death receptor and mitochondrial pathways, respectively (39, 42, 50).

In the present study, fluorometric assay was used to quantify caspase-3, -8, and -9 activities. Our data showed that all three caspases were significantly activated in H2S-treated pancreatic acini. When the corresponding inhibitors of the three caspases were used, the activity of these caspases was observed to be greatly attenuated, whereas no increase of PI staining was observed. Moreover, caspase-3 activity was blocked significantly by caspase-8 and caspase-9 inhibitors as well. These data indicate that both death receptor and mitochondrial pathways may be involved in activating caspase-3.

Mitochondria play a key role in controlling life and death by releasing cytochrome c into the cytosol, thereby activating caspases (14, 43). Permeability changes in the outer mitochondrial membrane, decrease of {Delta}{Psi}m, release of cytochrome c, and caspase activation are associated with the mitochondrial death pathway (44). The physiological importance of cytochrome c release and the subsequent activation of caspase-9 has been shown in Apaf-1 or caspase-9-knockout mice, which die perinatally with brain outgrowth and reduced apoptosis in the central nervous system (52). Also, cytochrome c-deficient mice die earlier during embryonic development, but this is presumably due to defective mitochondrial oxidative phosphorylation (52). Our studies show that H2S induces a significant caspase-9 activation, mitochondrial {Delta}{Psi}m reduction, and a significant release of cytochrome c by mitochondria as well. We did not observe any effect of caspase-9 inhibitor on mitochondrial membrane depolarization, which indicated that caspase-9 activation occurs downstream of mitochondrial membrane depolarization. These results support the conclusion that the mitochondrial pathway is involved in H2S-induced apoptosis of pancreatic acinar cells.

To clarify the possible involvement of the death receptor pathway, which is suggested by the activation of caspase-8, we analyzed the production of TNF-{alpha} and Fas ligand level in pancreatic acinar cells after treatment with H2S, but no increase in either of these was found (data not shown). We also evaluated the effect of caspase-8 inhibitor on mitochondrial {Delta}{Psi}m, but no blocking effect was observed.

To further investigate the processes underlying our observations of pancreatic acinar cell apoptosis, we characterized the protein expression of a range of molecules that are known to influence the apoptotic pathway. Active caspase-8 cleaves a narrow range of substrates, including effector caspases and BID. In some cells (type I cells), the caspase-8-mediated activation of effector caspases is sufficient for robust apoptosis induction, and BID cleavage, which can lead to apoptogenic activation of the mitochondrial pathway, attains no relevance for Fas-induced apoptosis. However, in another cell type (type II cells) BID cleavage and apoptogenic activation of the mitochondria contribute measurably to Fas-induced cell death (4). The function of Bax as a proapoptotic protein is regulated directly or indirectly by BH3-only Bcl-2 family members (2, 12). Bax is known to induce mitochondrion-driven apoptosis; irradiation-induced apoptosis was considered to have occurred through a p53-Bax pathway. However, the discovery of BID and a p53-responsive element in the Fas gene suggested the participation of a Fas-mediated apoptotic pathway after irradiation (51). Insertion of Bax into the membrane is dependent of Bid, because the COOH-terminal domain of Bax, which enhances targeting and insertion into mitochondria in vivo, might be required for Bid to trigger Bax insertion into membranes (47, 54). Our results show activation of Bax in treated cells but not Bid. Also, the antiapoptotic protein did not show any activation in pancreatic acinar cell apoptosis. Bcl-2 and Bcl-XL can inhibit apoptosis; Bax can dimerize with Bcl-2 or Bcl-XL and inhibit their function and thereby promote apoptosis. An increase in the Bax-to-Bcl-2 (or Bax to Bcl-XL) ratio fosters apoptosis (20). Low basal levels of Bcl-XL correlated with a greater tendency to undergo apoptosis, whereas cells with higher basal levels of Bcl-XL correlated with resistance to apoptosis (57).

On the other hand, downregulation of FLIP, the endogenous caspase-8 inhibitor in treated cells (30, 41, 49), suggests a role in the regulation of death responses in pancreatic acinar cell apoptosis. It has been shown that heterodimers of caspase-8 and FLIP are enzymatically active. FLIP associates with procaspase-8 and also competes with procaspases-8 and -10 for binding to Fadd (8, 37, 53).

The present results show, for the first time, that low concentrations of H2S induce apoptosis in pancreatic acinar cells in vitro, by activation of the mitochondrial pathway as well as the death receptor pathway.


   GRANTS
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 ABSTRACT
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This work was supported by the National Medical Research Council Grant No. R-184-000-078-213 and the Office of Life Sciences Cardiovascular Biology Program Grant No. R-184-000-074-712, National University of Singapore.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bhatia, Dept. of Pharmacology, National Univ. of Singapore, Yong Loo Lin School of Medicine, Bldg. MD2, 18 Medical Dr., Singapore 117597 (e-mail: mbhatia{at}nus.edu.sg)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Y. Cao and S. Adhikari contributed equally to this work. Back


   REFERENCES
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 DISCUSSION
 GRANTS
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