Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1tBHP caused a slow sustained increase in[Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 μmol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 μmol l-1),a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 μmol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.
Calcium ions (Ca2+) are a universal intracellular messenger,controlling a diverse range of cellular processes in birth, life and death,such as gene transcriptional activation, contraction and secretion, or cell differentiation and proliferation(Carafoli et al., 2001). Ca2+ concentration in the cytosolic environment changes in response to a variety of simultaneous signals, which differ in their origin (extra- or intracellular). In most cells, Ca2+ has a major signalling function when its concentration is elevated in the cytosolic compartment([Ca2+]c) (Berridge et al., 2000).
[Ca2+]c elevation has been mainly attributed to: (i)entry of external Ca2+ through plasma membrane channels(Putney, 1988); (ii)Ca2+ release from intracellular Ca2+ agonist-sensitive stores, which might be mediated by either inhibition of sarcoplasmic reticulum Ca2+-ATPase (SERCA) (Moreau et al., 1998) or by activation of several distinct types of messenger-activated channels [e.g. inositol-1,4,5-triphosphate(IP3)- and ryanodine-operated channels]. Additionally, mitochondria can trigger and perpetuate cytosolic Ca2+ signals viamitochondrial permeability transition activation(Duchen, 2000), contributing to Ca2+-induced Ca2+-release (CICR)(González and Salido,2001). [Ca2+]c is returned to basal levels by: (i) Ca2+ extrusion through plasma membrane pumps and exchangers(Camello et al., 1996) and(ii) Ca2+ reuptake into cytosolic and mitochondrial pools(Tepikin et al., 1992).
Reactive oxygen species (ROS) can be used as messengers in normal cell functions (Rosado et al.,2004). However, at oxidative stress levels they can disrupt physiological pathways and cause cell death. In addition, it has been shown that intracellular Ca2+ appears to play a role as a signal transducer in the mechanism of apoptosis(Distelhorst and Dubyak,1998). The effects of oxidants on Ca2+ signalling can vary from stimulatory to repressive, depending on the type of oxidants, their concentrations, and the duration of the exposure(Waring, 2005). However, it is generally reported that oxidants can cause a rapid increase in[Ca2+]c in diverse cell types(Rooney et al., 1991; Wang and Joseph, 2000), which can precede other morphological and functional alterations. Oxidants can also regulate the production of IP3 and Ca2+ release from the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR)(Doan et al., 1994). SERCA can be inhibited both by oxidation of its sulphydryl groups and by direct attack of oxidants on the ATP binding site(Castilho et al., 1996; Redondo et al., 2004). Plasma membrane ATPases are also inhibited by oxidants(Zaidi et al., 2003; Redondo et al., 2004). In addition, previous studies have evaluated the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on the cellular function of isolated rat pancreatic acinar cells, showing a rapid and sustained increase in [Ca2+]c(Klonowski-Stumpe et al.,1997).
Another known potent oxidant, menadione, evokes repetitive cytosolic Ca2+ spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis in isolated pancreatic acinar cells(Gerasimenko et al., 2002). Studies in our laboratories show that treatment of rat pancreatic acinar cells with hydrogen peroxide (H2O2) results in the release of Ca2+ from mitochondrial and non-mitochondrial intracellular Ca2+ stores, and this action is mediated by oxidation of sulphydryl groups of Ca2+-ATPases(Pariente et al., 2001). Additionally, H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2(González et al.,2005).
tBHP is a prototypical organic pro-oxidant and has been used to study the role of Ca2+ in oxidant-induced cell death(Jones et al., 1983; Liu et al., 1998). tBHP is an inducer of apoptosis and cellular damage through oxidative stress (Gorbunov et al.,1998). It has been previously shown that tBHP decreases the cell membrane resistance, triggering apoptosis(Lang et al., 2003), and induces lipid peroxidation and malondialdehyde formation(Rush et al., 1985),mobilising arachidonic acid from membrane phospholipids through a phospholipase A(2)-mediated mechanism(Martín et al., 2001; Masaki et al., 1989). Additionally, tBHP inhibits the plasma membrane Ca2+-pump ATPase (PMCA) (Rohn et al.,1993), and enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+ levels and onset of the permeability transition pore (Byrne et al.,1999).
The effects of tBHP on Ca2+ mobilisation in exocrine pancreas, however, have only been investigated in a few studies. In rat pancreatic acinar cells, tBHP disrupts repetitive Ca2+spiking in response to carbachol, leading to a sustained increase in[Ca2+]c (Sweiry et al., 1999). Nevertheless, the intracellular mechanisms underlying these effects remain unclear. Thus, the aim of the present study was to investigate the effect of tBHP on [Ca2+]c in collagenase-dispersed rat pancreatic acinar cells and to study the mechanisms involved, using an epifluorescence inverted microscope.
Materials and methods
Animals and chemicals
Adult male Wistar rats Rattus norvegicus albinus Berkenhaut (mass 120-150 g) were used throughout this study and obtained from the Animal Farm of the Faculty of Veterinary Sciences, University of Extremadura(Cáceres, Spain). Fura-2/AM was purchased from Molecular Probes Europe(Leiden, Netherlands). Collagenase CLSPA was obtained from Worthington Biochemical Corporation (Freehold, NJ, USA) and TPS from Alomone Labs(Jerusalem, Israel). All other reagents used were obtained from Sigma (Madrid,Spain).
Preparation of isolated rat pancreatic acinar cells
A suspension of single cells and small acini was obtained from isolate rat pancreas as described previously(Martínez et al.,2004). Briefly, after cervical dislocation of animals, the pancreas was rapidly removed, treated by enzymatic digestion with collagenase(Worthington, 40 U ml-1) and incubated at 37°C under gentle agitation. This enzymatic digestion was followed by mechanical dispersion, by gently pipetting the cell suspension. Acinar cells were suspended in a physiological salt solution (Na-Hepes buffer) containing: 0.1 mg ml-1 soybean trypsin inhibitor, 0.2% (w/v) bovine serum albumin and(in mmol l-1): 140 NaCl, KCl 4.7, MgCl2 1.1, N-2-hydroxyethylpiperazine-N′-2-sulphonic acid (Hepes)10, glucose 10 and CaCl2 1.2, pH adjusted to 7.4. All experiments were performed at room temperature (22-25°C). In experiments where Ca2+-free medium are indicated, Ca2+ was omitted and 1 mmol l-1 EGTA was added.
Cell loading and [Ca2+]c determination
After isolation, the cells were suspended in physiological solution (same composition as before) and loaded with the fluorescent ratiometric Ca2+ indicator fura-2 by incubation with 4 μmol l-1fura-2 acetoxymethyl ester at room temperature (23-25°C) for 25-30 min. Once loaded, the cells were washed and resuspended in fresh physiological solution and used within the next 2-4 h. Ca2+-dependent fluorescence signals were monitored in samples of fura-2-loaded cells placed on a thin glass coverslip attached to a Perspex perfusion chamber on the stage of an epifluorescence inverted microscope (Nikon diaphot T200, Kawasaki,Kanagawa, Japan). Perfusion (a flow rate of 1.5 ml min-1) at room temperature was started after a 5 min period to allow spontaneous attachment of the cell to the coverslip. No coating treatment was necessary to immobilize the cells. For quantification of fluorescence, samples were alternatively excited at 340 and 380 nm using a high-speed monochromator (Polychrome IV)with an integrated light source from a xenon lamp (UXL S/50 MO) (Tills Photonics GmbH, Munich, Germany). Fluorescence emission at 505 nm was detected using a high-speed cooled digital CCD camera (C-4880-81, Hamamatsu Photonics,Marimoto, Shizuoka, Japan) and recorded using dedicated software (Aquacosmos 2.5, Hamamatsu Photonics). Changes in [Ca2+]c were monitored using the fura-2 340/380 ratio and calibrated according to published methods (Grynkiewicz et al.,1985).
Cell viability was assessed using calcein-fluorescence and the Trypan Blue exclusion test. For calcein loading, cells were incubated for 30 min with 5μmol l-1 acetoxymethyl (calcein AM) at 37°C, centrifuged,and the pellet resuspended in fresh buffer. Fluorescence was recorded from 2 ml samples using a fluorescence spectrophotometer (Varian, Ltd., Madrid,Spain). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. After treatment with 1 mmol l-1tBHP or agonists, cells were centrifuged and resuspended in fresh buffer. The calcein fluorescence remaining in the cells after treatment with tBHP was the same as in controls, at least for the duration of our experiments,suggesting that under our conditions there was no cellular plasma membrane damage. The results obtained with calcein were confirmed using the Trypan Blue exclusion technique. 95% of cells were viable after treatment with tBHP similar to that observed in our resting acinar cells suspension. However, when the cells were perfused with 1 mmol l-1tBHP for a period longer than 40-45 min, their viability was reduced to 89% and the fura-2 fluorescence suddenly decreased, suggesting that during this period tBHP can damage cell permeability and the fluorescence from fura-2 is lost to the extracellular solution.
Analyses of statistical significance were performed using Student's t-test. Differences were considered significant at P<0.05.
In the presence of normal extracellular Ca2+ concentration([Ca2+]o =1.2 mmol l-1), perfusion of pancreatic acinar cells with 1 mmol l-1tBHP caused a slow and sustained [Ca2+]c increase, which reached a stable[Ca2+]c plateau after 20-25 min of perfusion(Fig. 1A). Fig. 1B shows that the increase of [Ca2+]c induced by tBHP was also observed in a Ca2+-free medium ([Ca2+]o=0 mmol l-1), reflecting the release of Ca2+ from intracellular store(s). The Ca2+ released by tBHP both in the presence and absence of extracellular Ca2+ was statistically similar(691±10 mol l-1versus 699±79 mol l-1, Fig. 1D). Fig. 1B also shows that tBHP caused a biphasic increase in [Ca2+]c,consisting of a slow initial rise observed within the first minutes and a second sustained rise later during the course of experiments. A significant proportion of the cells studied exhibited this biphasic transient increase in[Ca2+]c (36 of 53 examined cells, 67.92%). The Ca2+ release induced by 1 mmol l-1tBHP was reversed by an addition of 2 mmol l-1 of the reducing agent dithiothreitol (DTT) (Fig. 1C). This inhibition was reversible; removal of DTT allowed[Ca2+]c to return to the transient increase evoked by tBHP.
Pretreatment of cells with tBHP abolished the typical Ca2+ response both to the Ca2+-mobilising agonist CCK-8(1 nmol l-1) (Fig. 2A) and to TPS (1 μmol l-1), a specific inhibitor of SERCA (Fig. 2B), in the absence of extracellular Ca2+. However, when the agonist-releasable Ca2+ pools had previously been depleted by a maximal concentration(1 nmol l-1) of CCK-8 (Fig. 3A) or 1 μmol l-1 thapsigargin (TPS)(Fig. 3B) in a Ca2+-free solution, 1 mmol l-1tBHP was still able to induce Ca2+ release in 66 of 74 examined cells (89.18%) and in 41 of 58 examined cells (70.69%) from 12 and 10 experiments, respectively(Fig. 3), suggesting that tBHP is also able to release Ca2+ from an agonist-insensitive store. The amount of Ca2+ released by tBHP after treatment with CCK or TPS is shown in Fig. 3C.
In order to investigate the nature of the tBHP-releasable agonist-insensitive Ca2+ store we used FCCP, a mitochondrial uncoupler that collapses the mitochondrial membrane potential that drives Ca2+ uptake (Buckler and Vaughan-Jones, 1998). As shown in Fig. 4A, pretreatment of the acinar cells with 0.5 μmol l-1 FCCP in a Ca2+-free medium resulted in a sustained increase in [Ca2+]c due to release of Ca2+ from mitochondrial stores. Subsequent addition of 1 mmol l-1tBHP, the acinar cell suspension was still able to release Ca2+,presumably from agonist-sensitive stores (21 of 27 examined cells, 77.77%, from 7 experiments). Pretreatment with tBHP abolished Ca2+ release from mitochondria evoked by subsequent addition of FCCP (Fig. 4B) (in all 18 recorded cells, from 6 experiments), suggesting that mitochondrial stores are depleted by pretreatment with tBHP. By contrast, simultaneous addition of 1 μmol l-1 TPS and 0.5μmol l-1 FCCP (which deplete non-mitochondrial intracellular Ca2+ stores, e.g. endoplasmic reticulum and mitochondria,respectively) clearly abolished the tBHP-induced Ca2+increase in all 22 cells examined from 5 experiments (Figs 5, 3C). Taken together, these findings indicate that tBHP releases Ca2+ from both mitochondrial and non-mitochondrial Ca2+ pools.
Since it had been previously shown that oxidising reagents are able to sensitize IP3-induced Ca2+ release(Thorn et al., 1992; Wu et al., 1996), we also wanted to evaluate whether tBHP can release Ca2+ from agonist-mobilisable Ca2+ stores by sensitising the IP3-induced Ca2+ release. To test this possibility we employed 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release that does not interact with the IP3-binding site (Soulsby and Wojcikiewicz, 2002), and which was able to block the Ca2+ signal evoked by the Ca2+-mobilising agonist CCK-8(data not shown). As shown in Fig. 6A, application of 30 μmol l-1 2-APB to acinar cells, where the mitochondrial Ca2+ pool had been previously depleted by 0.5 μmol l-1 FCCP to avoid interference with mitochondrial Ca2+ release, was unable to suppress the increase in[Ca2+]c induced by 1 mmol l-1tBHP in 26 of 37 cells examined (70.27%) from three experiments.
The effects of ryanodine were also examined in order to investigate the putative implication of ryanodine receptors on tBHP-evoked Ca2+ mobilisation. In both excitable and nonexcitable cells,ryanodine at relatively low concentrations (10 nmol l-1-10 μmol l-1) is reported to cause activation of the Ca2+ release channel, whereas at higher concentrations (>10 μmol l-1)ryanodine blocks channel activation(Verkhratsky and Shmigol,1996). In our experimental conditions, pretreatment of pancreatic acinar cells, whose mitochondrial Ca2+ stores had been depleted using FCCP (0.5 μmol l-1), with 50 μmol l-1ryanodine (which blocked caffeine-evoked Ca2+ release, data not shown) abolished tBHP-evoked Ca2+ mobilisation in 35 of 46 examined cells (76.08%) from 3 experiments(Fig. 6B,C), suggesting that tBHP releases Ca2+ from non-mitochondrial Ca2+pools through ryanodine channels.
In this study we demonstrate that the membrane-permeant oxidant tBHP induces a [Ca2+]c increase in pancreatic acinar cells by Ca2+ release from intracellular stores, since this effect was observed in a Ca2+-free medium. By contrast,pretreatment of acinar cells with tBHP followed by the addition of either CCK-8 or TPS resulted in an abolition of the agonist-evoked rise in[Ca2+]c, whereas tBHP failed to increase[Ca2+]c in cells where non-mitochondrial and mitochondrial intracellular Ca2+ stores had previously been depleted by application of TPS plus FCCP, in a Ca2+-free solution. Our results are consistent with those previously described by us in isolated rat pancreatic acinar cells and platelets treated with H2O2 (Pariente et al., 2001; Redondo et al.,2004). In addition, our results seem to indicate that tBHP acts by mobilising Ca2+ from non-mitochondrial pools via an IP3-receptor independent mechanism that involves ryanodine channels.
On the basis of its ability to increase [Ca2+]c, ROS have been considered to be pathogenic factors in different tissues, including the pancreas (Weber et al.,1998). An increase in [Ca2+]c due to disturbance of Ca2+ homeostasis by ROS can cause morphological and functional alterations to the cells, and therefore, have been clearly established as contributing to disease and cell death(Jacobson and Duchen, 2002). Impairment of Ca2+ homeostasis and intrapancreatic activation of digestive enzymes have been proposed as critical events in the development of pancreatitis (Saluja et al.,1999). In addition, high levels of ROS have been implicated as important mediators in the pathogenesis of acute pancreatitis. Thus, it was of interest to analyse Ca2+ homeostasis in cells exposed to oxidative conditions.
In pancreatic acinar cells, the stimulatory effect of tBHP on resting [Ca2+]c and its inhibitory effect on agonist-induced Ca2+ mobilisation could be due to a direct effect on the Ca2+ release process and not a consequence of the opposing action in the Ca2+ pathway. Previous studies in different cell types, such as hepatocytes (Miyoshi et al., 1996; Byrne et al.,1999), erythrocytes (Lang et al., 2003), platelets(Elferink, 1999; Redondo et al., 2004), and endothelial cells (Elliot et al., 1989; Jornot et al., 1999), have reported that hydroperoxides and other sulphydryl reagents can induce Ca2+ mobilisation.
Other authors have shown that the sulphydryl group oxidising agents thimerosal (Thorn et al.,1992), vanadate (Pariente et al., 1999) and phenylarsine oxide(Lajas et al., 1999) are able to mobilise Ca2+ from intracellular stores in pancreatic acinar cells and that this effect is reversed in the presence of the thiol-reducing agent dithiothreitol. Similar results were obtained in thymus cells(Calviello et al., 1993) and hepatocytes (Nicotera et al.,1988) using tBHP as oxidising agent. Additionally, the depletion of intracellular stores by tBHP has been observed in other cell types, such as hepatocytes (Masaki et al., 1989), PC12 pheochromocytoma cells(Lu et al., 2002), alveolar macrophages (Hoyal et al.,1996), skeletal muscle (Silva et al., 1997), myeloid leukaemia U937 cells(Clementi et al., 1998) or neuronal cell line SH-SY5Y (Amoroso et al.,1999). However, evidence exists that tBHP increases[Ca2+]c exclusively via Ca2+ influx from the extracellular site (Kim et al.,1998). This Ca2+ entry can occur through voltage-dependent Ca2+ channels(Wahl et al., 1998). Other authors indicate that the tBHP-induced effect might be mediated both by Ca2+ influx from the extracellular medium and by intracellular store depletion (Bernardes et al.,1986; Teplova et al.,1998).
Our results show that tBHP releases Ca2+ from intracellular stores, suggesting that the failure of CCK-8 and TPS to induce Ca2+ mobilisation after tBHP is related to a partial or complete depletion of the stores by this agent. The tBHP-sensitive Ca2+ pools include those released by TPS (e.g. endoplasmic reticulum) and FCCP (e.g. mitochondria). This is shown by the failure of tBHP to increase [Ca2+]c after treatment with TPS plus FCCP in a Ca2+-free medium. Thus, when the non-mitochondrial agonist-releasable Ca2+ pools are previously depleted by CCK-8 or TPS, tBHP is able to induce Ca2+release from mitochondria in a Ca2+-free medium, whereas if the mitochondrial Ca2+ is released by treatment with FCCP, tBHP releases the Ca2+ from the TPS-sensitive pool. In this context, it is important to note that the existence of two major types of intracellular Ca2+ stores has been suggested: (i) the endoplasmic reticulum, which functions as a high-affinity, low-capacity Ca2+pool, and (ii) mitochondria, which are low-affinity, high-capacity Ca2+ pools (Carafoli,1987).
The existence of two intracellular Ca2+ pools could also explain the biphasic transient increase in [Ca2+]c induced by tBHP in the majority of our cells by sequential depletion of both pools. One of the two rises in [Ca2+]c could be due to mobilisation of Ca2+ from endoplasmic reticulum or mitochondria. The initial phase might be due to release of non-mitochondrial Ca2+, and the second to the release of mitochondrial Ca2+. It is also worth noting that once the non-mitochondrial pool is depleted, tBHP causes a slow Ca2+ release(corresponding to the mitochondrial store)(Fig. 3A,B), whereas when the mitochondrial store is already depleted the tBHP effect is much faster (Fig. 4A), as would be expected if the Ca2+ was released from the non-mitochondrial pool.
Our findings, in which tBHP releases Ca2+ from intracellular stores, are consistent with previous reports where the[Ca2+]c increase evoked by tBHP is accomplished by an inhibition of the PMCA (Hoyal et al., 1996) and/or by sensitisation of the sarcoplasmic reticulum Ca2+ release channels (Lang et al., 2003; Redondo et al.,2004). In fact, it has been reported that both tBHP metabolism to radical species and/or accumulation of oxidised glutathione can damage Ca2+-ATPase functions in the plasma membrane and the endoplasmic reticulum (Viner et al.,1997). Furthermore, the opening of these channels has been shown to be modulated by numerous factors, including phosphorylation, adenine nucleotides, thiol reactive compounds and pH(Bootman et al., 2001). Redox modulation of channel activity has been previously reported in various channels (DiChiara and Reinhart,1997). The endoplasmic reticulum, a key organelle in cytosolic Ca2+ signal generation, expresses two separate and related families of Ca2+-release channels, inositol 1,4,5-triphosphate(IP3R) and ryanodine (RyR) receptors(Ashby and Tepikin, 2002; Bootman et al., 2002), and it is largely responsible for mediating Ca2+ release from intracellular stores. One type of intracellular Ca2+ pool is sensitised by IP3, which activates IP3-induced Ca2+ release (IICR). Another is sensitised by ryanodine, leading to a Ca2+-induced Ca2+ release (CICR) process(Petersen and Wakui,1990).
It has been demonstrated in several cell types that the presence of different oxidising reagents `sensitise' RyR and IP3R, through blocker or stimulative mechanisms (Suko et al., 2000; Schultheiss et al.,2005). In pancreatic β-cells, thiol oxidation by the reactive disulphide 2,2′-dithiodipyridine causes a release of Ca2+from intracellular stores by mechanisms that do not involve activation of RyR,but occur from the IP3-sensitive intracellular Ca2+pools (Islam et al., 1997). Additionally, in pancreatic acinar cells it has been shown that free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine are able to mobilise Ca2+ from ryanodine-sensitive intracellular stores(Klonoswki-Stumpe et al., 1997). Our results using 2-APB (at a concentration of 30 μmol l-1, known to block the IP3R) indicate that tBHP releases Ca2+ from a non-mitochondrial Ca2+ pool by an IP3R-independent mechanism. Similar results were obtained previously by us(Pariente et al., 2001) and by others (Hoyal et al., 1996; Clementi et al., 1998). This conclusion is supported by our results showing that ryanodine (at a concentration of 50 μmol l-1, which blocks ryanodine receptors),abolishes tBHP-induced Ca2+-release from non-mitochondrial Ca2+ pools, thus suggesting that tBHP sensitises ryanodine receptors, at least in pancreatic acinar cells. In fact, it has been reported that other oxidising agents, like H2O2, release Ca2+ from intracellular stores by activation of the ryanodine receptor (Favero et al., 1995; Oba et al., 1998) and that sulphydryl groups (susceptible to oxidation) have a critical role in the ryanodine-sensitive Ca2+ channel(Oba et al., 1998). In addition, ryanodine shows `in vitro' sensitisation in the presence of the sulphydryl group oxidising agent thimerosal(Abramson et al., 1995; Wu et al., 1996).
In summary, our findings show that treatment of pancreatic acinar cells with tBHP results in the release of Ca2+ from mitochondrial and non-mitochondrial intracellular stores, viaryanodine-sensitive channels. From a physiological point of view, these results help us to understand the complex mechanism of intracellular Ca2+ homeostasis in pancreatic acinar cells.
cytosolic free Ca2+concentration
endoplasmic reticulum/sarcoplasmic reticulum
carbonyl cyanide p-trifluoromethoxy-phenylhydrazone
IP3-induced Ca2+ release
plasma membrane Ca2+-pump ATPase
reactive oxygen species
sarcoendoplasmic reticulum Ca2+ ATPase
This work was supported by the Spanish Ministry of Science and Technology(MCyT-DGI), grant no. BFI2002-02772 and by the Spanish Ministry of Science and Education (MEC-DGI), grant no. BFU2004-00165. The authors wish to thank Mrs Gómez Blázquez for her technical assistance. M.A.M.-B. is supported by a postdoctoral contract (ref. ARM/CSL) from the Junta de Andalucía.