Involvement of ryanodine-operated channels in tert-butylhydroperoxide-evoked Ca²⁺ mobilisation in pancreatic acinar cells

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.

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).

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.

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 IP<sub>3</sub>R, 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 Ca<sup>2+</sup>from intracellular stores by mechanisms that do not involve activation of RyR,but occur from the IP<sub>3</sub>-sensitive intracellular Ca<sup>2+</sup>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 Ca<sup>2+</sup> from ryanodine-sensitive intracellular stores(Klonoswki-Stumpe et al., 1997). Our results using 2-APB (at a concentration of 30 μmol l<sup>-1</sup>, known to block the IP<sub>3</sub>R) indicate that tBHP releases Ca<sup>2+</sup> from a non-mitochondrial Ca<sup>2+</sup> pool by an IP<sub>3</sub>R-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<sup>-1</sup>, which blocks ryanodine receptors),abolishes tBHP-induced Ca<sup>2+</sup>-release from non-mitochondrial Ca<sup>2+</sup> 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 H<sub>2</sub>O<sub>2</sub>, release Ca<sup>2+</sup> 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 Ca<sup>2+</sup> 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.

List of abbreviations
 
• [Ca²⁺]_c

  cytosolic free Ca²⁺concentration

 
• [Ca²⁺]_o

  extracellular Ca²⁺concentration

 
• 2-APB

  2-aminoethoxydiphenyl borane

 
• CCK-8

  cholecystokinin octapeptide

 
• CICR

  Ca²⁺-induced Ca²⁺release

 
• DTT

  dithiothreitol

 
• ER/SR

  endoplasmic reticulum/sarcoplasmic reticulum

 
• FCCP

  carbonyl cyanide p-trifluoromethoxy-phenylhydrazone

 
• H₂O₂

  hydrogen peroxide

 
• IICR

  IP₃-induced Ca²⁺ release

 
• IP₃

  inositol-1,4,5-triphosphate

 
• IP₃R

  inositol-1,4,5-triphosphate receptor

 
• PMCA

  plasma membrane Ca²⁺-pump ATPase

 
• ROS

  reactive oxygen species

 
• RyR

  ryanodine receptor

 
• SERCA

  sarcoendoplasmic reticulum Ca²⁺ ATPase

 
• tBHP

  tert-butylhydroperoxide

 
• TPS

  thapsigargin

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.