Pedro J. Camello

Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide.

Abstract. In the present study we have studied how [Ca2+]i is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+]i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca2+]i. In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+]i induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.

Mitochondrial production of oxidants is necessary for physiological calcium oscillations

Mitochondrial involvement in Ca2+ signaling is thought to be due to the effect of mitochondrial Ca2+ removal from and Ca2+ release to cytosolic domains close to ryanodine and IP3 Ca2+ channels. However, mitochondria are a source of low levels of endogenous reactive oxygen species, and Ca2+ release channels are known to be redox‐sensitive. In the present work, we studied the role of mitochondrial production of oxygen species in Ca2+ oscillations during physiological stimulation. Mitochondria‐targeted antioxidants and mitochondrial inhibitors quickly inhibited calcium oscillations in pancreatic acinar cells stimulated by postprandial levels of the gut hormone cholecystokinin. Confocal microscopy using different redox‐sensitive dyes showed that cholecystokinin‐induced oscillations are associated with mitochondrial production of reactive oxygen species. This production is inhibited by application of mitochondria‐targeted antioxidants and mitochondrial inhibitors. In addition, we found no correlation between inhibition of oscillations and mitochondrial depolarization. We conclude that low level production of reactive oxygen species by mitochondria is a necessary element in the development of Ca2+ oscillations during physiological stimulation. This study unveils a new and unexplored aspect of the participation of mitochondria in calcium signals. J. Cell. Physiol. 206: 487–494, 2006.

Melatonin restores impaired contractility in aged guinea pig urinary bladder

Abstract:  Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin‐treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca2+]i) was estimated by epifluorescence microscopy of fura‐2‐loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca2+]i in response to the stimuli, suggesting a reduced Ca2+sensitivity. Indeed, the agonist‐induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca2+ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI‐17 and MYPT1, and with the absence of CPI‐17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca2+ handling and Ca2+ sensitizations pathways. Moreover, the indoleamine restored age‐induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging‐related urinary bladder contractile impairment.

Sequential activation of different Ca2+ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells

1 We have studied capacitative calcium entry (CCE) under different experimental conditions in fura‐2‐loaded mouse pancreatic acinar cells by digital microscopic fluorimetry. CCE was investigated during [Ca2+]i decay after cell stimulation with a supramaximal concentration of ACh (10 μM) or during Ca2+ readmission in Ca2+‐depleted cells (pretreated with thapsigargin or ACh). 2 La3+ and Zn2+ (100 μM) inhibited CCE during Ca2+ readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 μM), an inhibitor of non‐selective cation channels, genistein (10 μM), a broad‐range tyrosine kinase inhibitor, and piceatannol (10 μM), an inhibitor specific for non‐receptor Syk tyrosine kinase, inhibited CCE during ACh decay but not during Ca2+ reintroduction. 3 Simultaneous detection of Mn2+ entry and [Ca2+]i measurement showed that, in the presence of extracellular calcium, application of 100 μM Mn2+ during ACh decay resulted in manganese influx without alteration of calcium influx, whilst when applied during Ca2+ readmission, Mn2+ entry was significantly smaller and induced a clear inhibition of CCE. 4 Application of the specific protein kinase C inhibitor GF109293X (3 μM) reduced CCE in Ca2+‐depleted cells, whereas the activator phorbol 12‐myristate, 13‐acetate (3 μM) increased Ca2+ entry. 5 Based on these results we propose that cholinergic stimulation of mouse pancreatic acinar cells induces Ca2+ influx with an initial phase operated by a non‐specific cation channel, sensitive to flufenamic acid and tyrosine kinase inhibitors but insensitive to lanthanum and divalent cations, followed by a moderately Ca2+‐selective conductance inhibited by lanthanum and divalent cations.

Role of mitochondria in Ca2+ oscillations and shape of Ca2+ signals in pancreatic acinar cells

Abstract We studied the role of mitochondria in Ca2+ signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca2+ signals under different experimental conditions. Spreading of Ca2+ waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca2+]i oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca2+ concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba2+ for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca2+ clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca2+ waves and in the development of [Ca2+]i oscillations.

NFATc3 Regulates Trypsinogen Activation, Neutrophil Recruitment, and Tissue Damage in Acute Pancreatitis in Mice.

BACKGROUND & AIMS The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

Role of proton gradients and vacuolar H+-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.

Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Abstract:  Information regarding age‐induced Ca2+ signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca2+ signals and mitochondrial potential, a parameter involved in both Ca2+ signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca2+ homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22–24 months old) mice by collagenase dispersion. Ca2+ signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca2+ oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca2+ homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca2+ responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca2+ signals and mitochondrial potential of aged exocrine cells.

XOD-catalyzed ROS generation mobilizes calcium from intracellular stores in mouse pancreatic acinar cells

In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) by release of Ca(2+) from intracellular stores. The ROS-induced Ca(2+) signals showed large variability in shape and time-course and resembled in part Ca(2+) signals in response to physiological secretagogues. ROS-induced Ca(2+) mobilization started at the luminal cell pole and spread towards the basolateral side in a wave manner. ROS-evoked Ca(2+) responses were not inhibited by the phospholipase C (PLC) inhibitor U73122 (10 microM). Neither 2-aminoethoxy-diphenylborate (2-APB) (70 microM) nor ryanodine (50 microM) suppressed ROS-evoked Ca(2+) release. ROS still released Ca(2+) when the endoplasmic reticulum Ca(2+)-ATPase was blocked with thapsigargin (1 microM), or when rotenone (10 microM) was added to release Ca(2+) from mitochondria. Our results suggest that pancreatic acinar cells ROS do not unspecifically affect Ca(2+) homeostasis. ROS primarily affect Ca(2+) stores located in the luminal cell pole, which is also the trigger zone for agonist-induced Ca(2+) signals. Release of Ca(2+) induces Ca(2+) waves carried by Ca(2+)-induced Ca(2+) release and produces thereby global Ca(2+) signals. Under oxidative stress conditions, the increase in [Ca(2+)](i) could be one mechanism contributing to an overstimulation of the cell which could result in cell dysfunction and cell damage.

Melatonin induces neural SOD2 expression independent of the NF-kappaB pathway and improves the mitochondrial population and function in old mice

Abstract:  Aging is commonly defined as a physiological phenomenon associated with morphological and functional deleterious changes in which oxidative stress has a fundamental impact; therefore, readjusting the oxidative balance should have beneficial effects. In our study, we tested the antioxidant melatonin in old mouse brains and showed positive effects at the cellular and mitochondrial levels. Melatonin attenuated β‐amyloid protein expression and α‐synuclein deposits in the brain compared to aged group. Furthermore, oxidative stress was increased by aging and induced the nuclear translocation of nuclear factor‐kappa B (NF‐κB), which was suppressed by melatonin treatment. The antioxidant mitochondrial expression, superoxide dismutase 2 (SOD2), was increased in both control and melatonin‐treated old mice, despite the different activation states of the NF‐κB pathway. The NF‐κB pathway was activated in the old mice, which may be explained by this group’s response to the increased oxidative insult; this insult was inhibited in melatonin‐treated animals, showing this group an increase in active mitochondria population that was not observed in old group. We also report that melatonin is capable of restoring the mitochondrial potential of age‐damaged neurons. In conclusion, melatonin’s beneficial effects on brain aging are linked to the increase in mitochondrial membrane potential and SOD2 expression, which probably reduces the mitochondrial contribution to the oxidative stress imbalance.