Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca2+ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca2+]i) signals. Several Ca2+ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca2+ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca2+]i signals by Ca2+ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca2+ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca2+ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca2+]i signals. Together, the data reviewed here indicate that Ca2+-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca2+]i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed.

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Most physiological agonists increase cytosolic free [Ca2+]c (cytosolic free Ca2+ concentration) to regulate a variety of cellular processes. How different stimuli evoke distinct spatiotemporal Ca2+ responses remains unclear, and the presence of separate intracellular Ca2+ stores might be of great functional relevance. Ca2+ accumulation into intracellular compartments mainly depends on the activity of Ca2+- and H+-ATPases. Platelets present two separate Ca2+ stores differentiated by the distinct sensitivity to thapsigargin and TBHQ [2,5-di-(t-butyl)-1,4-hydroquinone]. Although one store has long been identified as the dense tubular system, the nature of the TBHQ-sensitive store remains uncertain. Treatment of platelets with GPN (glycylphenylalanine-2-naphthylamide) impaired Ca2+ release by TBHQ and reduced that evoked by thrombin. In contrast, GPN did not modify Ca2+ mobilization stimulated by ADP or AVP ([arginine]vasopressin). Treatment with nigericin, a proton carrier, and bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, to dissipate the proton gradient into acidic organelles induces a transient increase in [Ca2+]c that was abolished by previous treatment with the SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) 3 inhibitor TBHQ. Depleted acidic stores after nigericin or bafilomycin A1 were refilled by SERCA 3. Thrombin, but not ADP or AVP, reduces the rise in [Ca2+]c evoked by nigericin and bafilomycin A1. Our results indicate that the TBHQ-sensitive store in human platelets is an acidic organelle whose Ca2+ accumulation is regulated by both Ca2+- and vacuolar H+-ATPases.

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 microm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 microm). 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.

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We have investigated the characteristics of cytosolic Ca 2؉ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca 2؉ waves and local apical Ca 2؉ spikes. This is the first evidence for local Ca 2؉ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca 2؉ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca 2؉ spikes in the apical pole more than 15 m away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca 2؉ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca 2؉ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca 2؉ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca 2؉ release channels.In response to agonist stimulation, many cell types produce transient elevations in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), which remain localized to a specific subcellular domain (1). In isolated pancreatic acinar cells, a low concentration of acetylcholine (ACh) 1 can evoke repetitive local cytosolic Ca 2ϩ spikes, which are confined to the granule containing apical pole (2). These local Ca 2ϩ spikes can activate exocytosis through the apical plasma membrane (3) and open Ca 2ϩ -dependent Cl Ϫ channels present exclusively in the apical plasma membrane, thereby regulating acinar fluid secretion (4).Agonist-receptor interaction causes activation of phospholipase C and the generation of inositol 1,4,5-trisphosphate (IP 3 ), which in turn opens Ca 2ϩ release channels in the endoplasmic reticulum membrane (5), thereby explaining the liberation of Ca 2ϩ from this intracellular store (6). Because the G-protein coupled receptors for cholecystokinin are localized in the basolateral plasma membrane as demonstrated in early autoradiographic studies on intact pancreas (7), the classical view of pancreatic acinar cell stimulation involves the binding of agonist to plasma membrane receptors located at the base. It is this end of the cell that is close to blood vessels and pancreatic nerve terminals from where physiologically released agonists approach the cell (8). If agonist-receptor interaction occurs at the base and Ca 2ϩ release occurs at the opposite (apical) pole, there is a need for a long distance Ca 2ϩ -releasing intracellular messenger. The established Ca 2ϩ -releasing messenger IP 3 (9, 10) as well as the more recently discovered Ca 2ϩ -liberating agents cyclic ADP-ribose and nicotinic acid adeni...

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.