Calcium leak from intracellular stores--the enigma of calcium signalling.

Wherever you travel through the cytoplasm of the cells you will find organelles with internal [Ca(2+)] levels higher than in the surrounding cytosol. This is particularly true of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells); such organelles serve as the main sources of releasable Ca(2+) for cytosolic cellular signalling. Calcium pumps of the SERCA family (sarcoplasmic and endoplasmic reticulum calcium ATP-ases) import calcium into the organelle lumen. The other mechanism that is responsible for the steady state calcium level within the lumen of ER or SR is a calcium leak that balances the influx created by the pumps. The leak remains the most enigmatic of the processes involved in calcium regulation. The molecular nature of the leak mechanism is not known. The basal leak is a relatively slow process, which is difficult to investigate and which is easily outmatched (both in the amplitude of calcium responses and in attractiveness to experimenters) by substantially faster second messenger-induced release. Nevertheless, information on the properties of the calcium leak, although thinly scattered through the pages of PubMed, has been slowly accumulating. In this review we will discuss the properties of the calcium leak and speculate about possible mechanisms, which could mediate this process.

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

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.

Role of proton gradients and vacuola 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.

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells.

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated for 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By contrast, glutathione had no effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.

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 microM) or during Ca2+ readmission in Ca2+-depleted cells (pretreated with thapsigargin or ACh). 2. La3+ and Zn2+ (100 microM) inhibited CCE during Ca2+ readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 microM), an inhibitor of non-selective cation channels, genistein (10 microM), a broad-range tyrosine kinase inhibitor, and piceatannol (10 microM), 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 microM 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 microM) reduced CCE in Ca2+-depleted cells, whereas the activator phorbol 12-myristate, 13-acetate (3 microM) 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.

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21rho, and the integrity of the actin cytoskeleton.

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.

Histamine does not potentiate cyclic AMP-mediated amylase secretion in the guinea-pig pancreatic acinar cells.

1. This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase in isolated guinea-pig pancreatic acinar cells. 2. Histamine caused does-related enhancement of amylase release. Similarly, incubation of acini with increasing concentrations of vasoactive intestinal peptide (VIP) resulted in a typical dose-dependent increase in amylase output. 3. When pancreatic acinar cells were incubated with histamine in combination with VIP, amylase secretion did not differ statistically from secretion induced by histamine or VIP alone and was significantly lower than theoretical additivity. Additionally, amylase secretion in the presence of histamine plus forskolin was significantly less than additive. The action of histamine was equally effective as VIP in causing cyclic adenosine monophosphate (cAMP) increase. 4. These results indicate that histamine may exert its secretory effects via the cyclic AMP pathway in the exocrine guinea-pig pancreas.