Pawel Ferdek

Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca2+ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca2+ responses because of the release of Ca2+ from both ER and acidic stores. Ca2+ responses to menadione were accompanied by elevation of Ca2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca2+ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca2+ with pericam. We conclude that elevated Ca2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca2+ concentration inside pancreatic acinar cells ([Ca2+]i), due to excessive release of Ca2+ stored inside the cells followed by Ca2+ entry from the interstitial fluid. The sustained [Ca2+]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca2+ release-activated Ca2+ channels (CRAC) would prevent sustained elevation of [Ca2+]i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca2+ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca2+. Ca2+ entry then occurred upon admission of Ca2+ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca2+ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC50 = 3.4 μM), but had little or no effect on the physiological Ca2+ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca2+]i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca2+]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.

Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors.

Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus–secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.

A novel role for Bcl-2 in regulation of cellular calcium extrusion.

Summary The antiapoptotic protein Bcl-2 [1, 2] plays important roles in Ca2+ signaling [3] by influencing inositol triphosphate receptors and regulating Ca2+-induced Ca2+ release [4–6]. Here we investigated whether Bcl-2 affects Ca2+ extrusion in pancreatic acinar cells. We specifically blocked the Ca2+ pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca2+ concentration after a period of enhanced Ca2+ entry. Because external Ca2+ was removed and endoplasmic reticulum Ca2+ pumps were blocked, Ca2+ extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca2+ level much faster than control cells. The enhanced Ca2+ extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na+/Ca2+ exchange activity, because removal of external Na+ did not influence the Ca2+ extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca2+ extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca2+ extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca2+ pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca2+ extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.

Inhibitors of Bcl-2 protein family deplete ER Ca2+ stores in pancreatic acinar cells

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca+2] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca+2] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca+2] and endoplasmic reticulum [Ca+2]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP3Rs and RyRs and substantially reduced by strong [Ca2+] buffering. Inhibition of IP3Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca+2] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.

Both RyRs and TPCs are required for NAADP-induced intracellular Ca2+ release

Graphical abstract

Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium-driven bile uptake.

Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas. Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored. Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells. Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells. Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis.

Nitric oxide signals are interlinked with calcium signals in normal pancreatic stellate cells upon oxidative stress and inflammation.

The mammalian diffuse stellate cell system comprises retinoid-storing cells capable of remarkable transformations from a quiescent to an activated myofibroblast-like phenotype. Activated pancreatic stellate cells (PSCs) attract attention owing to the pivotal role they play in development of tissue fibrosis in chronic pancreatitis and pancreatic cancer. However, little is known about the actual role of PSCs in the normal pancreas. These enigmatic cells have recently been shown to respond to physiological stimuli in a manner that is markedly different from their neighbouring pancreatic acinar cells (PACs). Here, we demonstrate the capacity of PSCs to generate nitric oxide (NO), a free radical messenger mediating, for example, inflammation and vasodilatation. We show that production of cytosolic NO in PSCs is unambiguously related to cytosolic Ca2+ signals. Only stimuli that evoke Ca2+ signals in the PSCs elicit consequent NO generation. We provide fresh evidence for the striking difference between signalling pathways in PSCs and adjacent PACs, because PSCs, in contrast to PACs, generate substantial Ca2+-mediated and NOS-dependent NO signals. We also show that inhibition of NO generation protects both PSCs and PACs from necrosis. Our results highlight the interplay between Ca2+ and NO signalling pathways in cell–cell communication, and also identify a potential therapeutic target for anti-inflammatory therapies.

BH3 mimetic-elicited Ca2+ signals in pancreatic acinar cells are dependent on Bax and can be reduced by Ca2+-like peptides.

BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2′ and HA14-1, induce Ca2+ release from intracellular stores followed by a sustained elevation of the cytosolic Ca2+ concentration. Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca2+ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax−/− cells, Ca2+ loss from the ER was reduced compared to WT cells. The Ca2+-like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca2+-binding proteins, significantly reduced excessive Ca2+ signals and necrosis caused by two BH3 mimetics: BH3I-2′ and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca2+ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca2+ leak in bax−/− cells as well as the fact that BH3 mimetics trigger substantial Ca2+ signals by liberating Bax, indicate that Bax may regulate Ca2+ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca2+-binding sites by CALPs can be used to ameliorate excessive Ca2+ signals caused by BH3 mimetics and shift necrotic death towards apoptosis.