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Ole H Petersen

Publications and source records attributed to Ole H Petersen.

At least 19 recordsLinked to original sources
PubMed2006

Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

Animals↗
PubMed2006

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors.

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.

Animals↗
PubMed2006

From Galvani to patch clamp: the development of electrophysiology.

The development of electrophysiology is traced from the early beginnings represented by the work of the Dutch microscopist, Jan Swammerdam, in the 17th century through the first notion of an aqueous transmembrane pore as a substrate of excitability made by Luigi Galvani in late 18th century to the invention late in the 20th century of the patch-clamp technique by Erwin Neher and Bert Sakmann.

Animals↗
PubMed2006

Failure of calcium microdomain generation and pathological consequences.

Normal physiological regulation depends on Ca(2+) microdomains, because there is a need to spatially separate Ca(2+) regulation of different cellular processes. It is only possible to generate local Ca(2+) signals transiently; so, there is an important functional link between Ca(2+) spiking and microdomains. The pancreatic acinar cell provides a useful cell biological model, because of its clear structural and functional polarization. Although local Ca(2+) spiking in the apical (granular) microdomain regulates fluid and enzyme secretion, prolonged global elevations of the cytosolic Ca(2+) concentration are associated with the human disease acute pancreatitis, in which proteases in the granular region become inappropriately activated and digest the pancreas and its surroundings. A major cause of pancreatitis is alcohol abuse and it has now been established that fatty acid ethyl esters and fatty acids, non-oxidative alcohol metabolites, are principally responsible for causing the acinar cell damage. The fatty acid ethyl esters release Ca(2+) from the endoplasmic reticulum and the fatty acids inhibit markedly mitochondrial ATP generation, which prevents the acinar cell from disposing of the excess Ca(2+) in the cytosol. Because of the abolition of ATP-dependent Ca(2+) pump activity, all intracellular Ca(2+) concentration gradients disappear and the most important part of the normal regulatory machinery is thereby destroyed. The end stage is necrosis.

Adenosine Triphosphate↗
PubMed2006

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers.

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.

Acetylcholine↗
PubMed2006

Calcium-dependent release of NO from intracellular S-nitrosothiols.

The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.

Acetylcholine↗
PubMed2006

NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area.

Inositol trisphosphate and cyclic ADP-ribose release Ca2+ from the endoplasmic reticulum via inositol trisphosphate and ryanodine receptors, respectively. By contrast, nicotinic acid adenine dinucleotide phosphate may activate a novel Ca2+ channel in an acid compartment. We show, in two-photon permeabilized pancreatic acinar cells, that the three messengers tested could each release Ca2+ from the endoplasmic reticulum and also from an acid store in the granular region. The nicotinic acid adenine dinucleotide phosphate action on both types of store, like that of cyclic ADP-ribose but unlike inositol trisphosphate, depended on operational ryanodine receptors, since it was blocked by ryanodine or ruthenium red. The acid Ca2+ store in the granular region did not have Golgi or lysosomal characteristics and might therefore be associated with the secretory granules. The endoplasmic reticulum is predominantly basal, but thin extensions penetrate into the granular area and cytosolic Ca2+ signals probably initiate at sites where endoplasmic reticulum elements and granules come close together.

Animals↗
PubMed2006

Ca2+ signalling and pancreatitis: effects of alcohol, bile and coffee.

Ca2+ is a universal intracellular messenger that controls a wide range of cellular processes. In pancreatic acinar cells, acetylcholine and cholecystokinin regulate secretion via generation of repetitive local cytosolic Ca2+ signals in the apical pole. Bile acids and non-oxidative alcohol metabolites can elicit abnormal cytosolic Ca2+ signals that are global and sustained and result in necrosis. Necrosis results from excessive loss of Ca2+ from the endoplasmic reticulum, which is mediated by Ca2+ release through specific channels and inhibition of Ca2+ pumps in intracellular stores, followed by entry of extracellular Ca2+. Reduction of the cellular ATP level has a major role in this process. These abnormal Ca2+ signals, which can be inhibited by caffeine, explain how excessive alcohol intake and biliary disease cause acute pancreatitis, an often-fatal human disease in which the pancreas digests itself and its surroundings.

Animals↗
PubMed2006

Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis.

BACKGROUND & AIMS: Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca(2+)](C)) implicated in pancreatitis. We sought to define the mechanisms of this elevation. METHODS: Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca(2+)](C) (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca(2+)](ER), Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10-100 micromol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. RESULTS: Intracellular delivery of ester induced oscillatory increases of [Ca(2+)](C) and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca(2+)](ER), not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca(2+)](C), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. CONCLUSIONS: Fatty acid ethyl ester increases [Ca(2+)](C) through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.

Adenosine Triphosphate↗
PubMed2006

Role of Ca2+ in pancreatic cell death induced by alcohol metabolites.

Whereas alcohol itself, even in high concentrations, has little effect on the functional performance of isolated pancreatic acinar cells, non-oxidative metabolites (fatty acid ethyl esters [FAEE] and fatty acids [FA]) can cause Ca(2+)-dependent necrosis. The mechanism of action of FAEE has been investigated using a combination of patch clamp whole-cell current recording and Ca(2+) imaging. At low stimulation intensities, FAEE evoke repetitive short-lasting cytosolic Ca(2+) spikes, which are inhibited by caffeine, used as an inositol trisphosphate receptor antagonist. With more intense stimulation, sustained elevations of the cytosolic Ca(2+) concentration are observed, which can be prevented by pharmacological inhibition of the conversion of FAEE to FA. It is therefore the FA and not the FAEE that cause necrosis. The effect of FA cannot be blocked by inositol trisphosphate receptor antagonists. Fatty acids elicit a marked reduction in the cytosolic adenosine triphosphate (ATP) level. The patch clamp experiments show that the toxic sustained Ca(2+) signal generation induced by FA can be prevented by adding ATP to the cell interior. The toxic alcohol effects are principally due to FAEE produced under non-oxidative conditions and their subsequent conversion to FA. The FA-induced necrosis is Ca(2+)-dependent. The destructive sustained Ca(2+) signals are due to inhibition of mitochondrial function with failure of ATP generation.

Animals↗
PubMed2005

Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells.

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.

Animals↗
PubMed2005

The NAADP receptor: new receptors or new regulation?

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent calcium mobilizing messenger yet discovered. Its action has now been reported in a large number of cell types from a diverse array of organisms, and in some cases linked to the transduction of specific cellular stimuli. However, what is controversial is the nature of its target calcium release channel, as well as the subcellular localization of its receptor. Some have proposed that NAADP activates a novel calcium release channel distinct from the two major classes of channels known, the inositol trisphosphate receptors and ryanodine receptors. However, others have suggested that it acts in a novel way to regulate a known calcium release channel, the ryanodine receptor.

Animals↗
PubMed2005

Twenty years of calcium imaging: cell physiology to dye for.

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca(2+) in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca(2+) signaling is accomplished. In excitable cells, the coordination of changing Ca(2+) concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca(2+) signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.

Animals↗
PubMed2004

Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration.

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.

Animals↗
PubMed2004

Dual sensitivity of sarcoplasmic/endoplasmic Ca2+-ATPase to cytosolic and endoplasmic reticulum Ca2+ as a mechanism of modulating cytosolic Ca2+ oscillations.

The effects of ER (endoplasmic reticulum) Ca2+ on cytosolic Ca2+ oscillations in pancreatic acinar cells were investigated using mathematical models of the Ca2+ oscillations. We first examined the mathematical model of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) to reproduce the highly co-operative inhibitory effect of Ca2+ in the ER lumen on ER Ca2+ uptake in the acinar cells. The model predicts that luminal Ca2+ would most probably inhibit the conversion of the conformation state with luminal Ca2+-binding sites (E2) into the conformation state with cytoplasmic Ca2+-binding sites (E1). The SERCA model derived from this prediction showed dose-response relationships to cytosolic and luminal Ca2+ concentrations that were consistent with the experimental data from the acinar cells. According to a mathematical model of cytosolic Ca2+ oscillations based on the modified SERCA model, a small decrease in the concentration of endoplasmic reticulum Ca2+ (approx. 20% of the total) was sufficient to abolish the oscillations. When a single type of IP3R (IP3 receptor) was included in the model, store depletion decreased the spike frequency. However, the frequency became less sensitive to store depletion when we added another type of IP3R with higher sensitivity to the concentration of free Ca2+ in the cytosol. Bifurcation analysis of the mathematical model showed that the loss of Ca2+ from the ER lumen decreased the sensitivity of cytosolic Ca2+ oscillations to IP3 [Ins(1,4,5)P3]. The addition of a high-affinity IP3R did not alter this property, but significantly decreased the sensitivity of the spike frequency to IP3. Our mathematical model demonstrates how luminal Ca2+, through its effect on Ca2+ uptake, can control cytosolic Ca2+ oscillations.

Binding Sites↗
PubMed2004

Ethanol toxicity in pancreatic acinar cells: mediation by nonoxidative fatty acid metabolites.

Ethanol causes pancreatic damage by an unknown mechanism. Previously, we demonstrated that a sustained rise of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes pancreatic acinar cell injury. Here we have investigated the effects of ethanol and its metabolites on Ca(2+) signaling in pancreatic acinar cells. Most cells exposed to ethanol (up to 850 mM) showed little or no increase in [Ca(2+)](i) (and never at concentrations <50 mM). During sustained exposure to 850 mM ethanol, acetylcholine (ACh) evoked a normal [Ca(2+)](i) elevation and following ACh removal there was a normal and rapid recovery to a low resting level. The oxidative metabolite acetaldehyde (up to 5 mM) had no effect, whereas the nonoxidative unsaturated metabolite palmitoleic acid ethyl ester (10-100 microM, added on top of 850 mM ethanol) induced sustained, concentration-dependent increases in [Ca(2+)](i) that were acutely dependent on external Ca(2+) and caused cell death. These actions were shared by the unsaturated metabolite arachidonic acid ethyl ester, the saturated equivalents palmitic and arachidic acid ethyl esters, and the fatty acid palmitoleic acid. In the absence of external Ca(2+), releasing all Ca(2+) from the endoplasmic reticulum by ACh (10 microM) or the specific Ca(2+) pump inhibitor thapsigargin (2 microM) prevented such Ca(2+) signal generation. We conclude that nonoxidative fatty acid metabolites, rather than ethanol itself, are responsible for the marked elevations of [Ca(2+)](i) that mediate toxicity in the pancreatic acinar cell and that these compounds act primarily by releasing Ca(2+) from the endoplasmic reticulum.

Acetylcholine↗
PubMed2004

Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim.

In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.

Animals↗