David N. Criddle

Reactive Oxygen Species Induced by Bile Acid Induce Apoptosis and Protect Against Necrosis in Pancreatic Acinar Cells

BACKGROUND & AIMS Oxidative stress is implicated in the pathogenesis of pancreatitis, but clinical trials of antioxidants have produced conflicting results. We examined the role of intracellular reactive oxygen species (ROS) in pancreatic acinar cell injury. METHODS Freshly isolated murine and human pancreatic acinar cells were studied using confocal microscopy to measure changes in intracellular and mitochondrial ROS concentrations ([ROS]I and [ROS]M), cytosolic and mitochondrial calcium concentrations ([Ca2+]C and [Ca2+]M), reduced nicotinamide adenine dinucleotide phosphate levels, and death pathways in response to taurolithocholate acid sulfate (TLC-S) or the oxidant menadione. Ca2+-activated Cl- currents were measured using whole-cell patch clamp, with or without adenosine triphosphate (ATP). RESULTS TLC-S induced prolonged increases in [Ca2+]C and [Ca2+]M, which led to dose-dependent increases in [ROS]I and [ROS]M, impaired production of ATP, apoptosis, and necrosis. Inhibition of the antioxidant reduced nicotinamide adenine dinucleotide phosphate quinine oxidoreductase by 2,4-dimethoxy-2-methylnaphthalene potentiated the increases in [ROS]I and apoptosis but reduced necrosis, whereas the antioxidant N-acetyl-L-cysteine reduced [ROS]I and apoptosis but increased necrosis. Inhibition of mitochondrial ROS production prevented apoptosis but did not alter necrosis; autophagy had no detectable role. Patched ATP prevented sustained increases in [Ca2+]C and necrosis. CONCLUSIONS Increases in [ROS]M and [ROS]I during bile acid injury of pancreatic acinar cells promote apoptosis but not necrosis. These results indicate that alternative strategies to antioxidants are required for oxidative stress in acute pancreatitis.

Signal transduction, calcium and acute pancreatitis

Evidence consistently suggests that the earliest changes of acute pancreatitis are intracellular, the hallmark of which is premature intracellular activation of digestive zymogens, accompanied by disruption of normal signal transduction and secretion. Principal components of physiological signal transduction include secretagogue-induced activation of G-protein-linked receptors, followed by generation of inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose. In response, calcium is released from endoplasmic reticulum terminals within the apical, granular pole of the cell, where calcium signals are usually contained by perigranular mitochondria, in turn responding by increased metabolism. When all three intracellular messengers are administered together, even at threshold concentrations, dramatic potentiation results in sustained, global, cytosolic calcium elevation. Prolonged, global elevation of cytosolic calcium is also induced by hyperstimulation, bile salts, alcohol and fatty acid ethyl esters, and depends on continued calcium entry into the cell. Such abnormal calcium signals induce intracellular activation of digestive enzymes, and of nuclear factor ĸB, as well as the morphological changes of acute pancreatitis. Depletion of endoplasmic reticulum calcium and mitochondrial membrane potential may contribute to further cell injury. This review outlines current understanding of signal transduction in the pancreas, and its application to the pathophysiology of acute pancreatitis.

Effect of niflumic acid on noradrenaline‐induced contractions of the rat aorta

1 The effects of niflumic acid, an inhibitor of calcium‐activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline‐evoked contractions in isolated preparations of the rat aorta. 2 The cumulative concentration‐effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration‐dependent manner. The degree of inhibition of the maximal contractile response to NA (1 μm) produced by 10 μm niflumic acid (38%) was similar to the effect of 1 μm nifedipine (39%). 3 Contractions to brief applications (30 s) of 1 μm NA were inhibited by 55% and 62% respectively by 10 μm niflumic acid and 1 μm nifedipine. 4 In the presence of 0.1 μm nifedipine, niflumic acid (10 μm) produced no further inhibition of the NA‐evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5 In Ca‐free conditions the transient contraction induced by 1 μm NA was not inhibited by niflumic acid (10 μm) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6 Niflumic acid 10 μm did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 μm levcromakalim but were unaffected by niflumic acid. 7 It was concluded that niflumic acid produces selective inhibition of a component of NA‐evoked contraction which is probably mediated by voltage‐gated calcium channels. These data are consistent with a model in which NA stimulates a calcium‐activated chloride conductance which leads to the opening of voltage‐gated calcium channels to produce contraction.

Fatty acids, alcohol and fatty acid ethyl esters: Toxic Ca2+ signal generation and pancreatitis

Pancreatitis, a potentially fatal disease in which the pancreas digests itself as well as its surroundings, is a well recognized complication of hyperlipidemia. Fatty acids have toxic effects on pancreatic acinar cells and these are mediated by large sustained elevations of the cytosolic Ca(2+) concentration. An important component of the effect of fatty acids is due to inhibition of mitochondrial function and subsequent ATP depletion, which reduces the operation of Ca(2+)-activated ATPases in both the endoplasmic reticulum and the plasma membrane. One of the main causes of pancreatitis is alcohol abuse. Whereas the effects of even high alcohol concentrations on isolated pancreatic acinar cells are variable and often small, fatty acid ethyl esters--synthesized by combination of alcohol and fatty acids--consistently evoke major Ca(2+) release from intracellular stores, subsequently opening Ca(2+) entry channels in the plasma membrane. The crucial trigger for pancreatic autodigestion is intracellular trypsin activation. Although there is still uncertainty about the exact molecular mechanism by which this Ca(2+)-dependent process occurs, progress has been made in identifying a subcellular compartment--namely acid post-exocytotic endocytic vacuoles--in which this activation takes place.

Bronchodilator activity of Mikania glomerata Sprengel on human bronchi and guinea‐pig trachea

The effects of aqueous extracts and hydro‐alcoholic extract (HAE), and of a dichloromethane fraction (MG1) obtained from the HAE of Mikania glomerata leaves on isolated respiratory and vascularsmooth muscle have been investigated. Aqueousextracts and HAE induced a significant inhibition on the histamine contractions on the isolated guinea‐pig trachea. HAE extract induced a concentration‐dependent relaxation on guinea‐pig trachea pre‐contracted with histamine (IC50 0.34 (0.29‐0.39) mg mL−1), acetylcholine (IC50 0.72 (0.67‐0.77) mg ml−1) or K+ (IC50 1.41 (1.18‐1.64) mg mL−1) and on isolated human bronchi precontracted with K+ (IC50 0.34 (0.26‐0.42) mg mL−1). The dichloromethane fraction induced a concentration dependent relaxation in guinea‐pig trachea precontracted with K+ (IC50 0.017 (0.012‐0.022) mg mL−1). The dichloromethane fraction had also a small vasodilator effect on the isolated mesenteric vascular bed and on the isolated rat aorta, and a significant reduction of the oedema induced by subplantar injections of Bothrops jararaca venom in mice. When tested on plasmid DNA, MG1 did not damage the DNA. Chromatographic analysis showed the presence of 11.4% w/w coumarin in MG1. The results supported the indication of M. glomerata products for the treatment of respiratory diseases where bronchoconstriction is present.

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat

The effects of niflumic acid, an inhibitor of calcium‐activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline‐ and 5‐hydroxytryptamine (5‐HT)‐induced pressor responses of the rat perfused isolated mesenteric vascular bed. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose‐dependent manner. Nifedipine (1 μm) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31±5%. Niflumic acid (10 and 30 μm) also inhibited the noradrenaline‐induced increase in perfusion pressure and 30 μm niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34±6%. The increases in perfusion elicited by 5‐HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 μm) in a concentration‐dependent manner and 30 μm niflumic acid inhibited responses to 0.3 and 3 nmol 5‐HT by, respectively, 49±8% and 50±7%. Nifedipine (1 μm) decreased the pressor response to 3 nmol 5‐HT by 44±9%. In the presence of a combination of 30 μm niflumic acid and 1 μm nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5‐HT (3 nmol) was not significantly greater than with niflumic acid (30 μm) alone. Thus the effects of niflumic acid and nifedipine were not additive. In Ca‐free conditions the transient contractions induced by 5‐HT (3 nmol) were not reduced by 30 μm niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5‐HT to its receptor. Niflumic acid 30 μm did not inhibit the pressor responses induced by KCl (20 and 60 μmol) which were markedly reduced by 1 μm nifedipine. In addition, 1 μm levcromakalim decreased pressor responses produced by 20 μmol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels. It is concluded that niflumic acid selectively reduces a component of noradrenaline‐ and 5‐HT‐induced pressor responses by inhibiting a mechanism which leads to the opening of voltage‐gated calcium channels. Our data suggest that the Ca2+‐activated chloride conductance may play a pivotal role in the activation of voltage‐gated calcium channels in agonist‐induced constriction of resistance blood vessels.

Mitochondrial injury in pancreatitis

Pancreatitis is an increasingly common disease that carries a significant mortality and which lacks specific therapy. Pathological calcium signalling is an important contributor to the initiating cell injury, caused by or acting through mitochondrial inhibition. A principal effect of disordered cell signalling and impaired mitochondrial function is cell death, either by apoptosis that is primarily protective, or by necrosis that is deleterious, both locally and systemically. Mitochondrial calcium overload is particularly important in necrotic injury, which may include damage mediated by the mitochondrial permeability transition pore. The role of reactive oxygen species remains controversial. Present understanding of the part played by disordered pancreatic acinar calcium signalling and mitochondrial inhibition offers several new potential therapeutic targets.

Myorelaxant and antispasmodic effects of the essential oil of Alpinia speciosa on rat ileum

The effects of the essential oil of Alpinia speciosa Schum (EOAS) on rat ileum were studied. EOAS (0.1–600 µg/mL) reversibly relaxed ileal basal tonus. Submaximal contractions induced by 60 mM KCl or acetylcholine were concentration dependently inhibited by EOAS with similar IC50 values (∼ 44 and 48 µg/mL, respectively). These results show that EOAS possesses both relaxant and antispasmodic actions in the ileum. Copyright

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis

Objective Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Design Intracellular calcium ([Ca2+]C), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Results Inhibition of OME with 4-MP converted predominantly transient [Ca2+]C rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. Conclusions A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.

Relaxant effects of the essential oil of Ocimum gratissimum on isolated ileum of the guinea pig

The effects of the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) on guinea pig ileum were studied. EOOG (0.1-1000 microg/ml) reversibly and concentration-dependently relaxed the basal tone of the ileum and reversed the tonic contractions induced by 60 mM KCl and 10 microM acetylcholine, with IC(50) values of 23.8+/-5.2, 18.6+/-4.0 and 70.0+/-4.6 microg/ml, respectively. Our results show that EOOG exerts relaxant effects on intestinal smooth muscle, consistent with the popular use of the plant to treat gastrointestinal disorders.