Classic Phytochemical Antioxidant and Lipoxygenase Inhibitor, Nordihydroguaiaretic Acid, Activates Phospholipase D through Oxidant Signaling and Tyrosine Phosphorylation Leading to Cytotoxicity in Lung Vascular Endothelial Cells.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.

Insulin protects pancreatic acinar cells from cytosolic calcium overload and inhibition of plasma membrane calcium pump.

Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria.

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase.

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca(2+)](i)) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca(2+)](i) signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca(2+)](i), and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca(2+)](i), which was sensitive to antioxidants and removal of external Ca(2+), and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca(2+)-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca(2+) uptake. This suggests that hydrogen peroxide promotes Ca(2+) release from the endoplasmic reticulum and the mitochondria and that it promotes Ca(2+) influx. Lower concentrations of hydrogen peroxide (10-100 muM) increased [Ca(2+)](i) and altered cholecystokinin-evoked [Ca(2+)](i) oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca(2+)](i) also upregulated the activity of the plasma membrane Ca(2+)-ATPase in a Ca(2+)-dependent manner, whereas higher concentrations (0.1-1 mM) inactivated the plasma membrane Ca(2+)-ATPase. This may be important in facilitating "Ca(2+) overload," resulting in cell injury associated with pancreatitis.

Curcumin induces electrical activity in rat pancreatic beta-cells by activating the volume-regulated anion channel.

Curcumin, the principal active component of turmeric, is reported to exert a number of therapeutic actions, including a hypoglycaemic/antidiabetic action. The underlying mechanisms to this action are essentially unknown. We have investigated the hypothesis that a direct stimulatory action on the pancreatic beta-cell could contribute towards the hypoglycaemic activity of this compound. Electrical and ion channel activity were recorded in rat beta-cells using the patch-clamp technique. beta-Cell volume was measured using a video-imaging technique. Insulin release was measured from intact islets by radioimmunoassay. Curcumin (2-10 microM) activated the volume-regulated anion channel in beta-cells. Single channel studies indicated that activation was the result of increased channel open probability. This effect was accompanied by depolarisation of the cell membrane potential, the generation of electrical activity and enhanced insulin release. Curcumin also decreased beta-cell volume, presumably reflecting loss of Cl(-) (and hence water) as a result of anion channel activation. These findings are consistent with the suggestion that Cl(-) fluxes play an important role in regulating beta-cell function. The stimulation of beta-cell function by curcumin could contribute to the hypoglycaemic actions of this compound, and these findings identify a novel potential therapeutic target for the treatment of type 2 diabetes mellitus.

Possible role of duration of PKC-induced ERK activation in the effects of agonists and phorbol esters on DNA synthesis in Panc-1 cells.

Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) have been implicated in the effects of regulatory peptides on proliferation. We studied how ERK was activated by PKC following regulatory peptide or phorbol ester stimulation and we also investigated the effect of ERK activation on proliferation in Panc-1 cells. Panc-1 cells transfected with CCK1 receptors were treated with cholecystokinin (CCK), neurotensin (NT), or phorbol 12-myristate 13-acetate (PMA). DNA synthesis was studied by measuring tritiated thymidine incorporation. PKC isoforms were selectively inhibited with Gö6983 and 200 nM Ro-32-0432, their translocation was detected by confocal microscopy and by subcellular fractionation followed by immunoblotting. ERK cascade activation was detected with phosphoERK immunoblotting and inhibited with 20 microM PD98059. PMA and CCK inhibited, NT stimulated DNA synthesis. These effects were inhibited by Ro-32-0432 but not by Gö6983 suggesting the involvement of PKCepsilon in proliferation control. Confocal microscopy and subcellular fractionation demonstrated that PMA, CCK, and NT caused cytosol to membrane translocation of PKCepsilon and ERK activation that was inhibited by Ro-32-0432 but not by Gö6983. ERK activation was prolonged following PMA and CCK, but transient after NT treatment. PMA, CCK, and NT all activated cyclinD1, while p21CIP1 expression was increased by only PMA and CCK, but not by NT; each of these effects is inhibited by PD98059. In conclusion, our results provide evidence for PKCepsilon-mediated differential ERK activation and growth regulation in Panc-1C cells. Identification of the mechanisms by which these key signaling pathways are modulated could provide a basis for the development of novel therapeutic interventions to treat pancreatic cancer.

Multiple fatty acid sensing mechanisms operate in enteroendocrine cells: novel evidence for direct mobilization of stored calcium by cytosolic fatty acid.

Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca(2+) ([Ca(2+)](i)) to stimulate cholecystokinin release from enteroendocrine cells. Using the murine enteroendocrine cell line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca(2+) directly from the intracellular store. STC-1 cells loaded with fura-2 were briefly (3 min) exposed to saturated FA above and below the threshold length (C(8), C(10), and C(12)). C(12), but not C(8) or C(10), induced a dose-dependent increase in [Ca(2+)](i), in the presence or absence of extracellular Ca(2+). Various signaling inhibitors, including d-myo-inositol 1,4,5-triphosphate receptor antagonists, all failed to block FA-induced Ca(2+) responses. To identify direct effects of cytosolic FA on the intracellular Ca(2+) store, [Ca(2+)](i) was measured in STC-1 cells loaded with the lower affinity Ca(2+) dye magfura-2, permeabilized by streptolysin O. In permeabilized cells, again C(12) but not C(8) or C(10), induced release of stored Ca(2+). Although C(12) released Ca(2+) in other permeabilized cell lines, only intact STC-1 cells responded to C(12) in the presence of extracellular Ca(2+). In addition, 30 min exposure to C(12) induced a sustained elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), but only a transient response in the absence of extracellular Ca(2+). These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (>/=C(12)) transiently induce Ca(2+) release from intracellular Ca(2+) stores. However, they also induce sustained Ca(2+) entry from the extracellular medium to maintain an elevated [Ca(2+)](i).

Human calcium-sensing receptor can be suppressed by antisense sequences.

We have evaluated the ability of an antisense cDNA sequence, directed to the amino-terminus of the human calcium-sensing receptor (CaR), to reduce the expression and function of an EGFP-tagged CaR (CaR-EGFP) in HEK293 cells. Confocal microscopy and Western blot analysis showed a significant and selective reduction of the expression of CaR-EGFP by the antisense construct. Measurements of changes in intracellular calcium induced by CaR agonists showed that CaR-EGFP function was significantly reduced by the antisense sequence, as was agonist-evoked phosphorylation of extracellular signal-regulated protein kinases (ERK1,2). A sense construct directed to the same region of the receptor had no effect, confirming the specificity of the antisense construct. Our results indicate that a CaR antisense cDNA reduces both the expression and function of the receptor. In the absence of strong, specific pharmacological inhibitors of CaR, the antisense approach will be helpful to elucidate contributions of the CaR to cell physiology.