Role of calcium independent phospholipase A2 in maintaining mitochondrial membrane potential and preventing excessive exocytosis in PC12 cells.

This study was carried out to elucidate the effects of calcium independent phospholipase A(2) (iPLA(2)) on mitochondrial function and exocytosis in neuroendocrine cells. iPLA(2) mRNA and protein were detected in cell lysates and mitochondria from PC12 cells. Treatment of cells with the iPLA(2) inhibitor, bromoenol lactone (BEL), resulted in reduction in the mitochondrial membrane potential. Increase in membrane capacitance and number of spikes at amperometry, indicating exocytosis, were detected from PC12 cells after treatment with BEL. The induced exocytosis was abolished by pre-incubation of cells with the antioxidant, glutathione monoethyl ester, spin-trap/free radical scavenger, PBN, or inhibitors of the mitochondrial permeability transition pore, cyclosporine A and bongkrekic acid. These findings indicate that inhibition of iPLA(2) results in excessive exocytosis through increased oxidative damage (or failure to repair such damage) and defects in mitochondrial function. A similar process may occur in neurons with mutations in iPLA(2), leading to neuronal injury.

Differential effects of lysophospholipids on exocytosis in rat PC12 cells.

Secretory phospholipase A2 (sPLA2) activity is present in the CNS and the sPLA2-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons. However, little is known about possible contributions of various lysophospholipid species to exocytosis in neuroendocrine cells. This study was therefore carried out to examine the effects of several lysophospholipid species on exocytosis on rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion, indicating exocytosis, was observed in PC12 cells after external infusion of lysophosphatidylinositol (LPI), but not lysophosphatidylcholine or lysophosphatidylserine by total internal reflection microscopy. Similarly, external infusion of LPI induced significant increases in capacitance, or number of spikes detected at amperometry, indicating exocytosis. Depletion of cholesterol by pre-incubation of cells with methyl beta cyclodextrin and depletion of Ca2+ by thapsigargin and incubation in zero external Ca2+ resulted in attenuation of LPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and intracellular Ca2+. Moreover, LPI induced a rise in intracellular Ca2+ suggesting that this could be the trigger for exocytosis. It is postulated that LPI may be an active participant in sPLA2-mediated exocytosis in the CNS.

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor.

AIMS: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. RESULTS: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. INNOVATION AND CONCLUSIONS: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo.

Expression profile of multiple secretory phospholipase A(2) isoforms in the rat CNS: enriched expression of sPLA(2)-IIA in brainstem and spinal cord.

Phospholipases A(2) (PLA(2)) are enzymes which cleave the sn-2 ester bond in membrane phospholipids to release free fatty acids and lysophospholipids. The present study aimed to elucidate the expression profile of multiple secretory phospholipase A(2) (sPLA(2)) isoforms in the normal rat CNS with focus on sPLA(2)-IIA in the brainstem and spinal cord. Quantitative RT-PCR analysis showed that sPLA(2)-IB expression was low throughout the CNS, sPLA(2)-IIA expression was high in the brainstem and spinal cord, sPLA(2)-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/hypothalamus, sPLA(2)-V expression was high in the olfactory bulb and cerebellum, and sPLA(2)-X was expressed at very low levels in the normal CNS. Of the isoforms, sPLA(2)-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway. Western blot analysis showed high level of sPLA(2)-IIA expression in the brainstem and cervical, thoracic and lumbar spinal segments but low level of expression in other parts of the brain. sPLA(2)-IIA was localized by immunohistochemistry to the spinal trigeminal and facial motor nuclei and dorsal- and ventral-horns of the spinal cord. The enzyme was found on the endoplasmic reticulum of neuronal cell bodies and small diameter dendrites or dendritic spines at electron microscopy. The expression of sPLA(2)-IIA in the dorsal horn and spinal trigeminal nucleus is consistent with previous results which showed an important role of CNS sPLA(2) in nociceptive transmission.

Effects of cholesterol oxidation products on exocytosis.

Increase in levels of oxysterols or cholesterol oxidation products have been detected in brain areas undergoing neuroinflammation after excitotoxic injury, and the present study was carried out to elucidate possible effects of these products on exocytosis in rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion with the cell membrane indicating exocytosis was observed by total internal reflection microscopy (TIRFM), and confirmed by capacitance measurements, after addition of 7 ketocholesterol, 24 hydroxycholesterol or cholesterol 5, 6 beta epoxide. 7 ketocholesterol induced exocytosis was attenuated by pretreatment with a disruptor of cholesterol-rich domains or "lipid rafts", methyl-beta-cyclodextrin (MbetaCD) as demonstrated by capacitance and amperometry measurements of neurotransmitter release. Moreover, treatment of cells with thapsigargin to deplete intracellular calcium, or treatment of cells with lanthanum chloride to block calcium channels resulted in attenuation of 7 ketocholesterol induced exocytosis. Fura-2 imaging showed that 7 ketocholesterol induced rapid and sustained increases in intracellular calcium concentration, and that this effect was attenuated in cells that were pre-treated with MbetaCD, thapsigargin or lanthanum chloride. Together, the results suggest that neurotransmitter release triggered by 7 ketocholesterol is dependent on the integrity of cholesterol rich lipid domains on cellular membranes and a rise in intracellular calcium, either through release from internal stores or influx via calcium channels. Increased cholesterol oxidation product concentrations in brain areas undergoing neuroinflammation may enhance exocytosis and neurotransmitter release, thereby aggravating excitotoxicity.