ABSTRACT
The protective potential of nortriptyline has been reported in a few experimental models of brain ischemia, both in vivo and in vitro. However, the detailed molecular mechanisms of the protective action of the drug are still unresolved. The aim of the present study was to determine whether treatment with low or medium concentrations of nortriptyline (0.1-10 µM) might have an effect on cPLA2 protein and/or mRNA expression in ischemic astrocytes, and whether this influence might be related to its potential positive influence on cell viability. On the 21(st) day in vitro, primary cultures of rat cortical astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation, OGD) for 24 h and exposed to nortriptyline. The drug at concentrations of 0.1 and 1 µM attenuated the expression of cPLA2 (both the phosphorylated and unphosphorylated forms) together with a significant decrease in the cPLA2 mRNA level in ischemic astrocytes. We have demonstrated that nortriptyline influences a decrease in cPLA2-mediated arachidonic acid (AA) release through a mechanism that appears to involve the attenuation of both PKC and Erk1/2 kinase expression. Nortriptyline also significantly prevented mitochondrial depolarization in ischemic astrocytes. Moreover, the antidepressant protected glial cells against OGD-induced apoptosis and necrosis. Our findings document a role for cPLA2 expression attenuation and AA release inhibition in the protective effect of nortriptyline in ischemic astrocytes.
Subject(s)
Astrocytes/drug effects , Glucose/deficiency , Neuroprotective Agents/pharmacology , Nortriptyline/pharmacology , Phospholipases A2, Cytosolic/antagonists & inhibitors , Animals , Apoptosis , Arachidonic Acid/metabolism , Astrocytes/cytology , Astrocytes/metabolism , Cell Hypoxia , Cell Survival/drug effects , Cells, Cultured , Glucose/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Necrosis , Phospholipases A2, Cytosolic/metabolism , Protein Kinase C/metabolism , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
Arachidonic acid (AA) is released from cells after nervous tissue injuries.We treated rat cortical neurons and astrocytes cultured under normoxic or simulated ischemic conditions with N-acetylcysteine (100 or 200 microM) or ebselen (10 or 20 microM). N-acetylcysteine decreased AA release in normoxic astrocytes, while ebselen decreased AA release from astrocytes in both conditions. N-acetylcysteine produced no changes in neuronal AA release. A low dose of ebselen significantly increased AA release from neurons in both conditions. The influence of N-acetylcysteine and ebselen on AA release might be implicated in their effects on astrocytes and neurons, however, the exact mechanisms have yet to be explained.
Subject(s)
Acetylcysteine/pharmacology , Arachidonic Acid/metabolism , Azoles/pharmacology , Ischemia/drug therapy , Organoselenium Compounds/pharmacology , Acetylcysteine/administration & dosage , Animals , Animals, Newborn , Astrocytes/drug effects , Astrocytes/metabolism , Azoles/administration & dosage , Cells, Cultured , Cerebral Cortex/cytology , Cytoprotection/drug effects , Dose-Response Relationship, Drug , Ischemia/physiopathology , Isoindoles , Neurons/drug effects , Neurons/metabolism , Organoselenium Compounds/administration & dosage , Rats , Rats, WistarABSTRACT
The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP(+)-induced cytosolic phospholipase A(2) (cPLA(2)) activation of dopaminergic neurons. MPP(+) activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca(2+)](i) level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA(2), and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF(3)), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA(2) on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA(2) phosphorylation at Ser505. Inhibitors of cPLA(2) and PKG increased viability and reduced MPP(+)-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA(2) phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson's disease may mediate dopaminergic neuron degeneration and death through activation of cPLA(2).
Subject(s)
Arachidonic Acid/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Nerve Degeneration/enzymology , Parkinsonian Disorders/enzymology , Phospholipases A2, Cytosolic/metabolism , Substantia Nigra/enzymology , Animals , Calcium/metabolism , Catalytic Domain/drug effects , Catalytic Domain/physiology , Cell Death/drug effects , Cell Death/physiology , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , In Vitro Techniques , Mice , Mice, Inbred C57BL , Models, Biological , Nerve Degeneration/chemically induced , Nerve Degeneration/physiopathology , Nitric Oxide Synthase Type I/drug effects , Nitric Oxide Synthase Type I/metabolism , PC12 Cells , Parkinsonian Disorders/physiopathology , Phospholipases A2, Cytosolic/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Serine/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Substantia Nigra/drug effects , Substantia Nigra/physiopathologyABSTRACT
Cytosolic phospholipase A2 (cPLA2) preferentially liberates arachidonic acid (AA), which is known to be elevated in Alzheimer's disease (AD). The aim of this study was to investigate the possible relationship between enhanced nitric oxide (NO) generation observed in AD and cPLA2 protein level, phosphorylation, and AA release in rat pheochromocytoma cell lines (PC12) differing in amyloid beta secretion. PC12 control cells, PC12 cells bearing the Swedish double mutation in amyloid beta precursor protein (APPsw), and PC12 cells transfected with human APP (APPwt) were used. The transfected APPwt and APPsw PC12 cells showed an about 2.8- and 4.8-fold increase of amyloid beta (Abeta) secretion comparing to control PC12 cells. An increase of NO synthase activity, cGMP and free radical levels in APPsw and APPwt PC12 cells was observed. cPLA2 protein level was higher in APPsw and APPwt PC12 cells comparing to PC12 cells. Moreover, phosphorylated cPLA2 protein level and [3H]AA release were also higher in APP-transfected PC12 cells than in the control PC12 cells. An NO donor, sodium nitroprusside, stimulated [3H]AA release from prelabeled cells. The highest NO-induced AA release was observed in control PC12 cells, the effect in the other cell lines being statistically insignificant. Inhibition of cPLA2 by AACOCF3 significantly decreased the AA release. Inhibitors of nNOS and gamma-secretase reduced AA release in APPsw and APPwt PC12 cells. The basal cytosolic [Ca2+](i) and mitochondrial Ca2+ concentration was not changed in all investigated cell lines. Stimulation with thapsigargin increased the cytosolic and mitochondrial Ca2+ level, activated NOS and stimulated AA release in APP-transfected PC12 cells. These results indicate that Abeta peptides enhance the protein level and phosphorylation of cPLA2 and AA release by the NO signaling pathway.
Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Arachidonic Acid/metabolism , Nitric Oxide/metabolism , Phospholipases A2, Cytosolic/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Arachidonic Acids/pharmacology , Blotting, Western , Calcium/metabolism , Cyclic GMP/metabolism , Humans , Mutation , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/metabolism , Nitroprusside/pharmacology , PC12 Cells , Phosphorylation , Rats , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , TransfectionABSTRACT
Both cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)) have been implicated in pathology of cerebral ischemia. However, which of PLA(2) isoforms in astrocytes is responsible for arachidonic acid (AA) release contributing to their ischemic injury remains to be determined. The aim of the present study was to investigate the time-dependent activation of cPLA(2) and sPLA(2) in astrocytes exposed to combined oxygen glucose deprivation (OGD) as well as to evaluate the effectiveness of their pharmacological blockage as a method of preventing ischemic damage of the glial cells. It was shown that exposure of cultured astrocytes to OGD (0.5-24h) causes an increase in cPLA(2) and sPLA(2) expression and activity. The role of AA liberated mainly by cPLA(2) in the process of apoptosis was also demonstrated. To confirm the specific role of cPLA(2) and sPLA(2) in the mechanism of cells injury by OGD exposure, the effect of AACOCF(3) as cPLA(2) inhibitor and 12-epi-scalaradial as sPLA(2) inhibitor on AA release was examined. It was proved that simultaneous pharmacological blockade of enzymatic activity of cPLA(2) and sPLA(2) during OGD by AACOCF(3) and 12-epi-scalaradial substantially improves survival of ischemic injured glial cells.
Subject(s)
Astrocytes/enzymology , Ischemia/pathology , Phospholipases A/metabolism , Animals , Apoptosis , Arachidonic Acid/metabolism , Astrocytes/metabolism , Caspase 3/metabolism , Cell Hypoxia , Cell Survival , Cells, Cultured , Enzyme Activation , Glucose/deficiency , Group II Phospholipases A2 , Group IV Phospholipases A2 , Ischemia/chemically induced , Phosphorylation , Rats , Rats, WistarABSTRACT
In the present study, we investigated whether the protective effect of FK506 and cyclosporin A (CsA) against in vitro ischemic injury of astrocytes might be mediated through attenuation of cytosolic isoform of phospholipase A(2) (cPLA(2)) expression and activity as well as inhibition of arachidonic acid (AA) release. On the 21st day in vitro, cultures of rat astrocytes were subjected to ischemia-simulating conditions (combined oxygen glucose deprivation) for 8 h and exposed to FK506 (10 - 1,000 nM) and CsA (0.25 - 10 microM). Obtained data suggest the cross-talk between the action of 0.25 - 10 microM CsA as well as 1 microM FK506 on calcineurin (CaN) and cPLA(2) in anti-apoptotic signal transduction pathways. Moreover, we have shown that immunosuppressants at these concentrations protected glial cells against ischemia-induced apoptosis through the increase of cell viability, mitochondrial function restoration, and attenuation of oxidative stress. Finally, in our study, low concentrations of FK506 (10 and 100 nM) exerted limited effects on the assessed parameters. Our findings document a key role either for CaN or cPLA(2) expression attenuation and AA release inhibition in the antiapoptotic effect of FK506 and CsA in ischemic astrocytes.
