Atrial natriuretic peptide effects on intracellular pH changes and ROS production in HEPG2 cells: role of p38 MAPK and phospholipase D.
OBJECTIVE
The present study was performed to evaluate Atrial Natriuretic Peptide (ANP) effects on intracellular pH, phospholipase D and ROS production and the possible relationship among them in HepG2 cells. Cancer extracellular microenvironment is more acidic than normal tissues and the activation of NHE-1, the only system able to regulate pHi homeostasis in this condition, can represent an important event in cell proliferation and malignant transformation.


METHODS
The ANP effects on pHi were evaluated by fluorescence spectrometry. The effects on p38 MAPK and ROS production were evaluated by immunoblots and analysis of DCF-DA fluorescence, respectively. RT-PCR analysis and Western blotting were used to determine the ANP effect on mRNA NHE-1 expression and protein levels. PLD-catalyzed conversion of phosphatidylcholine to phosphatydilethanol (PetOH), in the presence of ethanol, was monitored by thin layer chromatography.


RESULTS
A significant pHi decrease was observed in ANP-treated HepG2 cells and this effect was paralleled by the enhancement of PLD activity and ROS production. The ANP effect on pHi was coupled to an increased p38 MAPK phosphorylation and a down-regulation of mRNA NHE-1 expression and protein levels. Moreover, the relationship between PLD and ROS production was demonstrated by calphostin-c, a potent inhibitor of PLD. At the same time, all assessed ANP-effects were mediated by NPR-C receptors.


CONCLUSIONS
Our results indicate that ANP recruits a signal pathway associated with p38 MAPK, NHE-1 and PLD responsible for ROS production, suggesting a possible role for ANP as novel modulator of ROS generation in HepG2 cells.
Baldini
Patrizia Morena
PM
Department of Biology, University of Rome Tor Vergata, Italy. baldini@uniroma2.it
De Vito
Paolo
P
Vismara
Daniela
D
Bagni
Claudia
C
Zalfa
Francesca
F
Minieri
Marilena
M
Di Nardo
Paolo
P
D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D020404:Glycerophospholipids / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D015151:Immunoblotting; D007424:Intracellular Fluid / Q000187:drug effects* / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D020404:Glycerophospholipids; D009281:Naphthalenes; D017382:Reactive Oxygen Species; C051521:phosphatidylethanol; D009320:Atrial Natriuretic Factor; D048051:p38 Mitogen-Activated Protein Kinases; D010739:Phospholipase D; D017461:Receptors, Atrial Natriuretic Factor; C058819:calphostin C