Protective effects of noradrenaline against tumor necrosis factor-alpha-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression.
Từ khóa
trừu tượng
OBJECTIVE
To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes.
METHODS
Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers.
METHODS
Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels.
RESULTS
Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection.
CONCLUSIONS
These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.
