Effect of bradykinin on nitric oxide production, urea synthesis and viability of rat hepatocyte cultures.
BACKGROUND
It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-alpha (TNFalpha), interleukin-1. The control role of arginine in both urea and NO biosynthesis is well known, when NO is synthesized from arginine, by the NOS reaction, citrulline is produced. Thus, the urea cycle is bypassed by the NOS reaction. Many authors demonstrated in other cellular types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. The simultaneous increase of NO and ROS levels could cause peroxynitrite synthesis, inducing damage and reducing cell viability. The aim of this research is to study the effect of bradykinin, a proinflammatory mediator, on cell viability and on urea production in cultures of rat hepatocytes.


RESULTS
Hepatocytes were treated with bradykinin, that stimulates nitric oxide synthase (NOS). NO release was determined using 4,5 diaminofluorescein diacetate (DAF-2DA), as fluorescent indicator of NO. Addition of the NOS inhibitor, Ng-nitro-L-arginine methyl ester (L-NAME), to the culture medium inhibited the increase of NO production. Exposure of hepatocytes to bradykinin 0,1 mM for 2 hours resulted in a significant decrease of urea synthesis. Cell viability, instead, showed a significant decrease 24 hours after the end of bradykinin treatment as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. L-NAME addition recovered urea production and cell viability at control values.


CONCLUSIONS
The findings suggest that the cell toxicity, after bradykinin treatment, effectively depends upon exposure to increased NO levels and the effects are prevented by L-NAME. The results show also that the increased NO synthesis induces a reduced urea production, that is another index of cell damage.
Sesti
Settimio
S
Department of Cell Biology, University of Calabria, Italy. sesti@unical.it
Martino
Guglielmo
G
Mazzulla
Sergio
S
Chimenti
Rosa
R
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004396:Coloring Agents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D007202:Indicators and Reagents; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D014508:Urea / Q000378:metabolism*
C414689:4,5-diaminofluorescein diacetate; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009569:Nitric Oxide; D014508:Urea; C022616:thiazolyl blue; D001920:Bradykinin; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester