GW274150 inhibits nitric oxide production by primary cultures of rat proximal tubular cells.
BACKGROUND
Production of nitric oxide (NO) subsequent to expression of inducible NO synthase (iNOS) contributes to the development of ischemic renal injury and inflammation. Here we investigate the effects of GW274150, a potent, long-acting and highly selective inhibitor of iNOS activity, on NO production by primary cultures of rat proximal tubular cells (PTC).


METHODS
Pure populations of PTC were isolated from the cortex of kidneys obtained from male Wistar rats using a combination of collagenase digestion, sieving and Percoll centrifugation. Confluent PTC cultures were incubated for 1-24 h with MEM, interferon-gamma (IFN-gamma, 100 microg/ml), bacterial lipopolysaccharide (LPS, 10 microg/ml) in combination after which NO production was determined. PTC were also incubated with IFN-gamma (100 microg/ml) and LPS (10 microg/ml) and increasing concentrations of GW274150 or L-N6-(1-iminoethyl)lysine (L-NIL) (10 nM-1 mM) for 24 h after which nitrite levels in the incubation medium were measured.


RESULTS
IFN-g and LPS in combination produced a significant, time-dependent increase in NO production. Both GW274150 and L-NIL produced a significant and concentration-dependent inhibition of NO production. However, GW274150 was markedly more potent (EC50 approximately 100 nM) than L-NIL (EC50 approximately 10 microM).


CONCLUSIONS
GW274150 inhibits NO production by primary cultures of PTCs and may therefore be useful in conditions associated with nitrosative stress of the kidney.
Chatterjee
Prabal K
PK
Department of Experimental Medicine, Nephrology & Critical Care, William Harvey Research Institute, Queen Mary-University of London, UK.
Kvale
Espen O
EO
Patel
Nimesh S A
NS
Thiemermann
Christoph
C
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013440:Sulfides / Q000494:pharmacology; D013997:Time Factors
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; C090663:N(6)-(1-iminoethyl)lysine; D009566:Nitrates; D009573:Nitrites; D013440:Sulfides; C409004:GW 274150; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D008239:Lysine; D009584:Nitrogen; D019331:NG-Nitroarginine Methyl Ester