Altered nitric oxide production and exogenous nitric oxide do not affect the proliferation of rat mesangial cells.
OBJECTIVE
To examine the hypothesis that nitric oxide (NO) acts as an autocrine, antiproliferative regulator and that exogenous NO donor inhibitors the proliferation of cultured rat mesangial cells.


METHODS
The cellular effects of endogenous and exogenous NO were studied in rat mesangial cells in a two-dimensional culture of early mesangial cell passages. The proliferation of mesangial cells was determined by cell-counting and uptake of [3H]-thymidine. NO generation was induced by incubation with interleukin-1 beta (5 u/ml) or bacterial lipopolysaccharide (10 micrograms/ml) for 24 h. NO release by mesangial cells was assessed by measuring the accumulation of nitrite, the major stable end-product of NO, in mesangial cell supernates. In addition, cyclic GMP (cGMP) formation was measured by radioimmunoassay as an indicator for NO generation.


RESULTS
The formation of nitrite and cGMP was significantly increased after incubation of mesangial cells with interleukin-1 beta or lipopolysaccharide. This effect was greatly reduced by an inhibitor of NO synthesis. NG-monomethyl-L-arginine (L-NMMA; 0.1 mmol/l). The NO donor 3-morpholino-sydnonimine-HCl also increased the cGMP concentrations in the mesangial cells. The proliferation of mesangial cells was analysed in growth-arrested and mitogen-stimulated (platelet-derived growth factor, platelet-derived growth factor plus ATP and fetal calf serum) mesangial cells in the presence and absence of L-NMMA and the NO synthase substrate L-arginine (1 mmol/l). At 48 h platelet-derived growth factor (50 ng/ml), and platelet-derived growth factor (50 ng/ml) plus ATP (0.1 mmol/l) and fetal calf serum 5% each significantly increased the uptake of [3H]-thymidine in mesangial cells. These effects were not altered in the presence of L-NMMA or L-arginine. Pretreatment with interleukin-1 beta or with lipopolysaccharide also failed to affect the uptake of [3H]-thymidine in resting or proliferating mesangial cells. 3-Morpholino-sydnonimine-HCl (10(-3) to 10(-6) mol/l) did not suppress the mitogen-induced proliferation of mesangial cells, even when it was administered three times a day.


CONCLUSIONS
The present findings support recent observations that interleukin-1 beta and lipopolysaccharide strongly induce NO production in mesangial cells, as is shown indirectly by the greatly increased formation of nitrite and cGMP. However, these effects were not associated with antiproliferative action on mitogen-stimulated mesangial cells. Similarly, the exogenous NO donor 3-morpholino-sydnonimine-HCl induced cGMP formation but failed to inhibit proliferation of mesangial cells when used at a non-toxic dose. Our observations do not support the contention that the formation of NO and cGMP constitutes an autocrine downregulating mechanism in the control of the growth of mesangial cells. It remains to be seen what pathophysiological role the induction of NO release plays in the regulation of the behaviour of mesangial cells, e.g. during an inflammatory response to glomerular injury.
Mohaupt
M
M
Medizinische Klinik IV, Universität Erlangen-Nürnberg, Germany.
Schoecklmann
H O
HO
Schulze-Lohoff
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Sterzel
R B
RB
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019323:omega-N-Methylarginine
D007375:Interleukin-1; D008070:Lipopolysaccharides; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C002385:linsidomine; D001120:Arginine; D008981:Molsidomine; D006152:Cyclic GMP