Nitric oxide is involved in apoptosis induced by thapsigargin in rat mesangial cells.
The purpose of this study was to examine the mechanisms of thapsigargin-induced apoptosis in rat glomerular mesangial cells and the possible involvement of nitric oxide (NO) in this process. In mesangial cell monolayers incubated for 12 h in a medium without growth factors and with 10(-6) M thapsigargin, a known specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, a high percentage of cells showed typical nuclear features of apoptosis, assessed either by staining with propidium iodide (23 vs. 9% in control conditions) or by terminal desoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL; 17 vs. 5% in control conditions). When cells were maintained in a medium containing 10% fetal calf serum (FCS) or in a free-calcium medium, the thapsigargin-induced apoptosis rate was very low. In rat mesangial cells treatment with thapsigargin decreased the expression of BCL-2 protein and bcl-2 mRNA, whereas it did not alter the levels of BAX protein or bax mRNA. When mesangial cells were incubated with thapsigargin in the absence of FCS, we detected a significant increase in nitrite production (3.78 +/- 0.96 vs. 1.76 +/- 0.44 micromol/well). Furthermore, the treatment with the NO synthesis inhibitor L-NAME (10(-4) M) induced a significant decrease in the number of apoptotic cells (9%), whereas incubation with the NO donor SIN-1 (10(-5) M) induced a marked increase in the rate of apoptosis (29%). Western and Northern blot analysis of macrophage-type inducible NO synthase (iNOS) demonstrated that thapsigargin treatment induces the expression of the iNOS protein and iNOS mRNA. Treatment with L-NAME prevented the thapsigargin-induced BCL-2 decrease, whereas incubation with SIN-1 potentiated the effect of thapsigargin on BCL-2. Double labelling by immunohistochemistry for iNOS and TUNEL revealed that the same cells that suffered apoptosis were positive for iNOS. In summary, our results indicate that thapsigargin is able to enhance the apoptosis rate of rat mesangial cells by a mechanism that is mediated by an increase in cytosolic free calcium. Increased iNOS expression, and hence increased NO production, seems to be involved in this effect.
Rodríguez-López
A M
AM
Instituto Reina Sofía de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, España.
Martínez-Salgado
C
C
Eleno
N
N
Arévalo
M
M
López-Novoa
J M
JM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D009569:Nitric Oxide / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D003470:Culture Media; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D009569:Nitric Oxide; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium