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-ind uced apoptosis in rat glomerular mesangial cells and the possible involveme nt of nitric oxide (NO) in this process. In mesangial cell monolayers incub ated for 12 h in a medium without growth factors and with 10(-6) M thapsiga rgin, a known specific inhibitor of endoplasmic reticulum Ca2+-ATPase, a hi gh percentage of cells showed typical nuclear features of apoptosis, assess ed either by staining with propidium iodide (23 vs. 9% in control condition s) or by terminal desoxynucleotidyl transferase-mediated dUTP biotin nick e nd labelling (TUNEL; 17 vs. 5% in control conditions). When cells were main tained in a medium containing 10% fetal calf serum (FCS) or in a free-calci um medium, the thapsigargin-induced apoptosis rate was very low. In rat mes angial 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 bar 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 mu mol/well). Furthermore, the treatment wit h the NO syn thesis inhibitor L-NAME (10(-4) M) induced a significant decre ase in the number of apoptotic cells (9%), whereas incubation with the NO d onor SIN-1 (10(-5) M) induced a marked increase in the rate of apoptosis (2 9%). Western and Northern blot analysis of macrophage-type inducible NO syn thase (iNOS) demonstrated that thapsigargin treatment induces the expressio n of the iNOS protein and iNOS mRNA. Treatment with L-NAME prevented the th apsigargin-induced BCL-2 decrease, whereas incubation with SIN-1 potentiate d the effect of thapsigargin on BCL-2. Double labelling by immunohistochemi stry for iNOS and TUNEL revealed that the same cells that suffered apoptosi s we re positive for INOS. in summary, our results indicate that thapsigarg in is able to enhance the apoptosis rate of rat mesangial cells by a mechan ism that is mediated by an increase in cytosolic free calcium. Increased IN OS expression, and hence increased NO production, seems to be involved in t his effect. Copyright (C) 1999 S. Karger AG. Basel.