CHELATION OF INTRACELLULAR CALCIUM PREVENTS MESANGIAL CELL PROLIFERATIVE RESPONSIVENESS

Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsi veness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was s ufficient to elicit the same response. Rat primary mesangial cells (pa ssages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine ser um (FBS), then stimulated with 0.1 mu M endothelin-l, 1.9 mu M platele t-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 mu M ionomycin, wi th or without the intracellular calcium chelator -bis-(2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was meas ured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofl uorometry and in Fluo-3-loaded cells by confocal fluorescence laser mi croscopy. [H-3]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was anal yzed by Northern blot. All test agents stimulated a significant increa se in cytosolic and nuclear calcium, which were both effectively inhib ited with BAPTA. All agents stimulated a significant increase in [H-3] -thymidine incorporation and enhanced c-fos mRNA expression(no detecta ble c-fos mRNA was observed in quiescent cells). BAPTA prevented the e nhanced [H-3]-thymidine incorporation stimulated by endothelin-l and P DGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response t o these agents. Phorbol ester induction of c-fos mRNA in the absence o f raised cytosolic or nuclear calcium was also suppressed by BAPTA. Ce ll viability as measured by thiazolyl blue and trypan blue was not alt ered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.