HEPARIN INHIBITS CA2+ CALMODULIN-DEPENDENT KINASE-II ACTIVATION AND C-FOS INDUCTION IN MESANGIAL CELLS/

Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partiall y suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbo l ester or platelet-derived growth factor, this response appears to re sult from the ability of heparin to suppress activation of the extrace llular-signal-regulated kinase family of mitogen-activated protein kin ases. However, we have also shown that heparin suppresses c-fos expres sion in response to ionophores such as ionomycin, an event independent of mitogen-activated protein kinase [Miralem, Wang, Whiteside and Tem pleton (1996) J. Biol. Chem. 271, 17100-17106]. Here we identify this second heparin-sensitive pathway as involving Ca2+/calmodulin-dependen t kinase(CaMK) II. Ionomycin (100 nM) caused a transient rise in intra cellular Ca2+ concentration ([Ca2+](i)) in quiescent RMCs to 386 +/- 5 5 nM, with an increase in CaMK II activity that peaked 30 s later. The accumulation of c-fos mRNA that ensued 30 min later was prevented whe n the increase in [Ca2+](i) was prevented with the intracellular Ca2chelator, bis-(2-aminophenyoxy)ethane-N,N,N',N'-tetra-acetic acid. The broad-specificity CaMK inhibitor, KT 5926, inhibited ionomycin-depend ent c-fos induction at a concentration at which it was without effect on induction by serum or phorbol ester. The CaMK II-specific inhibitor , KN-93, likewise inhibited c-fos induction by ionomycin, but not by s erum or phorbol ester. ML-7, an inhibitor of the CaMK-related myosin l ight-chain kinase (MLCK), was without effect. Heparin (1 mu g/ml) supp ressed ionomycin-dependent c-fos induction. It was without effect on [ Ca2+](i), but inhibited the development of autonomous CaMK II activity . However, when heparin was added to the CaMK II assay solution in vit ro, it was without effect on autonomous activity. Furthermore, heparin did not prevent full activation of CaMK II by the Ca2+-calmodulin com plex in vitro. Heparin did not affect myosin light-chain phosphorylati on or RMC contraction, processes mediated by MLCK. We conclude that io nomycin induces c-fos in RMCs through the CaMK II pathway, and that he parin prevents CaMK II activation by an indirect process mediated by o ther cell components. Heparin does not affect activation of the closel y related CaMK, MLCK.