Effect of MgCl and GdCl on ORAI1 Expression and Store-Operated Ca Entry in Megakaryocytes

In chronic kidney disease, hyperphosphatemia upregulates the Ca channel ORAI and its activating Ca sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg and the calcium-sensing receptor (CaSR) agonist Gd. The present study explored whether sustained exposure to Mg or Gd interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl or 50 µM GdCl. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca concentration ([Ca]) by Fura-2 fluorescence and SOCE from the increase in [Ca] following re-addition of extracellular Ca after store depletion with thapsigargin (1 µM). As a result, Mg and Gd upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg and the CaSR agonist Gd interfere with phosphate-induced dysregulation of [Ca] in megakaryocytes.