Up-regulation of megakaryocytic Na(+)/Ca(2+) exchange in klotho-deficient mice

The active form of vitamin D, 1,25(OH)₂D₃, is a powerful regulator of cytosolic Ca(2+)-concentration ([Ca(2+)]i) in a variety of cell types. The formation of 1,25(OH)₂D₃ is inhibited by FGF23, an effect requiring presence of klotho. 1,25(OH)₂D₃ plasma levels are excessive in klotho-deficient mice (kl/kl). A previous study revealed that klotho-deficiency is followed by decreased activation of platelets, an effect at least in part due to blunted store operated Ca(2+) entry (SOCE). In other cell types 1,25(OH)₂D₃ has been shown to up-regulate the Na(+)/Ca(2+)-exchanger, which could, depending on cell membrane potential and cytosolic Na(+) concentration, either decrease or increase [Ca(2+)]i. The present study explored whether Na(+)/Ca(2+)-exchanger activity is different in megakaryocytes isolated from kl/kl mice than in megakaryocytes isolated from wild type mice. Na(+)/Ca(2+)-exchanger induced currents were determined by whole cell patch clamp and the Na(+)/Ca(2+)-exchanger induced alterations of [Ca(2+)]i by Fura-2 fluorescence. As a result, the inward current and the increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG were higher in kl/kl megakaryocytes than in wild type megakaryocytes, a difference abrogated by treatment of the mice with low Vitamin D diet. Pretreatment of wild type megakaryocytes with 1,25(OH)₂D₃ (100 nM, 48 h) was followed by enhancement of both, inward current and increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG. In conclusion, the present observations reveal a powerful stimulating effect of 1,25(OH)₂D₃ on Na(+)/Ca(2+)-exchanger activity in megakaryocytes.