Up-regulation of megakaryocytic Na⁺/Ca²⁺ exchange in klotho-deficient mice

The active form of vitamin D, 1,25(OH)<inf>2</inf>D<inf>3</inf>, is a powerful regulator of cytosolic Ca²⁺-concentration ([Ca²⁺]<inf>i</inf>) in a variety of cell types. The formation of 1,25(OH)<inf>2</inf>D<inf>3</inf> is inhibited by FGF23, an effect requiring presence of klotho. 1,25(OH)<inf>2</inf>D<inf>3</inf> 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 Ca²⁺ entry (SOCE). In other cell types 1,25(OH)<inf>2</inf>D<inf>3</inf> has been shown to up-regulate the Na⁺/Ca²⁺-exchanger, which could, depending on cell membrane potential and cytosolic Na⁺ concentration, either decrease or increase [Ca²⁺]<inf>i</inf>. The present study explored whether Na⁺/Ca²⁺-exchanger activity is different in megakaryocytes isolated from kl/kl mice than in megakaryocytes isolated from wild type mice. Na⁺/Ca²⁺-exchanger induced currents were determined by whole cell patch clamp and the Na⁺/Ca²⁺-exchanger induced alterations of [Ca²⁺]<inf>i</inf> by Fura-2 fluorescence. As a result, the inward current and the increase of [Ca²⁺]<inf>i</inf> 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)<inf>2</inf>D<inf>3</inf> (100 nM, 48 h) was followed by enhancement of both, inward current and increase of [Ca²⁺]<inf>i</inf> following replacement of extracellular Na⁺ by NMDG. In conclusion, the present observations reveal a powerful stimulating effect of 1,25(OH)<inf>2</inf>D<inf>3</inf> on Na⁺/Ca²⁺-exchanger activity in megakaryocytes.