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Probing differential subcellular calcium dynamics using targeted fluorescence calcium biosensors in rat pulmonary artery smooth muscle cells

Intracellular calcium (Ca2+) is involved in distinct cellular functions through global and local signaling in various subcellular compartments. To get better insight if the Ca2+ signals are independently regulated in subcellular compartments, we targeted genetically encoded Ca2+ indicator, D3cpv, into three different subcellular regions and used laser scanning confocal microscopy to study how Ca2+ signals are triggered in the plasma membrane (PM), cytoplasm and nucleus in cultured rat pulmonary artery smooth muscle cells (PASMCs). The in situ Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Application of IP3 or Endothelin‐1 to PASMCs caused rapid increase of fluorescence emission ratio in all three regions. The increase of cytoplasmic/PM Ca2+ preceded the nucleoplasmic Ca2+ and the response was more sustained in the nucleus. Platelet derived growth factor (PDGF) generated variable Ca2+ signals with Ca2+ increase occurred first in the cytoplasm followed by the nucleoplasm in some cells, while an early Ca2+ response in the nucleus was observed in the others. Similarly, the increase in PM Ca2+ was slower than that in the nucleoplasm in some PDGF treated cells. These data suggest a differential regulation of Ca2+ in the cellular compartments that may be essential for the independent regulation of contraction, cell signaling and gene expression in PASMCs.