Nicotinic acid adenine dinucleotide phosphate activates two-pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony-forming cells

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca²⁺ -releasing messenger that increases the intracellular Ca²⁺ concentration by mobilizing the lysosomal Ca²⁺ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca²⁺ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca²⁺ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca²⁺ store and TPC1 in circulating ECFCs by harnessing Ca²⁺ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca²⁺ sequestration by acidic organelles, caused endogenous Ca²⁺ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP₃ )-sensitive endoplasmic reticulum (ER) Ca²⁺ pool. Likewise, the amount of ER releasable Ca²⁺ was reduced by disrupting lysosomal Ca²⁺ content. Liposomal delivery of NAADP induced a transient Ca²⁺ signal that was abolished by disrupting the lysosomal Ca²⁺ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca²⁺ release also required ER-embedded InsP₃ receptors. Finally, NAADP-induced lysosomal Ca²⁺ release was found to trigger vascular endothelial growth factor-induced intracellular Ca²⁺ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca²⁺ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca²⁺ release can selectively be recruited to induce the Ca²⁺ response to specific cues in circulating ECFCs.

Keywords: Ca2+ signaling; NAADP; VEGF; endothelial colony-forming cells; two-pore channel 1.