NAADP Mediates [ca²⁺]i In Human Pulmonary Artery Smooth Muscle Cells Via Two Pore Channels

Objective To explore whether the pathway of NAADP induced calcium release through TPC1 and TPC2 expressed in human pulmonary artery smooth muscle cells, and study the basic mechanism of the pathway.Methods The expression of TPC1 and TPC2 in human pulmonary artery smooth muscle cells was measured by RT-PCR and protein imprinting;Using the cell permeant NAADP analogue, NAADP-AM to study the effect of calcium release with different concentration of NAADP;Research Ned-19 abolished the effect of NAADP-AM in human PASMCs;The effect of NAADP induced calcium release by calcium free;Effect of bafilomycin A1 on Ca²⁺ release by NAADP in human PASMCs;Effect of vacuolar H+-ATPase inhibitor bafilomycin A1 on Ca²⁺ release by NAADP-AM in human PASMCs;Research the effect of NAADP induced calcium release with Xestospongin C abolished Ins P3 R and Ryanodine abolished Ry R in human PASMCs.Results 1.Using RT-PCR assay for TPC1 and TPC2 m RNA, the expression of TPC1 and TPC2 m RNA in human pulmonary artery smooth muscle cells was observed.2.NAADP induced calcium release depend on concentration of NAADP,When the concentration of NAADP-AM was 0.25 ? M and 0.5 ? M cytosolic calcium concentration continued to increase,when the concentration is 1?M,caused a biphasic response, showed in came a period after an initial phase of internet growth peak;3.Pretreatment of human PASMCs with Ned-19?100?M? for 20 min eliminated the initial transient peak and significantly reduced the sustained phase of the NAADP-AM activated Ca²⁺ response? 285.1±25.2 n M?n=6?; Ned-19: 134.9±11.2 n M?n=6?, p< 0.01?.4.1?M NAADP cause calcium release reaction is not affected by calcium free.The peak of calcium release is 228.9±19.7?n = 5? and 222.1 + 18.4 nm?n = 5? in calcium free in human PASMCs.5.Bafilomycin A1 were pretreated for 1 hour in human pulmonary artery smooth muscle cells and almost completely blocked Ca2 + release in front of the average and plateau, the average peak control group was 186.4±16.9 n M?n =5?, Bafilomycin A1 pretreated human PASMCs group, the average peak was 81.4±8.8 n M?n=5; p<0.05?.6.Pretreatment of PASMCs with xestospongin C had no significant effect on the peak and the sustained Ca²⁺ response activated by NAADP-AM.7.Inhibition of Ry R with ryanodine caused significant reduction in the initial transient Ca²⁺ release,but did not affect the sustained phase of the Ca²⁺ response.Conclusion:1.TPC1 and TPC2 are expressed in human PASMCs,with TPC1 as the main.2.Application of NAADP activated concentration-dependent increase in [Ca²⁺]i3.NAADP mainly induced calcium release from intracellular calcium stores, and a great possibility that the release of calcium from within the lysosome system.4. NAADP induced calcium release can further cause endoplasmic reticulum calcium release.