Supplementary Material for: CNGA2 Contributes to ATP-Induced Noncapacitative Ca<sup>2+</sup> Influx in Vascular Endothelial Cells

<i>Background/Aims:</i> ATP can activate several Ca<sup>2+</sup> influx channels in vascular endothelial cells. For example, it stimulates TRPC channels via capacitative and noncapacitative Ca<sup>2+</sup> entry (CCE and non-CCE, respectively) mechanisms; it also directly acts on P2X purinoceptors, resulting in Ca<sup>2+</sup> influx. In the present study, we tested the hypothesis that cyclic nucleotide-gated (CNG) channels also contribute to ATP-induced non-CCE. <i>Methods:</i> Two selective inhibitors of CNG channels, <i>L</i>-<i>cis</i>-diltiazem and LY-83583, and CNGA2-specific siRNA were used to study the involvement of CNGA2 in ATP-induced non-CCE in endothelial cells. Ca<sup>2+</sup> influx was studied using Ca<sup>2+</sup>-sensitive fluorescence dyes Fluo-3 and Fluo-4. <i>Results/Conclusion:</i><i>L</i>-<i>cis</i>-diltiazem and LY-83583 markedly reduced ATP-induced non-CCE in 3 types of endothelial cells including the H5V endothelial cell line, the primary cultured bovine aortic endothelial cells and the endothelial cells within isolated mouse aortic strips. The CNGA2-specific siRNA also reduced the ATP-induced non-CCE in H5V endothelial cells. The Ca<sup>2+</sup> influx was inhibited by Rp-8-CPT-cAMPS, MDL-12330A, SQ-22536 and MRS-2179, but not by ODQ or NF-157. Taken together, the present study demonstrated that CNGA2 channels contribute to ATP-induced non-CCE in vascular endothelial cells. It is likely that ATP acts through P2Y<sub>1 </sub>receptors and adenylyl cyclases to stimulate CNGA2.