Figure 1.

SOCE-dependent effects on HASMC cAMP.A, model of the FRET cAMP sensor, mTurq2ΔEPACcp173Ven_Ven, showing the EPAC1 cAMP-binding domain (CD, ΔDEP, Q270E) positioned between a mTurquoise2 (donor domain, blue) and two Venus (acceptor, yellow) domains (top), and representing how cAMP binding increases the distance between the donor and acceptor domains, thus reducing FRET (bottom). B, representative trace of normalized FRET emission ratio measured in H134-expressing HASMCs under initial experimental conditions (Ca²⁺-free Krebs buffer, “A”) during ER(Ca²⁺) store depletion (Ca²⁺-free Krebs buffer supplemented with CPA [10 μM], “B”), during the early (blue, “C”) and late (yellow, “D”) phases of SOCE [Krebs buffer supplemented with CPA (10 μM)] and, lastly, during sensor saturation [Forskolin (10 μM) + IBMX (100 μM), Fsk/IBMX, “E”]. C, representative single cell traces of normalized FRET emission ratios measured in H134-expressing HASMCs treated as in B in the presence of either saline (black) or of SQ 22536 (1 mM). D–G, changes in normalized FRET emission ratios measured in control (n = 20) or SQ22536-treated (n = 10) HASMCs during ER(Ca²⁺) store depletions (D), the early phase of SOCE (E), or the late phase (F and G) (Student’s unpaired t test, ∗p = 0.0420 ∗∗p = 0.0083). H, representative single-cell traces of normalized FRET emission ratios measured in control (siCtrl) or ADCY6-silenced (siADCY6) HASMCs treated as in B. I–L, differences in normalized FRET emission ratios measured in control (n = 26) or ADCY6-silenced (n = 26) HASMCs during ER(Ca²⁺) store depletion (I), the early phase of SOCE (J), or the late phase (K and L) of SOCE, respectively (Student’s unpaired t test, ∗∗p = 0.0076 comparing cAMP increase in Ca²⁺ free Krebs with CPA, ∗∗p = 0.004 comparing cAMP decrease during initial phase of SOCE, ∗p = 0.01 comparing cAMP rate of increase during SOCE). M, representative single-cell traces of normalized FRET emission ratios measured in control (siCtrl) or ADCY8-silenced (siADCY8) HASMCs treated as in B. N–Q, differences in normalized FRET emission ratios measured in control (n = 32) or ADCY8-silenced (n = 22) HASMCs during ER(Ca²⁺) store depletions (N), during the early phase of SOCE (O) or the late phase (P and Q) of SOCE, respectively (Student’s unpaired t test, ∗p = 0.04, ∗∗p = 0.007). R, levels of ADCY6 mRNA in siCtrl and siADCY6 transfected HASMCs (Student’s unpaired t test, ∗∗∗∗p < 0.0001). S, levels of ADCY8 mRNA in siCtrl and siADCY8 transfected HASMCs (Student’s unpaired t test, ∗∗p = 0.0012). ADCY, adenylyl cyclase; cAMP, cyclic AMP; CPA, cyclopiazonic acid; EPAC1, exchange protein activated by cAMP-1; FRET, fluorescence resonance energy transfer; HASMC, human arterial smooth muscle cell; SOCE, store-operated calcium entry.