Figure 2

SOCE-generated cyclic AMP is abolished by inhibition of soluble adenylyl cyclase. (a) Primers designed to amplify adenylyl cyclase isoforms (1–10) were used to probe hCASMC cDNA. PCR products were separated on a 3% agarose gel. (b–e) hCASMCs transduced with adenoviruses encoding the cyclic AMP biosensor H187 and subjected to the same experimental scheme as described in Fig. 1. Cells were pre-incubated in either vehicle control (0.1% DMSO; black all traces); the transmembrane adenylyl cyclase inhibitor 2′-5′ dideoxyadenosine (DDA, 100 μM, n = 48 from 4 experimental repeats, (b)) or soluble adenylyl cyclase inhibitors 2-hydroxyestradiol (2-CE, 20 μM, n = 29 from 4 experimental repeats (d)) or 4-hydroxyestradiol (4-CE, 20 μM, n = 18 from 4 experimental repeats, (e)) for at least 10 minutes before addition of thapsigargin (TG, 2 μM). Inhibitors were then present until addition of forskolin (20 μM) and IBMX (500 μM) and the paired controls were conducted on the same day as the test. (c) As a positive control, addition of DDA (100 μM) induced a significant reduction in cyclic AMP generated in response to prostacyclin (PGI2; 1 nM, n = 24 from 4 experimental repeats). Error bars represent the standard error of the mean. (f) Store-operated Ca²⁺ entry recorded in Fluo-4AM-loaded hCASMCs under control conditions and in the presence of either 2-CE and 4-CE (20 μM). Cells were initially bathed in nominally Ca²⁺-free solution for 10 minutes before treatment with thapsigargin (TG, 2 μM). Ca²⁺ (1.8 mM) was then introduced into the extracellular solution (control, n = 90 cells; 2-CE, n = 65 cells; 4-CE, n = 89 cells). Error bars represent the standard error of the mean.