Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C094

Oral Communications

Soluble adenylyl cyclase links store-operated Ca2+ entry (SOCE) to Ca2+/cyclic AMP-response element binding protein (CREB) activation in human coronary artery smooth muscle cells

T. Parker1, C. Dart1

1. Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.


In vascular smooth muscle, Ca2+ influx through store-operated channels activates Ca2+/cyclic AMP-response element binding protein (CREB) and induces transcription of a distinct set of genes (1). CREB activation by store-operated Ca2+ entry (SOCE) is associated with early response/ proliferative gene transcription, although the physiological outcome and role of CREB activation in vascular repair or disease states is controversial (2). Here we show that the Ca2+ and HCO3--sensitive enzyme, soluble adenylyl cyclase (sAC), links SOCE with CREB activation in human coronary artery smooth muscle cells (hCASMCs) and may therefore represent an important regulatory point in the Ca2+-transcription pathway. In fura-2-loaded hCASMCs, internal Ca2+ stores were depleted by incubation with the sarcoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin (2μM) in Ca2+-free solutions. This induced a transient increase in cytosolic Ca2+ (Fig 1A; n=18 cells). SOCE was then induced by the addition of Ca2+ (1.8mM) to the extracellular solution. In hCASMCs expressing the FRET-based cyclic AMP biosensor H187, the same protocol produced changes in cyclic AMP that mirrored cytosolic [Ca2+] (Fig 1B; n=19). These cyclic AMP changes were blocked by the Orai1 inhibitor, GSK7975A (30μM; n=25). SOCE also activated the cyclic AMP effector protein kinase A (PKA). PKA activity was measured in hCASMCs expressing the PKA reporter, AKAR4-NES (n=19), and by immunoblot analysis to assess phosphorylation of the PKA substrate, vasodilator-stimulated phosphoprotein (n=3). Immunoblot analysis also revealed CREB phosphorylation (serine 133) in response to SOCE (n=3). Cell-permeant transmembrane adenylyl cyclase inhibitors, SQ22,536 (100μM; n=18) or dideoxyadenosine (DDA; 100μM; Fig 1C; n=28), had no effect on the SOCE-induced rise in cAMP. In contrast, sAC inhibitors 2-hydroxyestradiol (2-CE, 20μM; n=29) or 4-hydroxyestradiol (4-CE, 20μM; Fig 1D; n=18) completely abolished the generation of cAMP in response to SOCE. In immunoblots, another sAC inhibitor, KH7 (20μM), inhibited SOCE-induced phosphorylation of both VASP and CREB (n=3). 48-hour incubation with individual sAC inhibitors also significantly reduced viable hCASMC numbers compared to vehicle controls (2-CE, 39.2 ± 4.7% reduction; 4-CE, 44.7 ± 7.2%; KH7, 36.1 ± 1.8%; n=3; mean ± SEM; P<0.05 one-way ANOVA; Tukey post-hoc). Our data suggest that in hCASMCs SOCE activates sAC, which in turn activates the cAMP/PKA/CREB axis. sAC is directly activated by Ca2+ and HCO3- and responds to ATP (3). Since CO2 is in almost instantaneous equilibrium with HCO3-, sAC functions as a CO2/ HCO3- (and indirect pH) sensor. This ability to respond to several physiological signals suggests sAC acts as an integrator within signalling pathways and maybe an overlooked regulatory node in Ca2+-transcription coupling.

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