Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.Increasing evidence suggests that apoptosis of endothelial cells (EC)³ may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4–7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion.In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8, 9). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation.Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10). Because mitochondria play a fundamental role in apoptosis (11), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis.