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  • Sodium ascorbate On the other hand although


    On the other hand, although the cardiovascular effects of Epac have been investigated more intensely in recent years [31], there are hardly any studies that suggest or discard a participation of Epac in endothelium-dependent cAMP-induced relaxation. Only our previous results in rat Sodium ascorbate contractility and imaging in HUVEC [6], along with those obtained by Roberts et al. [32] in rat mesenteric arteries suggest that the endothelial component of cAMP-induced relaxation may be partially mediated by Epac activation. To confirm a possible involvement of Epac in eNOS activation, we have previously studied its expression in HUVEC, as well as that of its main effector Rap 1 protein. Our RT-PCR experiments demonstrate the presence of a cDNA band for Epac-1 and Rap 1, but not for Epac-2. Next, we measured the effects of the 8-pCPT-2′-OMe-cAMP on eNOS phosphorylation in HUVEC. As with PKA, selective activation of Epac did phosphorylate eNOS at Ser-1177. These results suggest that Epac may activate eNOS, a hypothesis supported by our imaging experiments in HUVEC monolayers, in which 8-pCPT-2′-O-Me-cAMP induces NO release, and ESI-09, a membrane-permeant Epac inhibitor [33], attenuates forskolin-induced NO generation. To verify or discard Ca2+-dependence of cAMP-induced NO release, we measured NO release by HUVEC in total absence of Ca2+. The results obtained do not support increase in [Ca2+]c as a necessary event for the generation of NO by cAMP. Two hypotheses could explain these results: i) cAMP induced [Ca2+]c rise may be not of enough intensity to generate a significant NO increase; ii) in the total absence of Ca2+, there are compensatory mechanisms that activate eNOS by different pathways. In this connection, we have studied the participation of Ca2+/calmodulin-CaMKII pathway using KN 93, a selective inhibitor of CaMKII. This agent did not significantly modify forskolin-induced NO release, suggesting that there is no activation of this pathway by forskolin in HUVEC, and that cAMP-induced [Ca2+]c increases may be uncoupled to eNOS. At this point, it should be remembered that Ser 1177 can also be phosphorylated by several kinases without an increase in [Ca2+]c [21]. Several authors have shown that phosphoinositide 3-kinase (PI3K)/Akt and AMPK are also involved in Ser 1177 eNOS phosphorylation induced by cAMP-elevating agents [28], [34], [35]. In our experiments, PI3K inhibition with LY-294,002 [36] significantly reduces forskolin-induced NO release in HUVEC, suggesting an implication of this kinase in cAMP-mediated eNOS activation and the subsequent endothelium-dependent vasorelaxation. Accordingly, Ferro et al. [4] demonstrated the implication of this pathway in isoprenaline-induced NO generation in rat aorta. Also, LY294,002 inhibition of NO release is enhanced by Rp-cAMPs (data not shown), probably because PI3K/Akt pathway acts independently of PKA. Since Epac participates in forskolin-induced NO release, this protein could activate the PI3K/Akt pathway resulting in the phosphorylation of eNOS Ser 1177, as suggested by Zieba et al. [37]. However, further experiments would be necessary to confirm this possibility. On the other hand, Compound C, a selective inhibitor of AMPK [38], did not significantly modify forskolin-induced NO generation in HUVEC, ruling out a participation of this kinase in this process. Taken together, our results suggest that both cAMP effectors Epac and PKA may directly activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway and independently of AMPK or CaMKII activation or a [Ca2+]c increase. This action explains, at least in part, the endothelium-dependent vasorelaxant effect of cAMP. Our study provides interesting data on how cAMP regulates vascular physiology and on the possible use of new molecules that act on cAMP regulation and/or signalling as potential therapeutic agents in cardiovascular diseases such as hypertension, heart failure or cardiac ischemia.