Considering these reports and with the aim
Considering these reports, and with the aim of further investigating the mechanism by which the cAMP-Epac/PKA pathway activates eNOS, we have performed imaging experiments evaluating the effect of drugs that increase cAMP or modify its signalling pathways (PKA or Epac activators and inhibitors) on basal [Ca2+]c levels and NO release in human umbilical vein endothelial cells (HUVEC). We have also measured the implication of cAMP signalling in the phosphorylation of eNOS at Ser 1177. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) experiments were performed to check for the presence of important elements of cAMP signalling pathways in our cells. Finally, we have also performed contraction-relaxation studies in isolated rat thoracic Chlorpropamide synthesis rings intact or deprived of endothelium to explore a functional correlation to our results using cells.
Materials and methods
Discussion We have previously reported that cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity . According to this, in the present work, the NO release induced by forskolin, an adenylyl cyclase activator that significantly elevates cAMP in HUVEC , was reduced in the presence of the eNOS inhibitor L-NAME. It is generally accepted that [Ca2+]c rises within endothelial cells control many processes that participate in the regulation of vascular tone, including eNOS activation , . Since agents that elevate intracellular cAMP may cause an increase in [Ca2+]c in vascular smooth muscle , ,  we thought that a similar effect in endothelial cells could participate in the activation of eNOS. To verify this possibility, we have first carried out imaging experiments to monitor basal [Ca2+]c variations in response to cAMP-elevating agents. In our experiments with isolated HUVEC, both forskolin and db-cAMP, a cell-permeant cAMP analogue, induced a significant increase in basal [Ca2+]c, but only in ∼30% and ∼25% of the cells, respectively. Selective activation of PKA with 6-Bnz-cAMP  or Epac with 8-pCPT-2′-O-Me-cAMP ,  did not significantly modify basal [Ca2+]c. However, a combined activation of both proteins did significantly increase [Ca2+]c (∼18% of the cells). A similar synergic effect was also described in vascular myocytes . These results seem to indicate that eNOS activation could take place, at least in part, by a modification of endothelial Ca2+ signalling mediated by a joint activation of Epac and PKA. However, results in HUVEC monolayers do not support this hypothesis, since neither cAMP-elevating agents nor the joint activation of Epac and PKA significantly increase [Ca2+]c in these conditions, suggesting that the low% of isolated HUVEC responding is not enough to induce a measurable [Ca2+]c increase in a population of cells. In any case, it should be also borne in mind that, in both cases, our experimental conditions may be insufficient to detect small increases in [Ca2+]c, especially if they occur in highly specific cell areas rich in eNOS, such as caveolae . In spite of the differences found here between isolated cells and monolayers when measuring [Ca2+]c, these differences do not exist between NO measurements performed in both conditions. In fact, we had reported a significant increase in NO generation induced by cAMP-elevating agents in isolated HUVEC under the same conditions used for fura-2 Ca2+ measurements in the present study . To further study the mechanism of eNOS activation by cAMP, we have measured eNOS phosphorylation at Ser 1177 residue, which is considered an indicator of eNOS activation in human endothelial cells and that can be induced by a rise of [Ca2+]c , , . Our results have shown that forskolin increases eNOS phosphorylation at Ser-1177 without increasing total eNOS expression, an effect reproduced by db-cAMP or 6-Bnz-cAMP. In concordance, the flavonoid quercetin phosphorylates eNOS at Ser-1177 through the cAMP/PKA pathway, thus increasing NO production and vasorelaxation . Also, both cilostazol, a selective inhibitor of phosphodiesterase type 3 (PDE3) that increases cAMP, and forskolin increase NO production by eNOS phosphorylation of Ser-1177 and dephosphorylation at Thr-495 via stimulation of the cAMP/PKA and PI3K/Akt pathways in human aortic endothelial cells . In addition, phosphorylation of eNOS on Ser-1177 or Ser-633 residues by PKA may increase eNOS activity  and activated PKA following an increase in cAMP may phosphorylate eNOS resulting in an increased activity . All these results suggest that PKA is involved in the activation of eNOS during cAMP-induced endothelium-dependent relaxation. In good agreement, 6-Bnz-cAMP induced here a significant NO release from HUVEC, and the forskolin-induced NO release was reduced by Rp-cAMPs, a selective PKA inhibitor . As a previous step, we carried out RT-PCR experiments, which demonstrated the expression of the PKA regulatory subunit RII (PKA-RIIα) in our cells.