eNOS activity has been considered for many years

eNOS activity has been considered for many years to be dependent on cytoplasmic Ca2+ ([Ca2+]c) rise and the subsequent binding of the Ca2+/CaM complex to the enzyme [12]. However, eNOS can be activated by different stimuli, the most important being activation of Akt by shear stress, without an increase in [Ca2+]c [8], [10], [13]. 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 Amphotericin B australia rings intact or deprived of endothelium to explore a functional correlation to our results using cells.
Materials and methods
Results
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 [6]. According to this, in the present work, the NO release induced by forskolin, an adenylyl cyclase activator that significantly elevates cAMP in HUVEC [20], 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 [9], [21]. Since agents that elevate intracellular cAMP may cause an increase in [Ca2+]c in vascular smooth muscle [1], [22], [23] 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 [5] or Epac with 8-pCPT-2′-O-Me-cAMP [24], [25] 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 [1]. 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 [26].