br Transparency document br Introduction The cyclic

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Introduction The cyclic nucleotide adenosine 3′,5′-cyclic monophosphate (cAMP) exerts both an endothelium-dependent and an endothelium independent vasorelaxant action in rat AG957 [1]. cAMP direct, endothelium-independent vasorelaxant effects have been attributed to several mechanism, such as a reduction of cytoplasmic Ca concentration ([Ca]c), a minor sensitivity of contractile filaments to [Ca]c, or an opening of several types of K+ channels (for detailed review see, i.e., Morgado et al. [2]). However, despite the large number of studies, cAMP-[Ca]c crosstalk in vascular myocytes is still not fully understood. Using endothelium-deprived rat aorta, we have previously shown that nanomolar concentrations of the selective adenylyl cyclase activator forskolin, and the subsequent increase in the intracellular levels of cAMP ([cAMP]i), inhibit the contractions induced by phenylephrine in a Ca-free external solution and the Ca-induced contraction after intracellular stores depletion with thapsigargin or phenylephrine [3]. These results suggest, but do not prove, that the mechanism of cAMP-induced vasorelaxation could consist, in part, of a lowering of Ca release from intracellular Ca stores and a reduction of the following transmembrane store-operated Ca entry (SOCE) in rat aortic smooth muscle cells. In the last decade, research on the cAMP target Epac (exchange protein activated by cAMP), have demonstrated several roles for cAMP which are not mediated by the activation of its traditional effector cAMP-dependent protein kinase (PKA) [4], [5], [6]. Several authors have described the ability of Epac activators to directly relax vascular and non-vascular smooth muscle preparations [5], [7]. Also, it has been reported that activation of Epac induces endothelial-dependent relaxation in rat mesenteric arteries [8]. In a previous work, we have demonstrated that Epac exerts a direct vasorelaxant effect on vascular myocytes and that the endothelium-dependent vasorelaxant effect of cAMP may also be mediated in part by activation of this protein [1]. However, despite the great number of studies on this subject, the specific roles of PKA and Epac have not been extensively investigated in vascular smooth muscle relaxation [6], [9]. This work was conducted in order to further study the effects of cAMP-elevating agents on Ca signalling in rat aortic smooth muscle cells (RASMC). We also pretend to clarify the role of the two main cAMP-targets, PKA and Epac in the crosstalk between Ca and cAMP. For this purpose, we have performed contraction-relaxation studies in isolated rat thoracic aorta rings deprived of endothelium and [Ca]c imaging experiments in isolated RASMC. This work have shown, for the first time, that activation of PKA and Epac is involved in the cAMP-induced depletion of Ca from thapsigargin-sensitive intracellular reservoirs in vascular myocytes, thus reducing the amount of Ca released by a subsequent administration of an agonist. Also, both proteins contribute to cAMP-induced vascular smooth muscle relaxation by SOCE inhibition.
Materials and methods
Results
Discussion In the present study, the selective adenylyl cyclase activator forskolin, at concentrations that increases [cAMP]i in RASMC [3], induced a slow, significant rise of basal [Ca]c in the absence of extracellular Ca. This effect was potentiated by inhibition of cyclic nucleotide phosphodiesterases with rolipram and milrinone or IBMX, and reproduced by db-cAMP, a membrane-permeable cAMP analogue, suggesting that enhanced [cAMP]i releases Ca from intracellular stores. The cAMP-induced increase of basal [Ca]c in vascular myocytes has already been reported [14], [15], and related to the activation of L-type voltage-operated Ca channels [16], [17]. However, this is the first study to demonstrate that this increase is mediated in part by depletion of intracellular Ca reservoirs in smooth muscle cells. A cAMP-mediated release of stored Ca has been already reported in other biological models, such as human T lymphocytes [18], hippocampal neurons [19] or the soil-dwelling amoeba Dictyostelium discoideum [20].