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  • In SLO permeabilized human sperm the AR elicited by


    In SLO-permeabilized human sperm, the AR elicited by calcium, persistently active Arf6 [41] and Rab3A, 8-pCPT-2′-O-Me-cAMP [8], diacylglycerol and a non-hydrolyzable analog [35], is sensitive to the PLC blocker U73122. Furthermore, 8-pCPT-2′-O-Me-cAMP elicits a calcium signal in non-permeabilized sperm; this signal is abrogated by U73122 [8]. Here, we moved several steps forward and identified the presence and localization of the ε isoform of PLC in human sperm (Fig. 6A). PLCε is required for the AR triggered by calcium and 8-pCPT-2′-O-Me-cAMP (Fig. 6B, C). Furthermore, 8-pCPT-2′-O-Me-cAMP failed to mobilize intra-acrosomal calcium in sperm treated with U73122 (Fig. 7C) or anti-PLCε MK2 Inhibitor IV (Fig. 7D). Likewise, experiments conducted with pancreatic islets from PLCε−/− mice demonstrated the requirement of this enzyme downstream of Epac2/Rap1 for the potentiation of glucose-induced insulin release and intracellular calcium mobilization [18], [19]. There is also pharmacological evidence of the requirement of a PLC for Epac1/Rap2-regulated chlorine secretion in human intestinal epithelial cells [24]. Thus, it is tempting to interpret the relationship between Epac, Rap1, and PLCε as molecular links between the two universal second messengers calcium and cAMP.
    Conclusions Our findings indicate that Epac activation by cAMP leads to the exchange of GDP for GTP on Rap1 in the acrosomal region of the sperm, with the subsequent recruitment and/or activation of a PLCε activity. This enzyme hydrolyzes PIP2 to generate IP3, which binds IP3-sensitive channels and promotes the release of calcium stored in the acrosomal granule. cAMP also mediates the activation of endogenous Rabs 3 and 27, mandatory events in the exocytotic cascade. The efflux of calcium from the acrosome and the assembly of the fusion machinery converge to accomplish the fusion of the acrosome to the plasma membrane (Fig. 8). The contribution of this paper to the sperm biology field is the characterization of signaling pathways through which the physiological trigger progesterone achieves the AR; of particular relevance is the finding that it requires endogenous cAMP and three small GTPases. The contribution of this paper to the exocytosis field is to have generated direct evidence for the role of each and every component of the signaling module cAMP/Epac/Rap1/PLCε in dense-core granule secretion. The following are the supplementary data related to this article.
    Funding This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT2010-0342, Argentina) and Secretaría de Ciencia y Técnica-Universidad Nacional de Cuyo (grant number 06/J416, Argentina) to C.N.T.
    Conflict of interest
    Transparency document
    Introduction In healthy vessels, VSMC exhibit very low rates of proliferation [1]. However, VSMC proliferation rates are dramatically elevated in response to vessel injury. Mechanisms responsible for MK2 Inhibitor IV this include release of growth factors and remodelling of the vascular extracellular matrix [2], [3] that activate cell signalling pathways required for cell-cycle progression. VSMC proliferation is also subject to negative regulation [4]. cAMP, a major second messenger produced by adenylate-cyclase has a well documented role as an inhibitor of VSMC proliferation. cAMP synthesis in response to endothelial-derived prostacyclin is an important mechanism promoting VSMC quiescence and vascular healing following injury. For example, elevated cAMP levels have been shown to inhibit VSMC proliferation in vitro and after vascular injury-induced proliferation in vivo, ultimately leading to a reduction in restenosis [4], [5]. The mechanisms underlying these growth-inhibitory properties are only partially understood. Elevated cAMP levels inhibit expression of the G1-S phase cell-cycle regulators, Cyclin D and Skp2 [4], [6], which accounts at least in part for cAMP-mediated G1-arrest and reduction in neointima development [7], [8], [9]. However, upstream signalling pathways responsible for these important biological effects of cAMP remain unclear.