Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens

Abstract

Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

Dynamic Expression, Differential Regulation and Functional Diversity of the CNGC Family Genes in Cotton

“…Adenylate cyclase is the sole enzyme responsible for the cellular production of cAMP, which stimulates cAMPdependent protein kinase A (PKA) to phosphorylate cAMP response-element bindingprotein (CREB), and subsequently activates the transcription of a variety of target genes, resulting in multiple physiological functions [81]. Both DDA and forskolin are commonly used in biological process or pathway studies involving adenylyl cyclase activity and cAMP pool modulation, whereas 8-Br-cAMP is often used to imitate cAMP activation agents, both in plants and animals [73,[82][83][84]. Based on our RNA sequencing results, 13 GhCNGCs showed differential expression at the threshold of an absolute value of log2(fold change) > 1 and adjusted p < 0.05 under treatments of DDA or forskolin, or 8-Br-cAMP during ovule culture (Table 1), which included GhCNGC39 being the closest homolog of AtCNGC16 that has been found to be critical for pollen tube growth by specifically impacting cell walls or membrane dynamics [76].…”