Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach

Abstract

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant (K d ) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS− rhodamine conjugate by sildenafil; the K i values were 3.6 ± 0.3 nM (IC 50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC 50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

β-arrestin 2 Is a Prognostic Factor for Survival of Ovarian Cancer Patients Upregulating Cell Proliferation

“…Although this study had limitations related to low specificity of the anti-gonadotropin receptor antibodies employed ( 62 ), it suggests that hCGβ and its receptor might be linked to survival in these patients. These data would be in line with the relatively high potency of hCGβ in inducing LHCGR-mediated intracellular cAMP increase ( 27 ), as an effect linked to the steroidogenic potential of the hormone and to pro-apoptotic effects ( 24 ). On the other hand, these data would be in contrast with the fact that hCGβ has been used as a marker of certain tumors, such as the choriocarcinoma ( 63 ), which reflects the involvement of β-arrestin 2 in the hCGβ-mediated intracellular signaling ( 29 ) and suggests that the connection between the two molecules in cancer merits further investigations.…”

“…Although this study had limitations related to low specificity of the anti-gonadotropin receptor antibodies employed (62), it suggests that hCGβ and its receptor might be linked to survival in these patients. These data would be in line with the relatively high potency of hCGβ in inducing LHCGRmediated intracellular cAMP increase (27), as an effect linked to the steroidogenic potential of the hormone and to proapoptotic effects (24). On the other hand, these data would Brightness/contrast of Western blotting pictures were adjusted uniformly in all panels.…”

“…Cell lines were maintained in an incubator at 37 • C and 5% CO 2 . The transfection protocol was described previously (25)(26)(27)(28). Briefly, 3.5 × 10 4 cells were transiently transfected in a 96well plate using increasing amount (range: 0-200 ng/well) of β-arrestin 2-encoding pcDNA3.1 plasmid (29) or commercial siRNA probes against β-arrestin 2 (#14387; Life-Technologies, Carlsbad, CA, USA) (21), using an electroporator (Gene Pulser MXcell; Bio-Rad Laboratories Inc., Hercules, CA, USA) in accordance with established protocol and settings (21,30).…”

GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors

“…GnRH-induced intracellular cAMP and Ca 2+ increase was assessed by the BRET technique in biosensor- and GnRHR-expressing cells, as previously described [57,59,61,62], in the presence or in the absence of GnRH antagonist. To evaluate Ca 2+ increase, cells were incubated in Hank’s Balanced Salt Solution (HBSS) with Ca 2+ and Mg 2+ , 1 mM Hepes and 5 µM Coelenterazine H (NanoLight Technologies, a division of Prolume, Ltd., Pinetop, AZ, USA), for 45 minutes, together with Cetrorelix, Ganirelix, Teverelix or vehicle, as appropriate.…”

“…BRET experiments were performed using a CLARIOstar plate reader equipped by a monocromator (BMG Labtech, Ortenberg, Germany), as previously described [56,59,60,61,62,63].…”