ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies

ARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 contains very low intrinsic GTP binding activity and depends on GEFs (guanine nucleotide exchange factors) such as CYTH3 (cytohesin 3) to bind GTP. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues.The expression of ARF6, ARF1, and CYTH1-4 was investigated by measuring mRNA (using RT-PCR) and protein levels (using immunoblotting) in samples of myometrium obtained from non-pregnant women, and women with normal pregnancies, before or after the spontaneous onset of labour. We also analysed myometrial samples from women with spontaneous preterm labour and from women with complicated pregnancies requiring emergency preterm delivery. The GST)-effector pull down assay was used to study the presence of active ARF6 and ARF1 in all myometrial extracts.ARF6, ARF1 and CYTH3 but not CYTH1, CYTH2 and CYTH4 were expressed in all samples and the levels did not change with pregnancy or labour. However, ARF6 and CYTH3 but not ARF1 levels were significantly reduced in complicated pregnancies. The alterations in the expression of ARF6 and its GEF in human myometrium indicate a potential involvement of this signalling system in modulating the response of myometrial smooth muscle in complicated pregnancies. The levels of ARF6-GTP or ARF1-GTP did not change with pregnancy or labour but ARF6-GTP levels were significantly decreased in women with severe complications of pregnancy.We have demonstrated a functional ARF6 system in human myometrium and a correlation between ARF6 level and activity in uterine and abnormal pregnancy

Regulation of expression of A-kinase anchoring proteins in rat granulosa cells

FSH action on granulosa cells involves the generation of cAMP and subsequent activation of the cAMP-dependent protein kinase (PKA). The PKA holoenzyme is targeted to specific subcellular sites through the interaction of the regulatory subunits with A-kinase anchoring proteins (AKAPs). We previously reported that FSH regulates expression of AKAPs. In this report we examine the relationship between AKAP expression and cell shape. Granulosa cells cultured in the absence of FSH tend to spread and flatten. Cell spreading is accompanied by an increased expression of a 140-kDa AKAP. This spreading/flattening phenotype is independent of the specific extracellular matrix proteins (fibronectin, polylysine, and gelatin) on which cells are plated. Addition of FSH prevents both cell spreading and induction of AKAP 140. Culturing cells on poly (2-hydroxyethyl methacrylate), a surface-coating agent that inhibits cell spreading and adhesion, also inhibits expression of AKAP 140. Addition of phorbol myristate acetate, an agent known to antagonize FSH actions, blocks FSH regulation of both cell shape and AKAP 140 expression. Addition of dexamethasone plus FSH causes a synergistic increase in progesterone levels but has no effect on cell shape or induction of AKAP 140. Dexamethasone produces a dose-dependent increase in AKAP 80 expression, which is blocked by FSH, suggesting cross talk between the glucocorticoid and FSH receptor signaling pathways. These data suggest that expression of AKAP 140 is linked to regulation of cell shape, and that changes in the expression of AKAPs are regulated by several different signaling pathways

Cloud point extraction for analysis of antiretrovirals in human plasma by UFLC-ESI-MS/MS

AbstractAn analytical methodology based on cloud point extraction (CPE) coupled to Ultra-Fast Liquid Chromatography and electrospray tandem mass spectrometry (UFLC-MS/MS) was developed for analysis of Abacavir (ABC), Efavirenz (EFV), Lamivudine (3 TC) and Nelfinavir (NFV) in human plasma. It is the first time that CPE was used for extraction of antiretrovirals (ARV) from plasma. The effects of relevant physic-chemical variables on analytical response of each ARV, including pH, surfactant concentration, equilibration time and temperature, were study and optimized; as well as its coupling to UFLC-ESI-MS/MS. Under optimized conditions, the resulting methodology was as follows: a 500 μL aliquot of human plasma was diluted with 2 mL deionized water in a 10 mL centrifuge tube. A 500 μL aliquot Triton X-114 5% w/v was added and homogenized using a vortex stirrer. The resulting cloudy solution was kept at 65 °C for 20 min for promoting the condensation of surfactant micelles. Then it was centrifuged at 3000 × g for 5 min for separation of the surfactant-rich phase. After discarding the aqueous supernatant, 400 μL ACN were added to the remaining surfactant rich phase and centrifuged in order to precipitate proteins and separate them. A 150 μL aliquot of the supernatant was transferred to 2 mL vial and further diluted with 400 μL deionized water. A 30 μL aliquot of the so-prepared solution was injected and analyzed into the UFLC-MS/MS. The method detection limits for ABC, EFV, 3 TC and NFV under optimized conditions were 31, 77, 57 and 21 ng mL−1, respectively. The RSD% for the studied analytes were <15%, except at the LOQ, which were <19%. Recovery values ranged from 81 to 107%. The proposed methodology was successfully applied for the analysis of ABC, EFV, 3 TC and NFV in human plasma within the concentration range of 43–6816, 125–4992, 81–3248 and 49–7904 ng mL−1, respectively. Under optimized working conditions the proposed analytical methodology meets standard requirements of international guidelines, which makes it suitable for pharmacokinetic studies of the four ARV, as well as for therapeutic monitoring of HIV patients

A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes

The luteinizing hormone/choriogonadotropin (LH/CG) receptor (R) is a heptahelical R that, upon agonist binding, activates the stimulatory guanine nucleotide-binding protein (G s ) and the downstream effector adenylyl cyclase (AC). Like other G protein-coupled Rs, the LH/CG R subsequently exhibits reduced agonist-dependent effector activity, or desensitization, in response to saturating agonist. Unlike desensitization of many other G protein-coupled Rs, the in vivo desensitization response of LH/CG R-stimulated AC activity of ovarian follicles to the preovulatory surge of LH can be mimicked under cell-free conditions. Based on evidence that porcine ovarian follicular membranes unexpectedly contained β-arrestin-1, the role of arrestins in desensitization of the LH/CG R was investigated. Results showed that neutralizing arrestin antibodies blocked the development of desensitization and that desensitization was rescued with a synthetic peptide corresponding to the antibody-binding epitope on β-arrestin-1. These results suggest that endogenous β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R. Addition of recombinant purified β-arrestin-1 mimicked human chorionic gonadotrophin to promote desensitization of human chorionic gonadotrophin-stimulated AC activity, in the presence of the ATP phosphorylation antagonist adenylyl-imidodiphosphate, with an ED 50 of ≈0.1 nM. Increased levels of an 87-kDa protein reactive with glycoprotein hormone R-reactive antibody, consistent with the LH/CG R, coimmunoprecipitated with follicular membrane β-arrestin-1 in response to LH/CG R activation compared with unactivated R. Taken together, these results show that ovarian follicles contain membrane-associated β-arrestin-1, that β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R, and that the trigger for β-arrestin-1 binding to the LH/CG R appears to be R activation