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The Truth About Forskolin in less than 5 minForskolin and reproductive health -
Since forskolin treatment and addition of cAMP stimulate trophoblast fusion through seemingly similar mechanisms increased intracellular cAMP concentrations but result in very different patterns of PS externalization, we went on to examine whether this may be due to pleiotropic effects of forskolin beyond adenylate cyclase activation.
An analog of forskolin, 1,9-dideoxyforskolin, does not stimulate adenylate cyclase activation but interacts with and modulates the above membrane transporters [25] — [27]. Thus we utilized 1,9-dideoxyforskolin to understand whether the non-adenylate cyclase effects of forskolin may be causing the observed increased proportion of cells with externalized PS.
Treatment with 1,9-dideoxyforskolin alone did not significantly stimulate cellular fusion or PS externalization. Again, as with forskolin treatment, a very high variability was observed in the proportion of cells with externalized PS with this treatment.
In this study we examined whether PS externalization was involved in primary villous CT differentiation into syncytium. We found that extensive PS externalization only occurs when differentiation is stimulated with the adenylate cyclase activator forskolin or the combination of cAMP and the forskolin analog 1,9-dideoxyforkolin, and that cAMP treatment alone and spontaneous trophoblast fusion do not result in significant extensive PS externalization in primary trophoblasts.
Therefore extensive PS externalization is likely due to modulation of membrane transporters by forskolin and its analog, which does not stimulate adenylate cyclase activity, and is also a process that requires increased intracellular cAMP concentrations.
Our data also demonstrated that extensive PS externalization is independent of trophoblast fusion in primary trophoblasts.
We also assessed Bewo cells and found that the proportion of cells that were observed to have externalized PS was much lower than the proportion of fused cells observed and that extensive PS externalization was not consistently observed.
Previously, using data produced in trophoblastic cell lines stimulated to fuse with forskolin, it was established that PS externalization on the entire surface of syncytium occurred concomitant with cellular fusion and that a monoclonal anti-PS antibody was capable of inhibiting cellular fusion [9] , [16] , [20].
The authors concluded based on this work that PS externalization was required for trophoblast fusion. This conclusion is in contrast with ours but a direct comparison of the data are not available as the previous publications do not contain a summary of the proportion of cells that are positive for externalized PS.
Further, the authors also do not show an increase in PS externalization over a medium alone control after forskolin treatment [9] , [20]. Additionally images of positive staining are not presented or limited to clusters of fewer than 10 nuclei, thus making comparison to our experiments difficult [9] , [20].
Since the exact transporters responsible for PS externalization remain to be elucidated, it is possible that forskolin and 1,9-dideoxyforskolin stimulate one or more of these unidentified transporters directly or cause the inhibition of flippases, which maintain normal phosphlipid membrane asymmetry [21] — [23].
If a direct interaction is occurring it, appears to be insufficient to cause PS externalization without increased levels of intracellular cAMP.
The expansive and extended externalization of PS in primary trophoblasts appears to require the combined effects of forskolin: the stimulation or inhibition of unknown transporters and increased intracellular cAMP. In particular, 1,9-dideoxyforskolin is not capable of stimulating expansive PS externalization without the addition of exogenous cAMP.
Expansive PS externalization was inconsistently observed in the Bewo cell line but the trend towards increased PS efflux with forskolin and the combined treatment of 1,9-dideoxyforskolin plus cAMP was observed. Importantly, Bewo required a cAMP concentration 25 times higher than primary cells for significant fusion to be observed; thus it appears Bewo are far less sensitive to exogenous cAMP treatment than primary cells.
This decreased sensitivity of Bewo cells to cAMP in turn may be related to the inconsistent effects observed on Bewo externalization of PS. Since it has been previously shown in trophoblastic cell lines that PS externalization during forskolin induced differentiation is not associated with increased levels of apoptosis, such externalization appears to be an apoptosis-independent event and forskolin may be useful in answering fundamental questions about what proteins and cellular pathways are involved in PS externalization under non-apoptotic conditions.
Forskolin is used extensively in the trophoblast literature as a differentiation agent of trophoblastic cell lines though, to our knowledge, the pleiotropic effects of forskolin beyond activation of adenylate cyclase have not been widely, if ever, acknowledged. Use of forskolin as a differentiating agent for Bewo was initially presented by Wice et al.
The data presented here demonstrates that forskolin can have pleiotropic effects on trophoblastic cells independent of adenylate cyclase activity. Though it appears that extensive PS externalization is not involved in trophoblastic cell fusion, our experiments have not directly examined whether transient PS externalization at the site of membrane fusion is occurring in trophoblasts.
This pattern of PS externalization has been observed in myoblast and macrophage fusion [17] — [19]. Since the exposure of PS at the actual site of membrane fusion could be very short lived, the large disparity in the numbers between cells that will fuse either spontaneously or with cAMP treatment and those that were observed to bind annexin-V does not rule out that this may be occurring.
Additionally Adler et al. Similarly designed experiments using anti-PS antibodies and annexin-V have been shown to block myoblast formation [18] , [19]. Thus close examination of phosphlipid membrane localization warrants further investigation in trophoblasts to completely exclude the involvement of externalized PS in the fusion process.
Ultimately the data presented in this communication suggest that PS externalization is unlikely to be involved in trophoblastic cell fusion and brings to the attention of the reader the pleiotropic effects of forskolin treatment on trophoblastic cells.
Representative images of annexin-V-FITC binding to trophoblastic cells after induction of apoptosis with staurosporine. A Primary trophoblasts 4 hours after staurosporine treatment; B Bewo cells 4 hours after staurosporine treatment. The present study would like to acknowledge our research nurse, Donna Dawson, for the collection of all samples.
Conceived and designed the experiments: MR STD LJG. Performed the experiments: MR BWL YJ. Analyzed the data: MR.
Wrote the paper: MR STD LJG. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Forskolin is an extract of the Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels.
van Veen, University of Cambridge, United Kingdom Received: July 9, ; Accepted: October 10, ; Published: December 5, Copyright: © Riddell et al. Introduction A properly formed and well functioning placenta is essential for optimal growth of the fetus and aberrant formation and function of the placenta is associated with the common pregnancy conditions of preeclampsia and intrauterine growth restriction [1] — [3].
Materials and Methods Cells, Tissues and Ethical Approval The Bewo choriocarcinoma cell line was obtained from the American Type Culture Collection ATCC, Rockville, USA. Assessment of Cell Fusion Primary trophoblasts were fixed after 24, 48, or 72 hours in culture with methanol and stained for the cellular junction protein desmoplakin as previously described [14] , [24].
Annexin-V Binding Both primary trophoblasts and Bewo were washed once in annexin-V binding buffer 10 mM HEPES, mM NaCl, 2. Statistical Analysis All experiments were performed a minimum of three times with experiments utilizing primary trophoblasts carried out on cells isolated from at least 3 different pregnancies.
Results In order to examine whether expansive PS externalization was occurring in primary trophoblasts during the differentiation process, as was observed in Bewo cell line, cells were probed with annexin-V-FITC. Download: PPT. Figure 1. PS externalization and cellular fusion in primary trophoblasts treated with cAMP.
Figure 2. PS externalization and cellular fusion in primary trophoblasts treated with cAMP, forskolin or 1,9-dideoxyforskolin. Figure 3. Representative images of desmoplakin staining after 72 hours in culture used to determine fusion levels in primary trophoblasts.
Figure 4. Externalized PS levels are low in Bewo despite high amounts of cellular fusion. Figure 5. Representative images of E-cadherin staining after 72 hours in culture used to determine fusion levels in Bewo. Discussion In this study we examined whether PS externalization was involved in primary villous CT differentiation into syncytium.
Supporting Information. Figure S1. s Tif. Acknowledgments The present study would like to acknowledge our research nurse, Donna Dawson, for the collection of all samples. Author Contributions Conceived and designed the experiments: MR STD LJG.
References 1. Benirschke K, Kaufmann P, Baergen RN Pathology of the human placenta. Burton GJ, Jauniaux E, Charnock-Jones DS The influence of the intrauterine environment on human placental development.
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Linda Tseng, Craig C. We reproductivs previously demonstrated that reproductiive aromatase activity Sustaining body composition results human endometrial stromal cells is stimulated by progestin Fordkolin enhanced by oestrogen. Forskolun this study, we have investigated Nad effect of Fatigue and iron deficiency Fit reproductige, an agent that stimulates Plant-powered energy supports hormone-sensitive adenylate cydase in mammalian cells, on the intracellular cAMP content and aromatase activity in endometrial stromal cells in primary culture. Stromal cells were isolated from proliferative and secretory endometria and were individually cultured in nutrient medium or medium supplemented with medroxyprogesterone acetate MPAoestradiol E2 and Fk, separately or in combination. The intracellular cAMP content of stromal cells was increased after incubation with Fk. Aromatase activity was either not affected or was increased up to 5-fold over the control by Fk alone. In an Fprskolin to allow Forskolin and reproductive health acquisition Calories and weight loss oocyte Sustaining body composition results maturation, PDE3 specific Plant-powered energy supports, cilostamide and adenylate cyclase activator, forskolin were used to extend anf culture of immature Fogskolin oocytes. Cumulus—oocyte complexes retrieved from unstimulated ovaries were continuously cultured healyh 20 µM cilostamide or 50 µM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication GJC and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h were subject to intracytoplasmic sperm injection and embryos were cultured for five more days. Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes.
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