The aim of the study was to evidence replicative senescence‐induced changes in human amniocytes via flow cytometry, quantitative reverse‐transcription‐polymerase chain reaction (qRT‐PCR) and automated/manual patch‐clamp. Both cryopreserved and senescent amniocytes cultured in BIO‐AMF‐2 medium featured high percentages of pluripotency cell surface antigens SSEA‐1, SSEA‐4, TRA1‐60, TRA1‐81 (assessed by flow cytometry) and expression of pluripotency markers Oct4 (Pou5f1) and Nanog (by qRT‐PCR). We demonstrated in senescent vs cryopreserved amniocytes decreases in mesenchymal stem cell surface markers. Senescence‐associated β‐galactosidase stained only senescent amniocytes, and they showed no deoxyuridine incorporation. The gene expression profile revealed a secretory phenotype of senescent amniocytes (increased interleukin (IL)‐1α, IL‐6, IL‐8, transforming growth factor β, nuclear factor κB p65 expression), increases for cell cycle‐regulating genes (p16INK4A), cytoskeletal elements (β‐actin); HMGB1, c‐Myc, Bcl‐2 showed reduced changes and p21, MDM2 decreased. Via patch‐clamp we identified five ion current components: outward rectifier K+ current, an inactivatable component, big conductance Ca2+‐dependent K+ channels (BK) current fluctuations, Na+ current, and inward rectifier K+ current. Iberiotoxin 100 nmol/L blocked 71% of BK fluctuations, and lidocaine 200 μmol/L exerted use‐dependent Na+ current block. Transient receptor potential (TRP)M7‐like current density at −120 mV was significantly increased in senescent amniocytes. The proinflammatory profile acquired by senescent amniocytes in vitro may prevent their use in clinical therapies for immunosuppression, antiapoptotic and healing effects.
However, retigabine validated opening of the hK v 7.2/7.3 channel as an antiepileptic strategy. Our aim is to develop selective hK v 7.2/7.3 channel openers with antiepileptic effects. We have developed derivatives of naturally occurring resin acids, some of them as potent as retigabine. The derivatives open the hK v 7.2/7.3 channel by an electrostatic mechanism, acting on the voltage sensor. This site of action is different from the retigabine-binding site and makes it possible to gain selectivity between hK v 7.2/7.3 and hK v 7.4. Small structural alterations of our derivatives had different effects on hK v 7.2/7.3 and hK v 7.4. To investigate potential off-target effects we tested our compounds on the cardiac hERG and hNa v 1.5 channels. At 10 mM, none of the compounds blocked the current nor shifted the voltage dependencies of the hERG or the hNa v 1.5 channel. To investigate the antiepileptic effect, we induced epileptic seizures in zebrafish larvae by a chemoconvulsant (PTZ) and recorded the electrical brain activity with extracellular electrodes. The epileptic seizures were prevented by retigabine (at 10 mM), and our potential lead compounds had similar effects at the same concentration. By opening the hK v 7.2/7.3 channel and preventing epileptic seizures in an in vivo model, our resin-acid derivatives thus show the potential to be developed into a new type of antiepileptic drug.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.