Citation
- Authors: Yart L. et al.
- Year: 2022
- Journal: J Cell Physiol 237 1980-1991
- Applications: in vitro / siRNA / INTERFERin
- Cell type: PHM1-41
Method
The genes encoding for the SOCE channels from the Orai and TRPC families were downregulated in PHM1‐41 cells using siRNA to investigate their implication in the nifedipine response. PHM1‐41 cells were seeded in a complete culture medium in black wall 96‐well plates with a clear bottom (7.5 × 103 cells/well) and in 6‐well plates (1.25 × 105 cells/well) for Ca2+ assay and RT‐qPCR analysis, respectively.
Twenty‐four hours after seeding, transfection of 10 nM of either siOrai1 (Microsynth), siOrai2 (Microsynth), siOrai3 (Microsynth), siTRPC1 (Qiagen), siTRPC4 (Santa Cruz Biotechnologies), siTRPC6 (Santa Cruz Biotechnologies), or control siRNA (siCTL, Microsynth) was carried out using InterferIN transfection reagent (Polyplus transfection). Cells were incubated with the transfection mix for 48 h before processing.
Abstract
Nifedipine, an L-type voltage-gated Ca2+ channel (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine-based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short-lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1-41 that does not express L-VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store-operated Ca2+ entry channels from the Orai and transient receptor potential-canonical (TRPC) families in PHM1-41 cells highlighted the implication of TRPC1 in nifedipine-induced Ca2+ entry. In addition, the use of 2-APB in combination with nifedipine on human myometrial strips tends to confirm that the pro-contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.