OXYTOCIN-STIMULATED RESPONSES IN A PREGNANT HUMAN IMMORTALIZED MYOMETRIAL CELL-LINE

Smooth muscle cells isolated from the myometrium of a pregnant woman a t term were infected with a replication-defective adenovirus vector ex pressing the E6/E7 proteins of human papilloma virus 16. A clonal line , PHM1-41, was selected by resistance to Geneticin and examined for ma intenance of smooth muscle phenotype and response to oxytocin. The imm ortalized cell line retained morphological characteristics of prolifer ating smooth muscle cells in culture for up to 22 passages and has bee n used for over 2 years. The cells expressed smooth muscle-specific al pha-actin and retained estrogen receptors. Oxytocin receptors were pre sent, as measured by whole cell binding assay using the oxytocin antag onist I-125-d(CH2)(5)[Tyr-(Me)(2), Thr(4), -Orn(8), Tyr(9)-NH2] as lig and and oxytocin as competitor. The data were best described by a one- site binding model, with a K-d of 0.36 nM and a binding site concentra tion of 37 fmol/mu g DNA. PHM1-41 cells responded to oxytocin with an increase in intracellular free calcium (EC(50) 15 nM) and an increase in phosphatidylinositol turnover. Oxytocin-stimulated phosphatidylinos itol turnover was inhibited by preincubation with the cAMP analog CPT- cAMP. This immortalized myometrial cell line should prove useful for s tudies relating to human myometrial function.