Abstract
The expression and modulation of myometrial gap junctions remain poorly understood. However, the availability of new molecular and biochemical tools including cDNAs and antipeptide antibodies permits extensive characterization of gap junctions. A myometrial cell culture system was developed for study of regulatory mechanisms governing gap junctional intercellular communication (GJIC). Primary myometrial cells, isolated by progressive enzymatic digestion of the uterine tissue were used to establish cell lines by serial passage and transfection with a pSV3neo plasmid. Steroid hormone assays by radioligand-hormone competition indicated that estrogen receptors were decreased in cell lines compared to primary cells. This limited the utility of cell lines. Primary cultures also expressed high levels of progesterone receptors. The smooth muscle origin of the cells was confirmed by immunofluorescence labelling for desmin, α-smooth muscle actin and myosin. Staining with antibody to α-gap junction protein confirmed the presence of connexin43 in these cultures. Functional and structural characterizations of GJIC were assayed by electron microscopy and a fluorescent recovery after photobleaching assay, gap FRAP. Contrary to the insitu uterus, cultured myometrial cells spontaneously formed gap junctions. Further, estradiol (10^-7M) increased GJIC, while progesterone only suppressed estradiol induced increases. Indomethacin treatment indicated that prostaglandins increased GJIC. In vivo, cAMP and its analogs decrease myometrial GJIC, in contrast, increases in GJIC was observed in cultured myometrial cells. Using a paradigm in which octanol was used to reduce GJIC to low levels, application of cAMP and its anologs caused rapid increases in GJIC. Similar results were obtained with SIGC and Clone 9 cells which also express connexin43. The rapidity of modulation by cAMP, within minutes, suggests gating of channels by phosphorylation. TPA induced protein kinase C was inhibitory on GJIC. An antipeptide antibody to a sequence of a-gap junction protein was produced, but proved ineffective for immunofluorescence..
Dookwah, Hugh Denroy (1993). Studies on the expression and modulation of gap junctions in rat myometrial cell cultures. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1475873.