REGULATION OF CELL-CELL COMMUNICATION MEDIATED BY CONNEXIN-43 IN RABBIT MYOMETRIAL CELLS

The aim of this study was to evaluate the rapid regulation of cell-cel l communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca2+-phospholipid-dependent pr otein kinase (protein kinase C), or the inhibitor proteins (PKI and CK I) that are, respectively, specific for each of these enzymes. Gap jun ction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). C ells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communicatio n was evaluated by monitoring Lucifer Yellow dye transfer. Cultures we re treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monoph osphate) or 10 mu M OAG(1-oleoyl-2-acetyl-sn-glycerol), which, respect ively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx4 3 was detected in myometrial cryosections and cultured cells by indire ct immunofluorescence, and its expression increased with gestation. Ex posure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of m id-pregnant uteri, and was maximal in late-pregnant cells. Treatment w ith 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment in hibited dye transfer in greater than 95% of the cells tested irrespect ive of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 mi n, with maximal transfer in 4-5 min. Protein kinase C inhibited commun ication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communi cation and that this process increases during pregnancy. Further, acti vators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.