CHANGES IN THE PHOSPHORYLATION STATES OF CONNEXIN43 IN MYOEPITHELIAL CELLS OF LACTATING RAT MAMMARY-GLANDS

Using specific antibodies and cDNA probe, we examined the expression p attern of a major gap junction protein, connexin43 (Cx43), in rat mamm ary glands during pregnancy and lactation, Double immunofluorescence r evealed that the labeling of Cx43 was superimposed in the alpha-smooth muscle actin-positive cells, suggesting that myoepithelial cells were interconnected by gap junctions formed of Cx43. Just after delivery t he Cx43-labeled plaques were enlarged and increased in a intensity Nor thern and Western blot analyses confirmed the dramatic induction of Cx 43 at both mRNA and protein levels on the day of parturition. Cx43 mRN A transcript immediately declined, while the increase of Cx43 protein continued for a few days, During pregnancy, immunoblots showed two ban ds of almost equal amounts at 43 and 45 kDa, Following delivery, the 4 5-kDa band gradually increased in intensity with a concomitant decreas e of the 43-kDa band, From the sixth day of lactation, Cx43 was always detected as a single band at 45 kDa, Alkaline phosphatase treatment o f immunoprecipitated Cx43 revealed that both bands represented phospho rylated forms, thus indicating that Cx43 was naturally phosphorylated and that it altered its phosphorylation states during lactation stages , These results suggest that the induction of Cx43 with the changes in the phosphorylation states plays an important role in the lactating f unction of myoepithelial cells in rat mammary glands, This is the firs t report on the changes of Cx43 phosphorylation states during physiolo gical stages in vivo.