CYCLIC-AMP MODIFIES THE CELLULAR-DISTRIBUTION OF CONNEXIN43 AND INDUCES A PERSISTENT INCREASE IN THE JUNCTIONAL PERMEABILITY OF MOUSE MAMMARY-TUMOR CELLS

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular respons es to external signals, We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumo r cell line, MMT22, Junctional permeance was quantitatively assessed u sing dye microinjection and video microscopy, An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 m inutes of treatment and attaining a fourfold higher level of permeance by 24 hours, This elevated level was maintained with continuous expos ure to 8-bromo-cAMP for seven days, The permeability change was accomp anied by an increase in gap junctions as shown by freeze-fracture elec tron microscopy and by confocal microscopy using antibodies directed a gainst the gap junction protein, connexin43, The amount of detergent-i nsoluble connexin43 also increased with 8-bromo-cAMP treatment, and mo st the increase could be attributed to an increase of slower migrating (i,e, phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyse s of northern and western blots, We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation, Since these changes were re latively slow to develop and persisted with prolonged exposure to 8-br omo-cAMP, it is possible that the junctional permeability of these mam mary tumor cells is linked to the 'basal' level of cAMP, i,e, levels m aintained by the cells in accordance with a particular cell state.