Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

Abstract

The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×10⁴ cells/cm², where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells). Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.