Reassembly of the occluding junctions in a renal cell line with characteristics of proximal tubular cells.

LLC-PK1 cells from trypsin-treated confluent cultures formed a continuous monolayer when plated at high cell density on collagen-coated Nuclepore filters. These monolayers developed a significant transepithelial electrical resistance that reached a maximum at 20 h. At 48 h, the resistance decreased to a value usually one-half the value obtained at 20 h. These changes were associated with an increase in the cell density of the monolayers. The drop in electrical resistance at 48 h was not observed when cell growth was arrested with excess thymidine. A hyperbolic relationship was demonstrated between cell density and electrical resistance. Although the increase in cell density was associated with an increase in the unidirectional flux of mannitol across the monolayers, selectivity studies indicated that the intrinsic properties of the occluding junctions were similar in the high and low cell density monolayers. These results indicate that, when cell growth is not arrested, changes in transepithelial electrical resistance observed after plating correspond to an increase in cell density and not to changes in the intrinsic properties of the occluding junctions. The development of transepithelial electrical resistance was delayed when the cells were in exponential growth. No such delay was observed, however, when exponential growth was synchronized. These findings and results obtained with the antimicrotubular agent Nocodazole indicate that the delay in the development of transepithelial electrical resistance is due to the asynchronous transit of the cells through the mitotic phase of the cell cycle: a time when most of the intercellular junctions are probably disrupted. Further investigation revealed that inhibition of protein but not mRNA synthesis blocked the development of occluding junctions in cells from confluent and exponentially growing cultures alike. These results indicate that, in contrast to MDCK cells, regulation of the occluding junctions in exponentially growing LLC-PK1 cells occurs at the translational not at the transcriptional level of protein synthesis.