Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells
Ladino, C.; Schneeberger, E.E.; Rabito, C.A.; Lynch, R.D.
European Journal of Cell Biology 55(2): 217-224
1991
ISSN/ISBN: 0171-9335 PMID: 1657607 Document Number: 383024
The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble solutes across epithelia is an energy-dependent process. The administration of N-formylhydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values. No difference in mannitol flux across Hadacidin-treated and control monolayers was observed at either time point. While freeze fracture replicas revealed a decrease in the number of parallel strands in the tight junctions that formed in the presence of Hadacidin, no discontinuities were noted. These data suggest that reductions in the level of cAMP, caused by Hadacidin, are associated with subtle changes in tight junctions which alter their electrical conductivity without affecting their barrier function to larger polar solutes such as mannitol.