Calcium Site Specificity : Early Ca(2+)-related Tight Junction Events

The molecular mechanisms by which Ca(2+) and metal ions interact with the binding sites that modulate the tight junctions (TJs) have not been fully described. Metal ions were used as probes of these sites in the frog urinary bladder. Basolateral Ca(2+) withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca(2+) is readmitted, and is followed by a complete recovery of the TJ seal. Mg(2+) and Ba(2+) were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg(2+) causes a reversible concentration-dependent inhibition of the Ca(2+)-induced TJ recovery. The effects of extracellular Ca(2+) manipulation on the TJs apparently is not mediated by changes of cytosolic Ca(2+) concentration. The transition elements, Mn(2+) and Cd(2+), act as Ca(2+) agonists. In the absence of Ca(2+), they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca(2+) withdrawal. In addition, Mn(2+) promotes an almost complete recovery of the TJ seal. Cd(2+), in spite of stabilizing the TJs in the closed state and halting TJ opening, does not promote TJ recovery, an effect that apparently results from a superimposed toxic effect that is markedly attenuated by the presence of Ca(2+). The interruption of TJ opening caused by Ca(2+), Cd(2+), or Mn(2+), and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La(3+) (2 μM) to the basolateral Ca(2+)-containing solution causes an increase of TJ permeability that fully reverses when La(3+) is removed. This effect of La(3+), observed in the presence of Ca(2+) (1 mM), indicates a high La(3+) affinity for the Ca(2+)-binding sites. This ability of La(3+) to open TJs in the presence of Ca(2+) is a relevant aspect that must be considered when using La(3+) in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.