Calcium Site Specificity : Early Ca2+-related Tight Junction Events

The molecular mechanisms by which Ca²⁺ 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 Ca²⁺ withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca²⁺ is readmitted, and is followed by a complete recovery of the TJ seal. Mg²⁺ and Ba²⁺ were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg²⁺ causes a reversible concentration-dependent inhibition of the Ca²⁺-induced TJ recovery. The effects of extracellular Ca²⁺ manipulation on the TJs apparently is not mediated by changes of cytosolic Ca²⁺ concentration. The transition elements, Mn²⁺ and Cd²⁺, act as Ca²⁺ agonists. In the absence of Ca²⁺, they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca²⁺ withdrawal. In addition, Mn²⁺ promotes an almost complete recovery of the TJ seal. Cd²⁺, 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 Ca²⁺. The interruption of TJ opening caused by Ca²⁺, Cd²⁺, or Mn²⁺, and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La³⁺ (2 μM) to the basolateral Ca²⁺-containing solution causes an increase of TJ permeability that fully reverses when La³⁺ is removed. This effect of La³⁺, observed in the presence of Ca²⁺ (1 mM), indicates a high La³⁺ affinity for the Ca²⁺-binding sites. This ability of La³⁺ to open TJs in the presence of Ca²⁺ is a relevant aspect that must be considered when using La³⁺ in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.