Calcium Site Specificity Early Ca 2 1-related Tight Junction Events

The molecular mechanisms by which Ca 2 1 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 1 withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca 2 1 is readmitted, and is followed by a complete recovery of the TJ seal. Mg 2 1 and Ba 2 1 were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg 2 1 causes a reversible concentrationdependent inhibition of the Ca 2 1 -induced TJ recovery. The effects of extracellular Ca 2 1 manipulation on the TJs apparently is not mediated by changes of cytosolic Ca 2 1 concentration. The transition elements, Mn 2 1 and Cd 2 1 , act as Ca 2 1 agonists. In the absence of Ca 2 1 , they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca 2 1 withdrawal. In addition, Mn 2 1 promotes an almost complete recovery of the TJ seal. Cd 2 1 , 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 1 . The interruption of TJ opening caused by Ca 2 1 , Cd 2 1 , or Mn 2 1 , and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La 3 1 (2 m M) to the basolateral Ca 2 1 -containing solution causes an increase of TJ permeability that fully reverses when La 3 1 is removed. This effect of La 3 1 , observed in the presence of Ca 2 1 (1 mM), indicates a high La 3 1 affinity for the Ca 2 1 -binding sites. This ability of La 3 1 to open TJs in the presence of Ca 2 1 is a relevant aspect that must be considered when using La 3 1 in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives. key words: tight junction • calcium • cadmium • lanthanum • E-cadherin i n t r o d u c t i o n Ca 2 1 is essential for cells to maintain intercellular contacts. When the extracellular Ca 2 1 is removed, the cell– cell connections generally become loose and multicellular organizations are destroyed. A number of studies emphasize the role of extracellular Ca 2 1 on the stability of mature tight junctions (TJs) 1 in natural epithelia (Sedar and Forte, 1964; Hays et al., 1965; Galli et al., 1976; Meldolesi et al., 1978; Pitelka et al., 1983; Palant et al., 1983) and on the development of new TJs in cell cultures in confluence (Martinez-Palomo et al., 1980; Cereijido et al., 1980, 1981; González-Mariscal et al., 1985). The removal of extracellular Ca 2 1 causes the opening of previously formed TJs and prevents de novo formation of TJs in confluent cell monolayers. Notwithstanding several studies addressing the role of extracellular Ca 2 1 in the dynamics of the TJs, major questions are still pending. The relative importance of extracel– lular (Gorodeski et al., 1997; Contreras et al., 1992; González-Mariscal et al., 1990) versus intracellular (Bhat et al., 1993; Jovov et al., 1994; Stuart et al., 1994) Ca 2 1 concentration on the control of TJs is not yet clearly characterized. The cell adhesion molecule E-cadherin (uvomorulin) (Gumbiner et al., 1988), which is particularly rich at the zonula adhaerens (Boller et al., 1985), plays a key role as the extracellular Ca 2 1 binding molecule that modulates the formation and maintenance of the epithelial junctional complex (Gumbiner et al., 1988). Ca 2 1 influences the conformation of E-cadherin and stabilizes it in its adhesive state (Ringwald et al., 1987). In addition, the interaction of Ca 2 1 with E-cadherin is transduced across the cell membrane by a cascade of reactions involving phospholipase C, G proteins, protein kinase C, and calmodulin (Balda et al., 1991, 1993). The structural and electrostatic mechanisms used by the Ca 2 1 binding sites of E-cadherin to provide Ca 2 1 specificity are not yet fully understood, as compared with the knowledge on the EF-hand-like sites (Snyder et al., 1990), in part because insufficient information is available regarding the ion specificity of the Address correspondence to F. Lacaz-Vieira, Institute of Biomedical Sciences—USP, Department of Physiology and Biophysics, 05508-900 São Paulo, SP, Brazil. Fax: 55-11-818.7285; E-mail: lacaz@bmb.icb1.