Evidence that a Ca2+ chelator and a calmodulin blocker interfere with the structure of inter-Sertoli junctions

Ca2+ dependence of tight junction structure has been well documented in cultured epithelial tissues, and regulatory mechanisms have been identified. To analyse the possible control exerted on inter-Sertoli junctions, we exposed guinea-pig seminiferous tubules to the presence of a Ca2+ chelator (EGTA) and to a calmodulin blocker (Trifluoperazine, TFP) in vitro, for times ranging from 30 to 120 min. We observed the morphology of junctional complexes and the basal cytoplasmic regions in sections and replicas. Sertoli cell response to Ca2+ depletion involved several events: retraction of cells toward the base of the tubule and a consequent stretching of the points of fusion, augmented density of the cytoplasm, and destabilization of the array of intramembrane particles. Exposure of tubules to TFP resulted in disruption of the interactions between actin filaments and membrane junctional specialization, as well as a disorganization of other cytoskeletal elements. Thus, in vitro, junction integrity appears to be related to Ca2+ level, and Ca2+ depletion apparently interferes with Ca2+ distribution inside the cell and on microfilaments involved in junction regulation. Our results do not provide direct evidence for any particular mechanism of action of TFP, but a multiple effect is evident. TFP, which affects Ca2+ regulation and membrane fluidity, probably acts indirectly on junction-associated filaments. Both the experimental conditions tested suggest a Ca2+-mediated regulatory role of microfilaments of this complex junction.