The localization of cathepsin L or type I collagen in the osteoclasts (rat femur) treated with or without E-64 (control) was examined immunocytochemically to investigate how E-64 affects the osteoclasts. Using a light microscope in the E-64-treated osteoclasts, the immunoreactivity for cathepsin L was extracellularly very weak compared with that in the control osteoclasts, but was strong intracellularly. The intracellular immunoreactivity for type I collagen was found in the vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteoclasts. On the other hand, the extracellular immunoreactivity along the resorption lacunae of the E-64-treated osteoclasts was somewhat weaker than that of the control osteoclasts. Using electron microscopy in the E-64-treated osteoclasts, only a small number of extracellular immunoreaction products for cathepsin L were seen along the resorption lacunae. In addition, intracellular cathepsin L was deposited in the endosome-lysosomal vacuoles which were well-developed by E-64. Gold particles indicating type I collagen appeared on the bone matrix, and they were also detected in the vacuoles and vesicles in the E-64-treated osteoclasts. However, they were not detected in the organelles of the control osteoclasts. Thus, in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen. In addition, the undegraded collagen seemed to be endocytosed. The above findings thus suggest that bone resorption is inhibited by this incomplete degradation of collagen.
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