The nature and localization of acid phosphatase during the early phases of urodele limb regeneration

Abstract

Summary This study is concerned with both histochemical and biochemical changes in the acid hydrolase, acid phosphatase, during the dedifferentiation phase of the regeneration of the adult urodele forelimb. We find acid phosphatase in a variety of tissues in the normal limb. Structures which stain most prominently are slime glands, perineurium, epidermis, and the vascular tree. This location is generally the same in regenerates except for increased activity in nerve, and more dramatically for the evolution of activity in dedifferentiating skeletal muscle. The morphologically homogeneous blastema reveals a positive reaction for acid phosphatase in a small proportion of the total number of cells. The vascular bed has the most intense acid phosphatase activity even when it is in the process of dedifferentiating. Biochemically, it is found that two major fractions of acid phosphatase exist in the normal and regenerate urodele limb. On the basis of tartrate-inhibition studies, it is concluded that 80–95% of acid phosphatase is lysosomal in nature, while the rest is “soluble”. An acetate-soluble fraction of acid phosphatase is also present in normal and regenerating limbs. This fraction is comprised of a small amount of “soluble” enzyme as well as structurally nonlatent lysosomal component. On the basis of time studies performed during the first 16 days of regeneration, it is concluded that: (1) changes in acid phosphatase activity occur in the limb during wound healing and dedifferentiation phases; (2) these changes take the form of a sequential rise in the activities of the total, lysosomal, and the structurally nonlatent fractions of acid phosphatase to levels significantly higher than normal values; (3) the sequential increase in activity reaches its peak in all fractions just prior to the period of maximum dedifferentiation observable in the regenerate. Moreover, these activities drop just prior to the end of the dedifferentiation phase of regeneration; and (4) no significant change occurs in the level of the “soluble” fraction. The possible role of structurally latent and structurally nonlatent lysosomal acid phosphatase are discussed particularly with respect to the availability of active lysosomal enzyme during the processes of cellular dedifferentiation and tissue remodeling in amphibian limb regeneration. Finally, it is concluded that the lysosomal hydrolases, as exemplified by acid phosphatase, appear to play a significant role in the phenomenon of dedifferentiation through a variety of mechanisms.