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The dipeptide Leu-Ala, which inhibits ubiquitin-mediated protein degradation, has been shown to act in vitro as an inhibitor of neurite outgrowth of PC12 cells (Hondermarck et al. [1992] Biochem. Biophys. Res. Commun. 189:280). Using agarose beads as vehicles, we tested, in vivo, the effect of this dipeptide (and the inactive inverse, Ala-Leu, as a control) on limb regeneration in the newt (Triturus cristatus), a nerve-dependent developmental process. Leu-Ala inhibited the growth of mid-bud blastemas without altering blastema differentiation, while Ala-Leu had no effect. Cytological observations of dipeptide-treated blastemas using Bodian staining or neurofilament antibodies showed that all the blastema tissues were unmodified except with regard to innervation. Leu-Ala-treated blastemas were devoid of nerve fibers in the epidermal cap, while the mesenchyme distal to the dipeptide impregnated bead exhibited fewer nerve fibers than did Ala-Leu-treated blastemas, which were similar to the control nontreated blastemas. Thus, Leu-Ala, in reducing blastema innervation, inhibits its growth in the same manner as surgical denervation.

Substance P-like immunoreactivity was found in Hydra attenuata mainly but not exclusively in the nerve and interstitial cells, localized in the cytoplasm and on the cell surface membranes.