ACTIVATION OF PROTEIN-KINASE-C DURING NEWT LIMB REGENERATION - EFFECTOF DENERVATION

Blastema cell proliferation during newt limb regeneration is a nerve-d ependent process. The present study was undertaken to determine whethe r or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles waif. Analysis included evalu ation of PKC activity and its subcellular localization at various stag es of regeneration, both in vivo and in vitro. The data reveal an incr ease in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correla tes with blastema cell proliferation rate. Denervation significantly r educes blastema cell proliferation and also causes a reduction in memb rane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic red uction in activity 96 h after denervation. In contrast, PKC activity i n the epidermal cap did not change. Cultured whole blastemas likewise; express a decrease in particulate PKC activity and therefore mimic den ervated blastemas in this parameter. Co-culture of blastemas with spin al ganglia partially reduces the decline in PKC activity, and the phor bol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimu lating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regener ation.