Experiments were performed in vitro to assess the mitogenic activity of brain homogenates and cyclic nucleotides on adult newt limb blastemal cells. Early cone stage blastemata were explanted into organ culture, and thus denervated. The mitotic index (M.1.) of each was calculated and shown to decline steadily in vitro. The inclusion in the medium of newt brain homogenate (NBH) or dibutyryl CAMP (dbcAMP) delayed the loss of mitotic activity. Cyclic GMP alone or in combination with theophylline was ineffective as a mitogen. Bovine serum albumin and fetal calf serum were also ineffective, thus the mitogenic activity of brain homogenate appears to be distinct from a simple enrichment of the culture medium. Preliminary characterization of the active principles in NBH revealed a mitogenic activity that was both heat labile and trypsin sensitive. In related experiments, control cultures were maintained for 54 hours, roughly equivalent to one urodele blastemal cell cycle. When the decline in M.I. had stabilized, NBH or dbcAMP were added to the medium.
Limb regeneration in the newt, Notophthalmus viridescens, was retarded by treatment with acetylcholine or imidazole, whereas mitotic activity and regenerative growth were stimulated by cyclic AMP, its dibutyryl derivative, and theophylline. It is proposed that cyclic nucleotides are implicated in amphibian limb regeneration and may be related to the neural factor postulated by others.
Cultured amphibian limb blastemas at both early and late stages of regeneration were treated with acetylcholine (ACH). Incorporation of radioactive leucine or thymidine was measured after 24 h. A significant reduction in leucine incorporation was observed in experimental cultures, particularly at the earlier stage of regeneration, whereas DNA synthesis was apparently unaffected. It is suggested that ACH may be involved in the regulation of protein synthesis during regeneration of the newt limb in vivo.
Dibutyryl cyclic AMP or sodium fluoride was found to stimulate 14C-leucine incorporation by newt limb regeneration blastemas in vitro. The increased amino acid incorporation was observed in both the mound stage blastema and the more advanced palette stage regenerate. Imidazole, theophylline or cyclic phosphodiesterase had no significant effect on the cultured blastemas. Some regenerating limbs were denervated 72 h before explantation of the blastema. Mound stage blastemas obtained from these limbs incorporated significantly higher amounts of leucine, after dibutyryl cyclic AMP treatment, than did control blastemas. Palette stage regenerates which are no longer dependent on neurotrophic influences did not respond differently from the control group. Sodium fluoride did not cause an increase in leucine incorporation by these denervated blastemas. The results were interpreted to support the hypothesis of an involvement of the cyclic AMP-adenylate cyclase system in amphibian limb regeneration, probably in connection with the postulated neurotrophic factor.
Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.
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