EFFECTS OF SELECTIVE-INHIBITION OF PROTEIN-KINASE-C, CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, AND CA2-CALMODULIN-DEPENDENT PROTEIN-KINASE ON NEURITE DEVELOPMENT IN CULTURED RAT HIPPOCAMPAL-NEURONS()

A variety of experimental evidence suggests that calmodulin and protei n kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiati on. However, the results are somewhat contradictory. Further, the role s of calmodulin and protein kinases on many aspects of neurite develop ment, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used thi s culture system and the new generation of highly specific protein kin ase inhibitors to investigate the roles of protein kinases and calmodu lin in neurite development. Neurons were cultured for 2 days in the co ntinuous presence of calphostin C (a specific inhibitor of protein kin ase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN6 2 (inhibitor of Ca2+-calmodulin-dependent protein kinase II), or calmi dazolium (inhibitor of calmodulin), each at concentrations from approx imately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-ace tate (an activator of protein kinase C) and 4alpha-phorbol 12,13-didec anoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurit e initiation and axon branching without significantly affecting the nu mber of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4alpha- phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no e ffect on any aspect of neurite development, but at high concentrations , calmidazolium inhibited every parameter that was measured (including viability). These results suggest that these three protein kinases se lectively modulate different aspects of neurite development. The unive rsality of effects caused by calmodulin inhibition make it impossible to determine if there are specific targets of calmodulin action involv ed in neurite development. Finally, our data indicate that some superf icially similar characteristics of neuronal differentiation, such as n eurite initiation and branching, may be controlled by quite different molecular mechanisms.