Calmodulin-dependent cyclic nucleotide phosphodiesterase in adult and developing chick spinal cord

Abstract

We investigated the level and characteristics of "low Km" 3'-5' cyclic nucleotide phosphodiesterase (PDE) activity in adult and embryo chick spinal cord. The DEAE cellulose chromatography elution profile of Triton X-100 extracts showed a single peak of calmodulin-dependent cAMP/cGMP PDE activity. After two additional purification steps, this activity showed a five-fold activation by calmodulin (Ka = 1.5 nM) for cGMP hydrolysis, and a linear kinetic behaviour with a Km of 1.3 microM. Conversely, the activity showed a biphasic behaviour for cAMP hydrolysis, with Km values of 3.1 and 18.5 microM. The enzyme showed a Stokes radius of 4.5 nm. Western blot analysis of the purified enzyme revealed two immunoreactive bands with molecular mass of 59 and 65 kDa, respectively. Immunohistochemical staining showed motoneuron decoration both on cell soma and fibres. The developmental pattern of Ca2+-calmodulin-dependent PDE expression in spinal cord was also studied; the hydrolytic activity for both substrates has been found to increase constantly from E5 to post-hatching stages, when it appears 5.6-fold higher as compared to the early embryo levels. Furthermore, in cultured spinal cord neurons from E8 embryos, muscle extract has been shown to induce a two-fold increase of Ca2+-calmodulin-dependent cGMP activity. In conclusion, the studies reported here present three relevant findings: (1) the presence in adult and embryo chick spinal cord of PDE activities with characteristics similar to those of the mammalian PDE I enzyme; (2) its localization in the ventral horn motoneurons; (3) its regulated expression during embryogenesis that is possibly related to soluble epigenetic factors produced by the target cells.

Differential expression and localization of calmodulin‐dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion

“…Immunohistochemical localization also shows a distinct distribution of PDE isoforms. PDE1A on P5 DRG sections shows a non‐homogeneous staining of the cytoplasm, suggesting that the enzyme is associated to a subcellular compartment, similar to the immunostaining observed for PDE1C in chick spinal cord (Caniglia et al . 1997).…”

“…PDE1A on P5 DRG sections shows a non-homogeneous staining of the cytoplasm, suggesting that the enzyme is associated to a subcellular compartment, similar to the immunostaining observed for PDE1C in chick spinal cord (Caniglia et al 1997). This spotlike staining is specifically observed for PDE1A, which is also the only isoform revealed on neuron fibers.…”