Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion.
The level and characteristics of 3'-5'-cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5-day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5-fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti-mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight- and 3.6 fold, respectively) while a more limited increase (1.6- and 1.5-fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non-neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.
Giorgi
Mauro
M
Dipartimento di Biologia di Base e Applicata, Università di l'Aquila, Coppito, Italy.
Giordano
Daniela
D
Rosati
Jessica
J
Tata
Ada Maria
AM
Augusti-Tocco
Gabriella
G
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002642:Chick Embryo; D002645:Chickens; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D003720:Densitometry; D004789:Enzyme Activation / Q000187:drug effects; D005727:Ganglia, Spinal / Q000166:cytology / Q000196:embryology* / Q000201:enzymology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism; D009928:Organ Specificity; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014020:Tissue Extracts / Q000737:chemistry
D002147:Calmodulin; D007527:Isoenzymes; D014020:Tissue Extracts; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D006152:Cyclic GMP; D002118:Calcium