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Title: Differential regulation of cyclic nucleotide phosphodiesterases in anterior pituitary cells
Author: Ang, Kok Long
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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Cyclic nucleotide hydrolysis by phosphodiesterases (PDEs) is an important process determining the amplitude and duration of the responses to cAMP generating agonists. In adenohypophysial corticotrophs and the mouse pituitary corticotroph tumour AtT20 cells, ACTH secretion is known to be mediated through the cAMP pathway. The aim of this thesis is to identify the cAMP-hydrolysing phosphodiesterase isozymes expressed in rat adenohypophysis and AtT20 cells, and to study the regulation of their activities by cAMP-dependent pathway in AtT20 cells. Multifaceted analysis showed that Ca2+/calmodulin (CaM)-stimulated (PDE1) and cAMP-specific, rolipram-inhibitable (PDE4) PDE isotypes are expressed in AtT20 cells as well as rat adenohypophysis. RT-PCR analysis revealed the expression of mRNAs from the PDE1B, PDE1C, PDE4A, PDE4B and PDE4D subfamilies in AtT20 cells and PDE1C, PDE4B and PDE4D in rat adenohypophysis. Stimulation of cAMP pathway in AtT20 cells was found to enhance the activity of PDE4 and to reduce that of PDE1. Calyculin A, an inhibitor of protein phosphatase 1/2A, synergistically enhanced the effect of CPT-cAMP on PDE1 and had an additive effect on PDE4. Incubation of AtT20 cytosol with the catalytic subunit of PKA produced the same changes of PDE1 and PDE4 activities as the activation of PKA in intact cells. The reduction of PDE1 activity was associated with a markedly diminished response to Ca2+/CaM stimulation. Immunoprecipitation studies with isozyme-specific antibodies showed that PDE4D isozymes accounted for more than half of the control as well as CPT-cAMP and/or calyculin A activated PDE4 activity. Immunoblot analysis of the immunoprecipitate showed two distinct bands that on the basis of apparent molecular weights correspond to the PDE4D3 and PDE4D5 splice variants. The migration of both bands on SDS-PAGE were retarded upon CTP-cAMP and/or calyculin A treatments, which is consistent with the reported effect of PKA phosphorylation on PDE4D3. In sum, these data indicate that 1) Both Ca2+/CaM-dependent (PDE1) and Ca2+/CaM-independent (primarily PDE4) PDEs are expressed in rat adenohypophysis and AtT20 cells; 2) Low Km PDE1, plausibly PDE1C, is found in rat adenohypophysis as well as AtT20 cells; 3) The rapid and differential regulation of PDE1 and PDE4 by cAMP-dependent phosphorylation and dephosphorylation in AtT20 cells provides a means for fine tuning the Ca2+/CaM dependency and the extent of cAMP hydrolysis in relation to the momentary status of cAMP and Ca2+/CaM pathways.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available