Modulation of phosphodiesterases of the brain.

The rationale behind the work in this thesis has been to develop methods for the quantitation of adenosine 3',5'-cyclic monophosphate (cAMP) hydrolysis (catalysed by phosphodiesterases) in intact cells of the central nervous system. The rate of decay of cAMP after cessation of synthesis (by addition of antagonist) in previously stimulated tissue provides one way to measure cAMP elimination. However, in brain slices, the beta-adrenoceptor (beta-AR) stimulation of cAMP was too poor to allow thorough kinetic investigation. Although conditions were sought which would serve to enhance the stimulation, none were found that could be applicable to these studies. Attention was then turned to using homogeneous model cell systems such as the SH-SY5Y neuroblastoma and 1321N1 astrocytoma cell lines. In the neuroblastoma cells, an unexpected difference in the efficacy of a non-selective PDE inhibitor, l-methyl-3-isobutylxanthine (IBMX) was observed when compared to type IV PDE inhibitors. This was apparently due to another action of IBMX at a locus distinct from PDE, and whilst adenosine receptor antagonism was at least partially responsible for the disparity, the possiblity that IBMX was acting at a third site could not be eliminated. Such a complication, as well as the poor cAMP radiolabelling, rendered these cells an unattractive system for measuring agonist-stimulated cAMP turnover. In 1321N1 cells, the decay method was successfully applied to assess the relationship between cAMP accumulation and PDE activity in the intact cell. Using a range of -adrenoceptor agonist concentrations, whole cell Michaelis-Menten-type kinetics for cAMP elimination were derived, and suggested that cAMP disposal processes (possibly dominated by Ca2+/CaM-dependent PDE) were being saturated under conditions of beta-AR activation. The deduced nature of the PDE population then has implications for the profile of agonist-induced cAMP accumulation. This novel technique for performing intact cell kinetics has a variety of applications. Another technique to measure cAMP turnover utilising a dual isotopic pulse-radiolabelling regimen was also instigated, with studies focussing upon the 1321N1 cells (brain slices and SH- SY5Y cells were inadequate models). This method was compared to the decay approach under conditions of beta-AR activation; in conjunction with the examination of receptor- and non-receptor-mediated increases in cAMP turnover, the pulse-labelling protocol was then deemed to be an accurate description of receptor-induced cAMP turnover, and provided another way of assessing PDE activity in the glial-type cells