Intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels are under the stimulatory or inhibitory control of several hormones and neurotransmitters. Forskolin (FSK) is a diterpene that activates adenyl cyclase directly, causing an elevation in intracellular cAMP levels. Cyclic nucleotide phosphodiesterases (PDE) are a group of enzymes that degrade 3',5'- cAMP to 5' adenosine monophosphate (AMP). One of the PDE types is selectively inhibited by RO 20-1724 and Rolipram. Rolipram has been effectively used in treating major (DMS-III) and endogenous (ICD-9) depressions, suggesting that PDE activity in the brain of these patients may not be regulated properly. The purpose of this proposal is to initiate studies describing the mechanism of regulating the Rolipram sensitive PDE. My work indicates that brief ( < 30 min) incubation of astroglial cells with FSK causes a doubling of PDE activity and that this increase manifests itself in an increase in two PDE peaks separated by DEAE chromatography. One of these is very sensitive to RO 20-1724. The principal aims of this proposal are to describe the rapid increases in PDE activity in astroglial cells and to initiate experiments designed to determine the mechanisms(s) responsible for PDE regulation. I will first characterize the kinetics and the pharmacology of the PDEs activated by FSK treatment and determine their sensitivities to endogenous regulators such as Ca 2 +/calmodulin and cGMP. Results from these experiments will identify these PDEs according to established criteria. I will then determine if the FSK effect is mediated by cAMP. First, I will determine if PDE activity increases following isoproterenol-stimulated increases of intracellular cAMP. Second, I will determine if direct application of cAMP analogs increases PDE activity. Finally, I will determine if the PDE activity is increased by phosphorylation using cell homogenates and the catalytic subunit of protein kinase A. Results from these experiments will provide a strong foundation for future experiments describing the mechanism of PDE activation and its role in regulating cell function.
| Madelian, V; La Vigne, E (1996) Rapid regulation of a cyclic AMP-specific phosphodiesterase (PDE IV) by forskolin and isoproterenol in LRM55 astroglial cells. Biochem Pharmacol 51:1739-47 |
