Effects of Dexamethasone and Glucagon after Long-Term Exposure on Cyclic AMP Phosphodiesterase 4 in Cultured Rat Hepatocytes
Introduction
Glucagon and glucocorticoids play an important role in regulation of glycogen metabolism in rat liver. The action of glucagon is mediated by the second messenger cAMP. The intracellular cAMP level is a dynamic equilibrium maintained by synthesis via adenylyl cyclases and degradation via cAMP phosphodiesterases (PDE). PDE could be divided in seven types each with different subtypes 1, 2. Recently two further types were described 3, 4. PDE types show different expression patterns in various cells tissues and organs 1, 2. The subject of this study is the low Km cAMP-specific, rolipram-inhibited PDE 4 which could also be affected by hormones. In Sertoli cells PDE 4 was found to be stimulated by FSH [5] and in plasma membrane fractions of rat liver by insulin [6]. According to Heyworth et al. [7] PDE 4 in plasma membranes does not seem to be stimulated by short-term treatment with glucagon. On the other hand decreased PDE 4 activities after glucagon application in presence of GTP were observed by Robles-Flores et al. [8]. The rolipram-sensitive PDE 4 was characterized as the PDE type up-regulated by cAMP-increasing agents in rat Sertoli cells [5], in human U937 cells [9], or in rat sceletal myoblasts [10]. Long-term treatment with dexamethasone decreased a low Km PDE in homogenates of cultured hepatocytes 11, 12, but the PDE type was not specified.
The aim of our studies was to investigate effects of dexamethasone and glucagon on PDE 4 in primary cultured hepatocytes after long-term exposure. Our results show that dexamethasone decreased PDE 4 activity only after addition at the beginning of cultivation during the time of plating. Glucagon was found to be able to stimulate the cytosolic rolipram-inhibited PDE 4. The subtypes A, B , C, and D of PDE 4 could be detected in hepatocytes. Semiquantitative RT-PCR analysis showed that in presence of dexamethasone the transcription of PDE 4D was found to be decreased selectively whereas glucagon did not influence the transcription of PDE 4 subtypes. The translation of PDE 4D was also reduced. We would like to discuss the way that dexamethasone seems to suppress PDE 4D expression in combination with other factors such as cytokines during cell plating, whereas glucagon acts directly via phosphorylations.
Section snippets
Chemicals
3[H]-cAMP (24 Ci/mmol) was supplied by Amersham-Buchler (Braunschweig, Germany), M 199 medium with 25 mM HEPES by Serva (Heidelberg, Germany), and ICI 118233 by D.E. Riley and J. Bebbington, ICI Pharmaceuticals (Macclesfield, U.K.). Monoclonal PDE 4D specific antipeptide antibody was supplied by S. Wolda, ICOS Corporation (Bothell, Washington, USA), and rolipram was kindly given by Dr. Wachtel, Schering AG (Berlin, Germany). Glucagon was obtained from Novo Nordisk Pharma GmbH (Mainz, Germany),
Results
To get information about the subcellular distribution of PDE activity cytosol, plasma membrane, `dense vesicle' membrane and endoplasmatic reticulum membrane fractions were prepared by stepwise centrifugations of the cell homogenate [14]. The main part of PDE avtivity (67%) measured at 1 μM cAMP was found in the cytosol of hepatocytes after 24 h culturing (Table 1). PDE activity of the plasma membrane and `dense vesicle' membrane fractions were equal and reached up to 15% respectively. Only a
Discussion
In a previous study we have used primary culture of rat hepatocytes in order to investigate hormonal short-term and long-term effects on PDE 3 [13]. Pursuing our investigations on hormonal regulation of PDE, we have now studied dexamethasone and glucagon effects after long-term exposure on the PDE 4 in cultured rat hepatocytes. Type specific inhibitors were used to differentiate exactly between PDE 4 or PDE 3 in various subcellular fractions. As shown in Table 1, PDE 4 could be found
Acknowledgements
This study was supported by grant from Deutsche Forschungsgemeinschaft, Bonn, Germany.
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