Dibutyryl cyclic AMP-induced morphological differentiation of rat brain astrocytes increases α 1-adrenoceptor induced phosphoinositide breakdown by a mechanism involving protein synthesis

Elevation of intracellular cAMP levels by treatment of cultured astrocytes with dibutyryl cyclic AMP (dBcAMP) resulted in a dose-dependent morphological transformation from a flat, polygonal phenotype into a stellate-like cell shape. This morphological differentiation was accompanied by an increased in maximal inositolphosphate (InsP n)-accumulation after stimulation of phosphoinositide (PI)-breakdown by norepinephrine (NE). Maximal enhancement of NE-induced PI breakdown was observed after treatment of the cells with 0.15 mM dBcAMP for 7 days. While there was a clear effect of dBcAMP-induced differentiation on the maximal NE-induced PI-response, no effect on the dose-response relationship was detectable, resulting in similar EC 50-values for astrocytes cultured either in the absence or presence of dBcAMP. The enhancement of NE-stimulated InsP n-formation was dependent on the duration of dBcAMP-treatment. More than a 6 h incubation time was needed to observe an increase in NE-induced PI-breakdown. Furthermore, the enhancing effect of dBcAMP could be prevented by inclusion of the protein-synthesis inhibitor cycloheximide and the blocker of mRNA-transcription actinomycin D. Both the α 1-receptor antagonists prazosin and WB 4101 potently inhibited NE-mediated PI-breakdown. Pretreatment of astrocytes with 100 μM CEC, an α 1B-adrenoreceptor-specific, irreversible antagonist increased the EC 50 values for NE-induced InsP-accumulation in non-treated as well as in dBcAMP-treated cultures, indicating that both the α 1A and α 1B-adrenoceptor subtypes were expressed under both culturing conditions. Reduction of extracellular Ca 2+ or pretreatment of the cells with either 12- O-tetradecanoyl-phorbol-13-acetate (TPA), or pertusis toxin (PTX) resulted in a significant reduction of NE-stimulated InsP n formation. The effects of the tested effectors were similar under both culturing conditions indicating that the susceptibility of components of the signalling pathway via α 1-adrenoceptors to these modulators was not influenced by morphological differentiation. Different mechanistic aspects of dBcAMP-action on NE-mediated signal-transduction are discussed.