DIBUTYRYL CYCLIC AMP-INDUCED MORPHOLOGICAL-DIFFERENTIATION OF RAT-BRAIN ASTROCYTES INCREASES ALPHA-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 increase in maximal inositolphosphate (InsP(n))-accumulation after stimulation of phosphoinositide (Pl)-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 EC50-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 alpha1-adrenoceptor antagonists prazosin and WB 4101 potently inhibited NE-mediated PI-breakdown. Pretreatment of astrocytes with 100 muM CEC, an alpha1B-adrenoceptor-specific, irreversible antagonist increased the EC50 values for NE-induced InsP(n)-accumulation in non-treated as well as in dBcAMP-treated cultures, indicating that both the alpha1A- and alpha1B-adrenoceptor subtypes were expressed under both culturing conditions. Reduction of extracellular Ca2+ or pretreatment of the cells with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or pertussis 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 alpha1-adrenoceptors to these modulators was not influenced by morphological differentiation. Different mechanistic aspects of dBcAMP-action on NE-mediated signal-transduction are discussed.