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 (InsPn)-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 EC50-values for astrocytes cultured either in the absence or presence of dBcAMP. The enhancement of NE-stimulated InsPn-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 alpha 1-adrenoceptor antagonists prazosin and WB 4101 potently inhibited NE-mediated PI-breakdown. Pretreatment of astrocytes with 100 microM CEC, an alpha 1B-adrenoceptor-specific, irreversible antagonist increased the EC50 values for NE-induced InsPn-accumulation in non-treated as well as in dBcAMP-treated cultures, indicating that both the alpha 1A- and alpha 1B-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 InsPn formation. The effects of the tested effectors were similar under both culturing conditions indicating that the susceptibility of components of the signalling pathway via alpha 1-adrenoceptors to these modulators was not influenced by morphological differentiation. Different mechanistic aspects of dBcAMP-action on NE-mediated signal-transduction are discussed.
Fahrig
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Institute for Neurobiology, Troponwerke GmbH & Co.KG, Köln, FRG.
Sommermeyer
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D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D001921:Brain / Q000166:cytology*; D003994:Bucladesine / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009638:Norepinephrine / Q000494:pharmacology; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D003609:Dactinomycin; D004533:Egtazic Acid; D003994:Bucladesine; D003513:Cycloheximide; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate; D009638:Norepinephrine