ALPHA-1-ADRENERGIC RECEPTOR COUPLING WITH PHOSPHOLIPASE-C IS NEGATIVELY REGULATED BY PROTEIN-KINASE-C IN PRIMARY CULTURES OF HYPOTHALAMIC NEURONS AND GLIAL-CELLS

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha-1-receptor (alpha-1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha-1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in yound and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha-1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha-1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.