Using a new NT-3 specific enzyme-immunoassay, we showed that neonatal rat cortical and, for comparison, cerebellar astrocytes in primary culture can synthesize NT-3; the basal cellular content of NT-3 protein was 23.2 ± 0.4 pg NT-3/mg cell protein and 23.6 ± 0.9 pg NT-3/mg cell protein, respectively. The examined neurotransmitters, with the exception of serotonin, were able to potently and transiently increase NT-3 mRNA and NT-3 protein content; their maximal effects were dose- and time-dependent. Noradrenaline (1 μM), adrenaline (1 μM), and dopamine (100 μM) showed a maximal increase in NT-3 cellular content after 6 h treatment causing a 1.9-, 1.8- and 2.7-fold elevation, respectively. Prior to the observed increase in NT-3 protein levels, the examined catecholamines increased NT-3 mRNA levels with maximal effects observed after 1 h (noradrenaline) and 2 h (adrenaline and dopamine) of incubation causing 2.4-, 2.6- and 3-fold elevation, respectively. Screening different activators of basic intracellular second messenger systems for their influence on NT-3 synthesis revealed that forskolin (20 μM), dibutyryl cAMP (dBcAMP) (100 μM), as well as calcimycin (1 μM) (Ca2+ ionophore A23187) and phorbol 12-myristate 13-acetate (TPA) (100 nM), markedly increased the cellular level of NT-3 protein. Neurotransmitter-induced NT-3 levels were susceptible (to varying degrees) to inhibition by H-89 (protein kinase A inhibitor) or staurosporin (protein kinase C inhibitor), which led us to conclude that downstream signaling responsible for the stimulation of NT-3 synthesis by monoamines in astrocytes consists of multiple, complex intracellular mechanisms involving the cAMP/protein kinase A pathway, activation of protein kinase C, as well as mobilization of Ca2+ ions.
Our results indicate for the first time that monoaminergic neurotransmitters play an important role in the regulation of NT-3 synthesis in cultured rat astrocytes.