CHARACTERISTICS OF NITRIC-OXIDE SYNTHASE TYPE-I OF RAT CEREBELLAR ASTROCYTES

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formatio n through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the pre sent work we have used rat cerebellar astrocyte-enriched primary cultu res to identify and characterise the isoform of NOS expressed in these cells, The specific NOS activity in astrocyte homogenates, determined by conversion of [H-3] arginine to [H-3]citrulline, was ten times low er than in homogenates from cerebellar granule neurons. Upon centrifug ation at 100,000g, the astroglial activity was recovered in the supern atant, whereas in neurons around 30% of the activity remained particul ate. The cytosolic NOS activities of both astrocytes and granule neuro ns displayed the same K-m for L-arginine, dependency of calcium, and s ensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosol ic fractions was revealed by Western blot with a specific polyclonal a ntiserum against recombinant NOS-I, Double immunofluorescence labellin g using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I ant ibodies revealed that a minor population of the GFAP-positive cells, u sually in clusters, presented a strong NOS-I immunostaining that was p redominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. As trocytes of stellate morphology also showed immunoreactivity in the pr ocesses. Similar staining was observed with the avidin-biotin-peroxida se complex using different anti-NOS-I antisera, With this method the m ajority of cells showed a weak NOS-I immunoreactivity around the nucle i and cytosol, A similar pattern was observed with the NADPH-diaphoras e reaction. These results demonstrate that the NOS-I expressed in astr ocytes presents the same biochemical characteristics as the predominan t neuronal isoform but may differ in intracellular location. (C) 1996 Wiley-Liss, Inc.