Calcium-dependent nitric oxide formation in glial cells

We have previously demonstrated nitric oxide (NO)-dependent cyclic GMP (cGMP) formation in response to noradrenaline (NA) and glutamate (GLU) in astrocyte-enriched cultures from rat cerebrum. In the present work we show heterogeneity in agonist responses in astrocyte cultures from cerebellum, hippocampus and cortex. The response to NA was higher in cells from cerebellum, intermediate in cultures from hippocampus and low in cortical astrocytes. GLU had no significant effect in cortical and cerebellar cultures and presented lower effects than NA in cells from hippocampus. The NO donor sodium nitroprusside (SNP) produced much higher cGMP levels than agonists and the order of efficacies was cerebellum > cortex > hippocampus. Responses to NA and SNP in cerebellar astrocytes were sensitive to culture conditions decreasing when cells were seeded at low density or subcultured. Microglial cells were the main contaminants of the cerebellar astrocyte cultures but did not contribute to the NA or the SNP responses. No soluble guanylyl cyclase or calcium-dependent NO synthase (cNOS) activities were detected in microglial cultures. The effect of NA in cerebellar astrocytes was blocked by L-arginine analogues and by the alpha 1-adrenoceptor antagonist prazosin. The calcium ionophore A23187 mimicked the effect of NA and omission of calcium from the medium prevented both responses. NA did not elicit cGMP formation in granule cell cultures. These results support an astroglial location of the alpha 1-adrenoceptors and the cNOS that mediate NA stimulation of cGMP formation in cerebellum.