RAPID CA2-DEPENDENT NO-PRODUCTION FROM CENTRAL-NERVOUS-SYSTEM CELLS IN CULTURE MEASURED BY NO-NITRITE()OZONE CHEMOLUMINESCENCE/

We have established simple and reliable measurement of constitutive ni tric oxide (NO) synthase-dependent nitrite formation in supernatants f rom primary central nervous system (CNS) cells in culture using NO-ozo ne chemoluminescence. We found that: (1) astrocytes, endothelial cells and cerebellar granule cells produce NO upon stimulation with the cal cium ionophore A23187 (1 mu M); (2) application of 100 mu M glutamate for 2 min results in NO-production in cerebellar granule cells and cor tical neurons. NO-formation upon application of 50 mM KCl was found in cortical neurons; (3) in cultivated cerebral endothelial cells, an in ducible form of NO-synthase (iNOS) is found under standard culture con ditions. This induction was blocked by dexamethasone applied for at le ast 48 h and stimulation of constitutive NOS was detectable while iNOS was inhibited. The activity of iNOS was selectively inhibited by appl ication of aminoguanidine for 48 h. Our results suggest that all major CNS cells implied in cerebral blood flow regulation and neurovascular coupling are capable of rapidly producing the vasodilator NO upon int racellular increases of the universal second messenger calcium.