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Regulation of CO production by glutamate in cultured astrocytes from the cerebral cortex

Carbon monoxide (CO), a potent dilator in cerebral circulation, is a product of heme degradation by heme oxygenase (HO) that is highly expressed in the brain as a constitutive HO‐2 isoform, whereas inducible HO‐1 is not detectable. In newborn pigs, brain CO production is increased during glutamatic seizures without HO‐1 induction. Astrocytes that account for majority of the cell population in the cerebral cortex may contribute to overall brain CO production. We addressed the hypothesis that glutamate increases CO production in astrocytes via activation of constitutive HO‐2. Primary cultures of astrocytes from cerebral cortex of newborn piglets were grown on Costar plates or Cytodex microcarrier beads and were identified by immunostaining for glial fibrillary acidic protein and aquaporin‐4. CO production by cultured astrocytes was detected by gas chromatography/mass spectrometry. HO‐2 is the major isoform in quiescent astrocytes, although HO‐1 is also immunodetectable. Glutamate (100 mkM, 1h) increased basal CO production from 13±2 to 45±8 pmol/mg protein and stimulated HO catalytic activity (astrocyte CO formation from exogenous heme) 2‐fold. We upregulated HO‐1 expression ~ 10‐fold by a heme analog cobalt protoporphyrin (CoPP). In CoPP‐treated astrocytes, basal CO production was increased to 139±20 pmol CO/mg protein consistent with HO‐1 overexpression. In HO‐1 overexpressing astrocytes, glutamate did not stimulate HO catalytic activity and only mildly enhanced CO formation. We conclude that glutamate rapidly increases astrocyte CO production via posttranslational activation of HO‐2 but not HO‐1.