Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate.
Endogenous carbon monoxide (CO) contributes to vasodilator responses of cerebral microvessels in newborn pigs. We investigated the expression, intracellular localization, and activity of heme oxygenase (HO), the key enzyme in CO production, in quiescent cerebral microvascular endothelial cells (CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected by RT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 are membrane-bound proteins that have a strong preference for the nuclear envelope and perinuclear area of the cytoplasm. Betamethasone (10(-6) to 10(-4) M for 48 h) was associated with upregulation of HO-2 protein by approximately 50% and inhibition of Cox-2 but did not alter HO-1 or endothelial nitric oxide synthase expression in CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by 30-60%. HO activity as (14)CO production from [(14)C]glycine-labeled endogenous heme was inhibited by chromium mesoporphyrin (10(-6) to 10(-4) M). L-Glutamate (0.3-1.0 mM) stimulated HO activity 1.5-fold. High-affinity specific binding sites for L-[(3)H]glutamate suggestive of the glutamate receptors were detected in CMVEC. Altogether, these data suggest that, in cerebral circulation of newborn pigs, endothelium-derived CO may contribute to basal vascular tone and to responses that involve glutamate receptor activation.
Parfenova
H
H
Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. hparf@physio1.utmem.edu
Neff
R A
RA
Alonso
J S
JS
Shlopov
B V
BV
Jamal
C N
CN
Sarkisova
S A
SA
Leffler
C W
CW
D000818:Animals; D000831:Animals, Newborn; D001623:Betamethasone / Q000494:pharmacology; D002248:Carbon Monoxide / Q000378:metabolism; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000098:blood supply* / Q000502:physiology; D051546:Cyclooxygenase 2; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D005938:Glucocorticoids / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D013552:Swine
D005938:Glucocorticoids; D007527:Isoenzymes; D008565:Membrane Proteins; D018698:Glutamic Acid; D002248:Carbon Monoxide; D001623:Betamethasone; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006419:Heme Oxygenase (Decyclizing); D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases