Influence of heme and heme oxygenase-1 transfection of pulmonary microvascular endothelium on oxidant generation and cGMP.
Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.
Mingone
Christopher J
CJ
Department of Physiology, New York Medical College, Valhalla, New York 10595, USA.
Gupte
Sachin A
SA
Quan
Shuo
S
Abraham
Nader G
NG
Wolin
Michael S
MS
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001393:Azoles / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D051547:Heme Oxygenase-1; D006801:Humans; D054833:Isoindoles; D008168:Lung / Q000098:blood supply*; D008298:Maleates / Q000378:metabolism; D008565:Membrane Proteins; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019335:Nitroarginine / Q000378:metabolism; D016566:Organoselenium Compounds / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection
D000975:Antioxidants; D001393:Azoles; D004791:Enzyme Inhibitors; D054833:Isoindoles; D008298:Maleates; D008565:Membrane Proteins; D016566:Organoselenium Compounds; D016877:Oxidants; D017382:Reactive Oxygen Species; D019335:Nitroarginine; C042986:ebselen; D006418:Heme; D002374:Catalase; D019001:Nitric Oxide Synthase; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C014476:diethyl maleate; D006152:Cyclic GMP