Physiologic roles for the heme oxygenase products carbon monoxide, bilirubin and iron: links to neuroprotection in stroke and Alzheimer's disease David E. Baran Äano a , Sylvain Dore  a,e , Christopher D. Ferris a,b , Solomon H. Snyder a,c,d, * a Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA b Department of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA c Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA d Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA e Department of Anesthesiology and Critical Care Medicine, The John Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA Abstract Heme oxygenase (HO) cleaves the heme ring releasing iron, carbon monoxide (CO) and biliverdin, which is immediately reduced to bilirubin by the abundant biliverdin reductase. Recent research indicates major cellular roles for all three HO products. The constitutive isoform, heme oxygenase 2 (HO2), is concentrated in the brain with discrete localizations resembling soluble guanylyl cyclase, implying a role in regulating cyclic guanosine monophosphate (cGMP). HO2 is localized to myenteric plexus neurons of the intestine similar to neuronal nitric oxide synthase (nNOS). Mice with targeted deletion of HO2 or nNOS each display marked reductions in non-adrenergic-non- cholinergic neurotransmission and cGMP levels indicating a neurotransmitter role for the two gases acting as co-transmitters. HO2 is localized to the endothelial cell layer of blood vessels as well as neurons in the adventitial layer. HO inhibitors reduce nitric oxide (NO)- independent vasodilation implicating CO as an endothelial derived relaxing factor. Bilirubin, formed from HO2, appears to be a physiologic neuroprotectant. Neurotoxicity is augmented in brain cultures from HO2 gene knockout mice which also display augmented neural damage following cerebral vascular occlusion. Very low concentrations of bilirubin reverse the neurotoxicity. Heme oxygenase 1 (HO1) plays a role in iron ef¯ux from cells. Iron ef¯ux is diminished in cells from HO1 gene knockout mice, while HO1 transfection augments ef¯ux. Iron dependent cytotoxicity is worsened in HO1 gene knockout cells while HO1 transfection alleviates the toxicity. q 2001 Association for Research in Nervous and Mental Disease. All rights reserved. Keywords: Heme oxygenase; Neuroprotection; Bilibrubin; Iron; Carbon monoxide; Nitric oxide 1. Introduction Nitric oxide (NO) is increasingly appreciated as a major neurotransmitter, which has overturned many classic concepts of neurotransmission [1,2]. Being a diffusible gas, it cannot be stored in synaptic vesicles, nor released by exocytosis, nor bind to speci®c receptors on adjacent neuronal cell membranes. Instead, each molecule of NO must be synthesized as needed. NO diffuses from neurons into adjacent cells where it activates target proteins such as soluble guanylyl cyclase (sGC). Neurotransmitters come in chemical classes such as the biogenic amines, amino acids, and peptides. This prompted us to wonder whether there might be other gaseous neurotransmitters. The formation of carbon monoxide (CO) from heme by heme oxygenase (HO) had been well established prompting us to speculate about a transmitter role. Over the past years compelling evidence has emerged for CO as a neurotransmitter in the enteric nervous system and the brain. HO forms two other products, iron and biliverdin (Fig. 1). Biliverdin is almost instantaneously reduced to bilirubin by the abun- dant enzyme biliverdin reductase. We have established a physiologic neuroprotectant role for bilirubin. We have also shown that one form of HO, heme oxygenase 1 (HO1), is associated with the egress of iron from cells providing protection from the toxic effects of iron. In Clinical Neuroscience Research 1 (2001) 46±52 www.elsevier.nl/locate/clires 1566-2772/01/$ - see front matter q 2001 Association for Research in Nervous and Mental Disease. All rights reserved. PII: S1566-2772(00)00006-2 * Corresponding author. Tel.: 11-410-955-2380; fax.: 11-410-955- 3623. E-mail address: ssnyder@bs.jhmi.edu (S.H. Snyder).