Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate lizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cefl populations, though in differing proportions. In the hlppocampus, loalizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity.Hippocampal pyramidal hells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.Nitric oxide (NO) appears to be a biological messenger mediating immune responses of macrophages (1, 2) and endothelium-dependent blood vessel relaxation (3-5) and serving as a neurotransmitter in the peripheral and central nervous systems (6)(7)(8). NO is formed from arginine by NO synthase (NOS), which oxidizes a guanidino nitrogen of arginine, releasing NO and citrulline (5). Several distinct NOS enzymes have been cloned and localized (9, 10). Macrophage NOS is localized to macrophages throughout the body but also occurs in the brain in microglial cells, which are modified macrophages. By use of antibodies that recognize both neuronal and endothelial NOS, staining of the endothelium of blood vessels in the periphery and in the brain has been detected (11-15), whereas an antiserum selective for endothelial NOS (eNOS) has detected endothelium of blood vessels in the brain following ischemic injury (16). Extensive mapping with antiserum selective for eNOS has not been reported. Neuronal NOS (nNOS) is localized to neurons throughout the peripheral and central nervous system (11)(12)(13)17). Targeted disruption of nNOS in mice produces a >90% depletion of NOS catalytic activity in the brain (18). However, the residual NOS activity displays discrete regional distributions suggesting that neurons in some areas of the brain express a form ofNOS encoded by a different gene (18).Studies of the role of NO in brain function have implied a participation in long-term potentiation (LTP) in the hippocampus, a form of synaptic plasticity. NOS inhibitors block the induction of LTP in a fashion that is reversed by L-arginine (19)(20)(21)(22)(23) (19,26,27).Central to the notion that NO is the retrograde messenger of LTP is the assumption that hippocampal pyramidal cells possess abundant NOS. However, while immunohistochemical (13, 17) and in situ hybridization (13) studies demonstrate substantial NOS in small interneurons in the hippocampus, NOS is not readily evident in CA1 pyramidal cells (11,13). Some studies have shown faint, inconsistent staining of CA1 pyrami...

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with -10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelialderived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.Carbon monoxide (CO) has been implicated as a biological messenger molecule analogous to nitric oxide (NO) (1, 2). Two forms of heme oxygenase (HO-1 and HO-2) convert heme to biliverdin and CO (3, 4). HO-1 is highly expressed in liver and spleen and is easily induced by heme and by oxidative stress (5,6). A noninducible form, HO-2, is widely expressed with high concentrations in the brain (3). In the brain, the distribution of HO-2 closely parallels that of soluble guanylyl cyclase (SGC) (1). The ability of CO to directly activate SGC (7,8) and the depletion of cGMP levels in olfactory neurons treated with HO inhibitors (1) supports a role for CO as a modulator of cGMP. Like nitric oxide synthase (NOS), HO-2 occurs in discrete neuronal populations throughout the brain (1, 9) and both NO and CO have been implicated in long-term potentiation (10, 11), although the specificity of HO inhibitors used in these experiments has been questioned (12, 13).Although NO is well established as a mediator in the periphery, in both neurons and blood vessels (14-17) roles for CO have been less well characterized. HO-2 is localized to the glomus cells of the carotid body, where CO may mediate carotid body reactivity to hypoxia (18). HO inhibitors diminish esophageal motility (19), while hypoxia augments HO-1 in smooth muscle cells (20), and HO inhibitors decrease Na,KATPase activity in cerebellar Purkinje cells (21).We now report discrete localizations of HO-2 in vascular endothelium, nerves in the adventitia of blood vessels, and peripheral autonomic ganglia. Inhibition of HO-2 attenuates a component of endothelium-dependent vasodilation, implicating CO in vascular regulation. MATERIALS AND METHODSMaterials. Anti-neurofilament antibody, acetylcholine (ACh), phenylephrine, and NADPH were from Sigma, metalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Abbreviations: ACh, acetylcholine; HO, heme o...