Cell, Vol. 70, 705-707,
Se@tember 4, 1992, Copyright
0 1992 by Cell Press
Minireview
Nitric Oxide, A Novel Biologic Messenger Charles J. Lowenstein and Solomon H. Snyder The Johns Hopkins University School of Medicine Department of Neuroscience, Pharmacology and Molecular Sciences and Department of Psychiatry Baltimore, Maryland 21205
Nitric oxide (NO), is a noxious, stable, free radical gas. Recently, work in several disciplines has converged to establish N O as a major messenger molecule regulating immune function and blood vessel dilatation and serving as a neurotransmitter in the brain and peripheral nervous system. N O was first appreciated in mammalian physiology as a mediator of macrophage actions (Nathan and Hibbs, 1991). Macrophages produce nitrates that derive from NO, whose precursor is arginine. Arginine derivatives block the formation of NO, as does removal of arginine from the incubation medium. Both these treatments block the tumoricidal and bactericidal actions of macrophages, establishing N O as a crucial mediator of macrophage function (Figure 1). A role for N O in blood vessels stemmed from the discovery that the ability of acetylcholine and other agents to relax smooth muscle and hence dilate blood vessels is dependent on the presence of an intact endothelium, which releases a diffusible factor (Ignarro, 1990; Moncada et al., 1991). This endothelial-derived relaxing factor appeared to be extremely labile with a half-life of -5 s. Its identification as N O was facilitated by parallel studies that established N O as the active metabolite mediating the smooth muscle relaxant effects of nitroglycerin and other anti-angina1 organic nitrates. The identification of N O as endothelial-derived relaxing factor led to experiments showing that brain tissue can generate N O (Garthwaite, 1991). Subsequent evidence for N O functions in brain derived from knowledge of how N O acts in blood vessels. By binding to iron in the heme at the active site of guanylyl cyclase, N O activates the enzyme to generate cGMP, which stimulates cGMP-dependent protein kinase, resulting in muscle relaxation (Figure 1). In the brain, the highest densities of cGMP occur in the cerebellum, where the excitatory neurotransmitter glutamate elevates cGMP levels via the N-methyl-p-aspartate (NMDA) subtype of receptors. Glutamate or NMDA triple N O synthase activity (Bredt and Snyder, 1992). Addition of N O synthase inhibitors such as N-methyl-arginine or NG-nitro-arginine block both increased N O synthase activity and elevation of cGMP levels, establishing a role for N O in the neurotransmitter actionsof glutamate (Bredt and Snyder, 1992; Garthwaite, 1991). N O Synthase Because N O is so labile, direct measurements of N O are difficult, so that molecular advances have largely stemmed from studies of N O synthase. There appear to be at least three distinct forms of N O synthase. Under
basal conditions, N O synthase activity in macrophages is negligible, while stimulation with lipopolysaccharide and y-interferon produces massive enhancement of N O synthase in a few hours (Nathan and Hibbs, 1991). Inducible N O synthase also occurs in neutrophils. Activated neutrophils and macrophages form oxygen free radicals, which can combine with N O to form substances substantially more toxic than N O itself. Thus, N O combined with superoxide anion yields peroxynitrite that decomposes to hydroxide free radical and NO2 free radical. Since N O synthase inhibitors completely block macrophage cytotoxic actions, N O generation may be rate limiting, though substances formed by interaction of N O and oxygen free radicals are the cytotoxic effecters (Figure 2). In blood vessels and neurons, N O synthase is constitutive; no major enzyme induction has yet been demonstrated. Instead, the enzyme is activated by calcium, which binds calmodulin as an enzyme cofactor (Bredt and Snyder, 1990). In blood vessels, acetylcholine stimulates the phosphoinositide cycle generating inositol 1,4,5-trisphosphate to release calcium, which binds to calmodulin and activates N O synthase. In the brain, glutamate stimulation of NMDA receptors opens calcium channels that are part of the receptor protein, triggering calcium influx to elicit increases of N O synthase (see Figure 1). Localization and Function in the Nervous System Purification of N O synthase from the brain has permitted the production of antibodies and immunohistochemical mapping, while molecular cloning of brain, macrophage, and endothelial N O synthase has permitted localization of mRNA by in situ hybridization. Localization studies of
NEURON
I
ENDOTH.
erg + 0, -tit
MAC.
+ NO*
I
Figure 1. NO Synthesis and Mechanisms of Action as an intercellular Messenger in Macrophages, Blood Vessels, and Neurons MAC., macrophage; tit, citrulline; arg, arginine; NOS, NO synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
CAL
PUN
HP
CAL
FUN
H
CAL
HP
NEURONAL NOS
PAD
NADPH
PAD
NADPH
w
6
I obo
560
ENDOTHELIAL NOS
MACROPHAGE NOS Figure 2. Structure Synthases
1 6
of Neuronal,
fig I 1060
560
Endothelial,
and Macrophage
CAL, calmodulin; P, consensussiteforcAMP-dependent tion; H, consensus site for heme binding.
NO
phosphoryla-
macrophage NO synthase reveal that macrophage NO synthase mRNA is undetectable in tissues under basal conditions. Treatment of rats with endotoxin provokes high concentrations of macrophage NO synthase mRNA in the white pulp of the spleen, which is enriched in macrophages, as well as in the red pulp, where macrophages migrate to interact with T cells (Lowenstein et al., 1992). In blood vessels, immunohistochemical studies localize NO synthase to endothelial layers in large vessels (Bredt et al., 1990). Small arterioles and capillaries display very little NO synthase, fitting with physiologic evidence that NO primarily regulates large rather than small blood vessels. In large cerebral blood vessels as well as those in choroid layers of the eye and in the penis, NO synthase also occurs in nerve fibers in the outer, adventitial layers, where it is stored together with the neuropeptide vasoactive intestinal polypeptide (Bredt and Snyder, 1992). In the penis, dilatation of these vessels underlies penile erection. Stimulation of the pelvic nerves from which the NO synthase fibers arise causes penile erection, which is completely blocked by NO synthase inhibitors, establishing NO as the physiologic mediator of erections (Burnett et al., 1992). NO synthase immunoreactivity occurs in autonomic nerve fibers throughout the body, including the entire gastrointestinal pathway and the bronchial tree. In the intestine, electrical stimulation of the NO synthase-positive myenteric neural plexus evokes the relaxation component of peristalsis, which is blocked by NO synthase inhibitors, indicating that NO acts here as a neurotransmitter. In the brain, NO synthase occurs in discrete neuronal populations. In some areas, such as the cerebral cortex, hippocampus, and corpus striatum, it occurs in small interneurons comprising only 1%-2% of the total neuronal population. On the other hand, in the cerebellum, all granule cells and basket cells contain NO synthase, while the enzyme is absent from Purkinje cells. This unique pattern is identical to that of NADPH diaphorase, a histochemical stain whereby the dye nitro blue tetrazolium is reduced in the presence of NADPH but not NAD (Dawson et al., 199la). Neurons staining with NADPH diaphorase are resistant to various types of neurotoxicity. In stroke or in cerebral cultures treated with glutamate or NMDA, up to 9 0 % of neurons die, while NADPH diaphorase-staining
neurons survive. Similarly, in Huntington’s disease, up to 95O/bof neuronal cells in thecaudate nucleuscan degenerate while NADPH diaphorase staining is normal. NO synthase activity accounts for NADPH diaphorase staining, as transfection of NO synthase cDNA into kidney 293 cells leads to NO synthase and NADPH diaphorase staining in the same proportions as in neurons (Dawson et al., 1991 a). Since glutamate stimulates NO formation, it seemed paradoxical that NO synthase neurons should be resistant to glutamate neurotoxicity. A possible resolution of this paradox would be if glutamate stimulates NO synthase neurons to synthesize NO that diffuses to adjacent cells and kills them. This notion is in accord with experiments showing that NMDA toxicity in primary cerebral cultures is blocked by NO synthase inhibitors as well as by omission of argininefrom the medium (Dawson et al., 1991 b). NMDA toxicity is probably involved in neuronal damage following vascular strokes, since NMDA antagonists relieve stroke damage in animal models. Strikingly, the NO synthase inhibitor nitroarginine is more effective than the NMDA antagonist MK-801 in blocking stroke damage following ligation of cerebral arteries of mice (Nowicki et al., 1991). Structure of NO Synthase and Its Targets Molecular cloning of NO synthase has revealed much about its regulation. Neuronal NO synthase, the first cloned (Bredt et al., 1991), has multiple regulatory sites, including binding sites for NADPH, flavin adenine dinucleotide, and flavin mononucleotide (Figure 2). Additionally, NOsynthaseactivityrequirestetrahydrobiopterin. Purified NO synthase contains tightly bound flavin adenine dinucleotide and flavin mononucleotide. The purified enzyme binds heme tightly and absorbs at 450 nm following treatment with carbon monoxide, indicating that NO synthase is a cytochrome P-450 enzyme (McMillan et al., 1992). The use of such a large number of cofactors to handle the electron transfers involved in NO synthase activity is unprecedented. Another novel feature is the capacity of NO synthase to produce superoxide as well as NO (Pou et al., 1992). Purified neuronal NO synthase generates superoxide in the absence of arginine, while arginine addition reduces superoxide formation concomitant with enhanced NO formation. Conceivably, NO and superoxide, both produced by the same enzyme, combine to form the radicals that mediate cytotoxicity. Neuronal NO synthase has a recognition site for calmodulin that is also evident in endothelial NO synthase (Lamas et al., 1992; Janssens et al., 1992) and macrophage NO synthase (Lyons et al., 1992; Xie et al., 1992; Lowenstein et al., 1992) (Figure 2). However, in contrast with the dependence of endothelial and brain NO synthase upon calcium, macrophage NO synthase activity is calcium independent. Neuronal, macrophage, and endothelial NO synthase all. have consensus sites for CAMP-dependent phosphotylation. Neuronal NO synthase is phosphorylated by CAMP-dependent protein kinase, protein kinase C, and Ca2+/calmodulin protein kinase (Bredt et al., 1992). Protein kinase C phosphorylation markedly reduces NO synthase catalytic activity, while the effects of other types
Minireview 707
of phosphorylation are unclear. The three forms of N O synthase display about 50% identity in amino acid sequence; macrophage and endothelial N O synthase are shorter at the N-and C-termini than neuronal N O synthase. All three N O synthase isozymes display significant sequence homology to only one other mammalian enzyme, cytochrome P-450 reductase (CPR) (Figure 2). CPR donates electrons for cytochrome P-450 drug-metabolizing enzymes. The CPR-like sequence of N O synthase comprises the carboxyl half of the molecule so that N O synthase resembles a fusion of a cytochrome P-450 enzyme and CPR. Perhaps N O synthase and the cytochrome P-450 enzymes were linked in evolution, which fits with N O synthase displaying P-450 properties (McMillan et al., 1992). Besides cytochrome P-450 enzymes, CPR donates electrons for heme oxygenase, an enzyme that cleaves heme to form biliverdin and carbon monoxide. This relationship suggests that carbon monoxide may serve functions resembling NO. Interestingly, mRNA for 1 of the 2 forms of heme oxygenase is selectively localized to neuronal populations throughout the brain with a pattern of distribution closely resembling guanylyl cyclase (Verma et al., 1992). A role for carbon monoxide in regulating endogenous cGMP issupported by the ability of potent, selective inhibitors of heme oxygenase to deplete endogenous cGMP levels in brain tissue. The notion that carbon monoxide, not NO, is the major physiologic regulator of brain guanylyl cyclase suggests that other targets exist for NO. N O enhances ADP ribosylation of certain platelet proteins (Brune and Lapetina, 1990). In the brain and in red blood cells, the target of NO-stimulated ADP ribosylation is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) @hang and Snyder, 1992; Kots et al., 1992) (Figure 2). GAPDH is auto-ADP ribosylated, and N O enhances this process. Since ADP ribosylation involves the cysteine to which NAD binds in GAPDH catalysis, ADP ribosylation should inhibit GAPDH activity. Because of its crucial role in glycolysis, NO-stimulated GAPDH ADP ribosylation seems a fitting target to mediate N O neurotoxicity. References Bredt, D. S., and Snyder, S. H. (1990). Proc. Natl. Acad. Sci. USA 87, 662-665. Bredt, D. S., and Snyder, S. H. (1992). Neuron 8, 3-11. Bredt, D. S., Hwang, P. M., and Snyder, S. Ii. (1990). Nature 347,766770. Bredt, D. S., Hwang, P. H., Glatt, C., Lowenstein, Snyder, S. H. (1991). Nature 357, 714-716.
C.. Reed, R. R., and
Bredt, D. S., Ferris, C. D., and Snyder, S. H. (1992). J. Biol. Chem. 267, 10976-l 0961. Brune, B., and Lapetina, 266-290.
E. G. (1990). Arch. Biochem.
Biophys. 279,
Burnett, A. L., Lowenstein, C. J., Bredt, D. S., Chang, T. S. K., and Snyder, S. H. (1992). Science 257,401-403. Dawson, T. M., Bredt, D. S., Fotuhi, M., Hwang, P. M., and Snyder, S. H. (1991a). Proc. Natl. Acad. Sci. USA 88, 7797-7601. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., and Snyder, S. H. (1991b). Proc. Natl. Acad. Sci. USA 88, 6366-6371. Garthwaite, Ignarro,
J. (1991). Trends Neural. Sci. 74, 60-67.
L. J. (1990). Annu. Rev. Pharmacol.
Toxicol.
30, 535-560.
Janssens, S. P.,
Shimouchi, A., Quertermous, T., Bloch, D. B., and Bloch, K. D. (1992). J. Biol. Chem. 267, 14519-14522.
Kots, A., Ya., Skurat, A. V., Sergienko, E. A., Bulargina, Severin, E. S. (1992). FEBS Lett. 300, 9-12.
T. V., and
Lamas,S., Marsden,P. A., PrOC.
Li, G. K., Tempst, P., and Michel, T. (1992). Natl. Acad. Sci. USA 89, 6348-6352.
Lowenstein,C. J., Glalt, C. S., Bredt, D. S., and Proc. Natl. Acad. Sci. USA 89, 6711-6715. Lyons, C. R., Orloff, G. J., and Cunningham,
Snyder, S. H. (1992). J. M. (1992). J. Biol.
Chem. 267, 6370-6374.
McMillan, K., Bredt, D. S., Hirsch, D. J., Snyder, S. H., Clark, J. E., and Masters, B. S. S. (1992). Proc. Natl. Acad. Sci. USA, in press. Moncada, S., Palmer, R. M. J., and Higgs,
E. A. (1991). Pharmacol.
Rev. 43, 109-142.
Nathan, C. F., and Hibbs, J.
B., Jr. (1991). Curr. Opin.
Immunol. 3,
65-70. Nowicki, J. P., Duval, D., Poignet, H., and Scatton, B. (1991). Eur. J. Pharmacol. 204,339~340. Pou, S., Pou, W., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992). J. Biol. Chem., in press. Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V., and Snyder, S. H. (1992). Science, in press. Xie, Q.-W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992). Science 256, 225-226.
Zhang, J., and Snyder, press.
S. H. (1992). Proc. Natl. Acad. Sci. USA, in
Se@tember 4, 1992, Copyright
0 1992 by Cell Press
Minireview
Nitric Oxide, A Novel Biologic Messenger Charles J. Lowenstein and Solomon H. Snyder The Johns Hopkins University School of Medicine Department of Neuroscience, Pharmacology and Molecular Sciences and Department of Psychiatry Baltimore, Maryland 21205
Nitric oxide (NO), is a noxious, stable, free radical gas. Recently, work in several disciplines has converged to establish N O as a major messenger molecule regulating immune function and blood vessel dilatation and serving as a neurotransmitter in the brain and peripheral nervous system. N O was first appreciated in mammalian physiology as a mediator of macrophage actions (Nathan and Hibbs, 1991). Macrophages produce nitrates that derive from NO, whose precursor is arginine. Arginine derivatives block the formation of NO, as does removal of arginine from the incubation medium. Both these treatments block the tumoricidal and bactericidal actions of macrophages, establishing N O as a crucial mediator of macrophage function (Figure 1). A role for N O in blood vessels stemmed from the discovery that the ability of acetylcholine and other agents to relax smooth muscle and hence dilate blood vessels is dependent on the presence of an intact endothelium, which releases a diffusible factor (Ignarro, 1990; Moncada et al., 1991). This endothelial-derived relaxing factor appeared to be extremely labile with a half-life of -5 s. Its identification as N O was facilitated by parallel studies that established N O as the active metabolite mediating the smooth muscle relaxant effects of nitroglycerin and other anti-angina1 organic nitrates. The identification of N O as endothelial-derived relaxing factor led to experiments showing that brain tissue can generate N O (Garthwaite, 1991). Subsequent evidence for N O functions in brain derived from knowledge of how N O acts in blood vessels. By binding to iron in the heme at the active site of guanylyl cyclase, N O activates the enzyme to generate cGMP, which stimulates cGMP-dependent protein kinase, resulting in muscle relaxation (Figure 1). In the brain, the highest densities of cGMP occur in the cerebellum, where the excitatory neurotransmitter glutamate elevates cGMP levels via the N-methyl-p-aspartate (NMDA) subtype of receptors. Glutamate or NMDA triple N O synthase activity (Bredt and Snyder, 1992). Addition of N O synthase inhibitors such as N-methyl-arginine or NG-nitro-arginine block both increased N O synthase activity and elevation of cGMP levels, establishing a role for N O in the neurotransmitter actionsof glutamate (Bredt and Snyder, 1992; Garthwaite, 1991). N O Synthase Because N O is so labile, direct measurements of N O are difficult, so that molecular advances have largely stemmed from studies of N O synthase. There appear to be at least three distinct forms of N O synthase. Under
basal conditions, N O synthase activity in macrophages is negligible, while stimulation with lipopolysaccharide and y-interferon produces massive enhancement of N O synthase in a few hours (Nathan and Hibbs, 1991). Inducible N O synthase also occurs in neutrophils. Activated neutrophils and macrophages form oxygen free radicals, which can combine with N O to form substances substantially more toxic than N O itself. Thus, N O combined with superoxide anion yields peroxynitrite that decomposes to hydroxide free radical and NO2 free radical. Since N O synthase inhibitors completely block macrophage cytotoxic actions, N O generation may be rate limiting, though substances formed by interaction of N O and oxygen free radicals are the cytotoxic effecters (Figure 2). In blood vessels and neurons, N O synthase is constitutive; no major enzyme induction has yet been demonstrated. Instead, the enzyme is activated by calcium, which binds calmodulin as an enzyme cofactor (Bredt and Snyder, 1990). In blood vessels, acetylcholine stimulates the phosphoinositide cycle generating inositol 1,4,5-trisphosphate to release calcium, which binds to calmodulin and activates N O synthase. In the brain, glutamate stimulation of NMDA receptors opens calcium channels that are part of the receptor protein, triggering calcium influx to elicit increases of N O synthase (see Figure 1). Localization and Function in the Nervous System Purification of N O synthase from the brain has permitted the production of antibodies and immunohistochemical mapping, while molecular cloning of brain, macrophage, and endothelial N O synthase has permitted localization of mRNA by in situ hybridization. Localization studies of
NEURON
I
ENDOTH.
erg + 0, -tit
MAC.
+ NO*
I
Figure 1. NO Synthesis and Mechanisms of Action as an intercellular Messenger in Macrophages, Blood Vessels, and Neurons MAC., macrophage; tit, citrulline; arg, arginine; NOS, NO synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
CAL
PUN
HP
CAL
FUN
H
CAL
HP
NEURONAL NOS
PAD
NADPH
PAD
NADPH
w
6
I obo
560
ENDOTHELIAL NOS
MACROPHAGE NOS Figure 2. Structure Synthases
1 6
of Neuronal,
fig I 1060
560
Endothelial,
and Macrophage
CAL, calmodulin; P, consensussiteforcAMP-dependent tion; H, consensus site for heme binding.
NO
phosphoryla-
macrophage NO synthase reveal that macrophage NO synthase mRNA is undetectable in tissues under basal conditions. Treatment of rats with endotoxin provokes high concentrations of macrophage NO synthase mRNA in the white pulp of the spleen, which is enriched in macrophages, as well as in the red pulp, where macrophages migrate to interact with T cells (Lowenstein et al., 1992). In blood vessels, immunohistochemical studies localize NO synthase to endothelial layers in large vessels (Bredt et al., 1990). Small arterioles and capillaries display very little NO synthase, fitting with physiologic evidence that NO primarily regulates large rather than small blood vessels. In large cerebral blood vessels as well as those in choroid layers of the eye and in the penis, NO synthase also occurs in nerve fibers in the outer, adventitial layers, where it is stored together with the neuropeptide vasoactive intestinal polypeptide (Bredt and Snyder, 1992). In the penis, dilatation of these vessels underlies penile erection. Stimulation of the pelvic nerves from which the NO synthase fibers arise causes penile erection, which is completely blocked by NO synthase inhibitors, establishing NO as the physiologic mediator of erections (Burnett et al., 1992). NO synthase immunoreactivity occurs in autonomic nerve fibers throughout the body, including the entire gastrointestinal pathway and the bronchial tree. In the intestine, electrical stimulation of the NO synthase-positive myenteric neural plexus evokes the relaxation component of peristalsis, which is blocked by NO synthase inhibitors, indicating that NO acts here as a neurotransmitter. In the brain, NO synthase occurs in discrete neuronal populations. In some areas, such as the cerebral cortex, hippocampus, and corpus striatum, it occurs in small interneurons comprising only 1%-2% of the total neuronal population. On the other hand, in the cerebellum, all granule cells and basket cells contain NO synthase, while the enzyme is absent from Purkinje cells. This unique pattern is identical to that of NADPH diaphorase, a histochemical stain whereby the dye nitro blue tetrazolium is reduced in the presence of NADPH but not NAD (Dawson et al., 199la). Neurons staining with NADPH diaphorase are resistant to various types of neurotoxicity. In stroke or in cerebral cultures treated with glutamate or NMDA, up to 9 0 % of neurons die, while NADPH diaphorase-staining
neurons survive. Similarly, in Huntington’s disease, up to 95O/bof neuronal cells in thecaudate nucleuscan degenerate while NADPH diaphorase staining is normal. NO synthase activity accounts for NADPH diaphorase staining, as transfection of NO synthase cDNA into kidney 293 cells leads to NO synthase and NADPH diaphorase staining in the same proportions as in neurons (Dawson et al., 1991 a). Since glutamate stimulates NO formation, it seemed paradoxical that NO synthase neurons should be resistant to glutamate neurotoxicity. A possible resolution of this paradox would be if glutamate stimulates NO synthase neurons to synthesize NO that diffuses to adjacent cells and kills them. This notion is in accord with experiments showing that NMDA toxicity in primary cerebral cultures is blocked by NO synthase inhibitors as well as by omission of argininefrom the medium (Dawson et al., 1991 b). NMDA toxicity is probably involved in neuronal damage following vascular strokes, since NMDA antagonists relieve stroke damage in animal models. Strikingly, the NO synthase inhibitor nitroarginine is more effective than the NMDA antagonist MK-801 in blocking stroke damage following ligation of cerebral arteries of mice (Nowicki et al., 1991). Structure of NO Synthase and Its Targets Molecular cloning of NO synthase has revealed much about its regulation. Neuronal NO synthase, the first cloned (Bredt et al., 1991), has multiple regulatory sites, including binding sites for NADPH, flavin adenine dinucleotide, and flavin mononucleotide (Figure 2). Additionally, NOsynthaseactivityrequirestetrahydrobiopterin. Purified NO synthase contains tightly bound flavin adenine dinucleotide and flavin mononucleotide. The purified enzyme binds heme tightly and absorbs at 450 nm following treatment with carbon monoxide, indicating that NO synthase is a cytochrome P-450 enzyme (McMillan et al., 1992). The use of such a large number of cofactors to handle the electron transfers involved in NO synthase activity is unprecedented. Another novel feature is the capacity of NO synthase to produce superoxide as well as NO (Pou et al., 1992). Purified neuronal NO synthase generates superoxide in the absence of arginine, while arginine addition reduces superoxide formation concomitant with enhanced NO formation. Conceivably, NO and superoxide, both produced by the same enzyme, combine to form the radicals that mediate cytotoxicity. Neuronal NO synthase has a recognition site for calmodulin that is also evident in endothelial NO synthase (Lamas et al., 1992; Janssens et al., 1992) and macrophage NO synthase (Lyons et al., 1992; Xie et al., 1992; Lowenstein et al., 1992) (Figure 2). However, in contrast with the dependence of endothelial and brain NO synthase upon calcium, macrophage NO synthase activity is calcium independent. Neuronal, macrophage, and endothelial NO synthase all. have consensus sites for CAMP-dependent phosphotylation. Neuronal NO synthase is phosphorylated by CAMP-dependent protein kinase, protein kinase C, and Ca2+/calmodulin protein kinase (Bredt et al., 1992). Protein kinase C phosphorylation markedly reduces NO synthase catalytic activity, while the effects of other types
Minireview 707
of phosphorylation are unclear. The three forms of N O synthase display about 50% identity in amino acid sequence; macrophage and endothelial N O synthase are shorter at the N-and C-termini than neuronal N O synthase. All three N O synthase isozymes display significant sequence homology to only one other mammalian enzyme, cytochrome P-450 reductase (CPR) (Figure 2). CPR donates electrons for cytochrome P-450 drug-metabolizing enzymes. The CPR-like sequence of N O synthase comprises the carboxyl half of the molecule so that N O synthase resembles a fusion of a cytochrome P-450 enzyme and CPR. Perhaps N O synthase and the cytochrome P-450 enzymes were linked in evolution, which fits with N O synthase displaying P-450 properties (McMillan et al., 1992). Besides cytochrome P-450 enzymes, CPR donates electrons for heme oxygenase, an enzyme that cleaves heme to form biliverdin and carbon monoxide. This relationship suggests that carbon monoxide may serve functions resembling NO. Interestingly, mRNA for 1 of the 2 forms of heme oxygenase is selectively localized to neuronal populations throughout the brain with a pattern of distribution closely resembling guanylyl cyclase (Verma et al., 1992). A role for carbon monoxide in regulating endogenous cGMP issupported by the ability of potent, selective inhibitors of heme oxygenase to deplete endogenous cGMP levels in brain tissue. The notion that carbon monoxide, not NO, is the major physiologic regulator of brain guanylyl cyclase suggests that other targets exist for NO. N O enhances ADP ribosylation of certain platelet proteins (Brune and Lapetina, 1990). In the brain and in red blood cells, the target of NO-stimulated ADP ribosylation is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) @hang and Snyder, 1992; Kots et al., 1992) (Figure 2). GAPDH is auto-ADP ribosylated, and N O enhances this process. Since ADP ribosylation involves the cysteine to which NAD binds in GAPDH catalysis, ADP ribosylation should inhibit GAPDH activity. Because of its crucial role in glycolysis, NO-stimulated GAPDH ADP ribosylation seems a fitting target to mediate N O neurotoxicity. References Bredt, D. S., and Snyder, S. H. (1990). Proc. Natl. Acad. Sci. USA 87, 662-665. Bredt, D. S., and Snyder, S. H. (1992). Neuron 8, 3-11. Bredt, D. S., Hwang, P. M., and Snyder, S. Ii. (1990). Nature 347,766770. Bredt, D. S., Hwang, P. H., Glatt, C., Lowenstein, Snyder, S. H. (1991). Nature 357, 714-716.
C.. Reed, R. R., and
Bredt, D. S., Ferris, C. D., and Snyder, S. H. (1992). J. Biol. Chem. 267, 10976-l 0961. Brune, B., and Lapetina, 266-290.
E. G. (1990). Arch. Biochem.
Biophys. 279,
Burnett, A. L., Lowenstein, C. J., Bredt, D. S., Chang, T. S. K., and Snyder, S. H. (1992). Science 257,401-403. Dawson, T. M., Bredt, D. S., Fotuhi, M., Hwang, P. M., and Snyder, S. H. (1991a). Proc. Natl. Acad. Sci. USA 88, 7797-7601. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., and Snyder, S. H. (1991b). Proc. Natl. Acad. Sci. USA 88, 6366-6371. Garthwaite, Ignarro,
J. (1991). Trends Neural. Sci. 74, 60-67.
L. J. (1990). Annu. Rev. Pharmacol.
Toxicol.
30, 535-560.
Janssens, S. P.,
Shimouchi, A., Quertermous, T., Bloch, D. B., and Bloch, K. D. (1992). J. Biol. Chem. 267, 14519-14522.
Kots, A., Ya., Skurat, A. V., Sergienko, E. A., Bulargina, Severin, E. S. (1992). FEBS Lett. 300, 9-12.
T. V., and
Lamas,S., Marsden,P. A., PrOC.
Li, G. K., Tempst, P., and Michel, T. (1992). Natl. Acad. Sci. USA 89, 6348-6352.
Lowenstein,C. J., Glalt, C. S., Bredt, D. S., and Proc. Natl. Acad. Sci. USA 89, 6711-6715. Lyons, C. R., Orloff, G. J., and Cunningham,
Snyder, S. H. (1992). J. M. (1992). J. Biol.
Chem. 267, 6370-6374.
McMillan, K., Bredt, D. S., Hirsch, D. J., Snyder, S. H., Clark, J. E., and Masters, B. S. S. (1992). Proc. Natl. Acad. Sci. USA, in press. Moncada, S., Palmer, R. M. J., and Higgs,
E. A. (1991). Pharmacol.
Rev. 43, 109-142.
Nathan, C. F., and Hibbs, J.
B., Jr. (1991). Curr. Opin.
Immunol. 3,
65-70. Nowicki, J. P., Duval, D., Poignet, H., and Scatton, B. (1991). Eur. J. Pharmacol. 204,339~340. Pou, S., Pou, W., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992). J. Biol. Chem., in press. Verma, A., Hirsch, D. J., Glatt, C. E., Ronnett, G. V., and Snyder, S. H. (1992). Science, in press. Xie, Q.-W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992). Science 256, 225-226.
Zhang, J., and Snyder, press.
S. H. (1992). Proc. Natl. Acad. Sci. USA, in
