Blood Vessels 1991;28:67-73
Heme-Dependent Activation of Guanylate Cyclase by Nitric Oxide: A Novel Signal Transduction Mechanism1 Louis J. Ignarro Department o f Pharmacology, UCLA School o f Medicine, Center for the Health Sciences, Los Angeles, Calif., USA
Key Words. Cyclic GMP • Guanylate cyclase activation • Nitric oxide • Nitrogen oxides • Endothelium-dependent vasodilation • Endothelium-derived relaxing factor • Vasodilation • Endothelium Abstract. The interaction between nitric oxide (NO) synthesized in one cell and cytosolic guanylate-cyclase-bound heme located in adjacent target cells to generate the NO-heme adduct of guanylate cyclase represents a novel and widespread signal transduction mecha nism that links extracellular stimuli to the biosynthesis of cyclic GMP in target cells. A variety of chemical factors interact with selective extracellular receptors and trigger the bio synthesis of NO from L-arginine. The unique chemistry of NO endows this molecule with the capacity to diffuse rapidly into nearby cells and stimulate cyclic GMP formation. Cyclic GMP acts as a messenger in each cell type to trigger different but complementary cellular responses within a localized environment. This transcellular signaling is a form of rapid intercellular communication allowing the simultaneous local initiation of increased blood flow, inhibition of platelet-induced thrombosis and other cellular functions.
The concept of signal transduction and the involvement of G proteins and other macromolecules in linking extracellular ef fector molecules to elicitation of cellular re sponses was well appreciated long before an understanding developed of the physiologi cal importance of cyclic GMP. Neurotrans mitters, hormones, autacoids and other en dogenous substances interact with selective cell surface receptors to trigger a cascade of molecular events terminating in or constitut
ing the cellular response. In this manner, one or more extracellular effectors can turn on or turn off one or more cellular functions. Nit ric oxide (NO) is a different kind of effector molecule in that it is synthesized in one cell and diffuses into adjacent target cells to trig ger multicellular responses via a common signal transduction mechanism. The objec tive of this report is to provide an account of 1 Supported by grants HL350I4 and HL40922 from the National Institutes o f Health, and a grant from the Laubisch Fund for Cardiovascular Research.
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Introduction
the evidence that supports this unique signal transduction mechanism as well as an inter cellular modulatory role for NO. Activation of soluble guanylate cyclase by nitrogen oxide compounds including NO was first appreciated in the mid 1970s [1-3]. Employing crude enzyme preparations, the activation of guanylate cyclase by NO and nitroso compounds was reported to be hemedependent [3], and enzyme activation by hydroxylamine and azide was attributed to a catalase-dependent oxidation of these agents to NO [4], The knowledge that nitrovasodilators such as nitroprusside and nitroglyc erin stimulated cyclic GMP formation in tis sues [5] led to the original observations that authentic NO is a potent vascular smooth muscle relaxant [6]. Further studies revealed that organic nitrate and nitrite esters react with thiols or sulfhydryl compounds to gen erate NO via the intermediate formation of labile S-nitrosothiols [7], which themselves are potent activators of guanylate cyclase and vasodilators [8]. Soon thereafter, NO and S-nitrosothiols were found to inhibit hu man platelet aggregation [9, 10]. Six years after the discovery of endothe lium-derived relaxing factor (EDRF) [11], the proposal was made that EDRF is NO or a labile nitroso precursor [12, 13]. Our pro posal was based on the observations that the vascular responses to EDRF were virtually indistinguishable from those of authentic NO and that both substances directly acti vated purified soluble guanylate cyclase [12, 14]. Shortly thereafter, EDRF generated from arterial endothelial cells and perfused pulmonary artery and vein was identified chemically as NO [15-17], Subsequently, the formation and release of NO from other cell types or tissues such as activated macro phages [18-20], neutrophils [21, 22], hepatic
Ignarro
Kupffer cells [23] and brain tissue [24-26] were demonstrated. Thus, NO has turned out to be a fairly widespread endogenous molecule with biological actions that may include vascular smooth muscle relaxation, inhibition of platelet function, modulation of phagocytic cell function, stimulation or enhancement of cytotoxicity and modula tion of neurotransmission or other brain functions.
Activation of Guanylate Cyclase by NO NO activates cytosolic guanylate cyclase and is responsible for enzyme activation caused by nitroglycerin, nitroprusside and other nitrogen-containing substances includ ing EDRF. Guanylate cyclase activation causes cyclic GMP accumulation in vascular and nonvascular smooth muscle, endothelial cells, platelets, macrophages, neutrophils and tissues from many organs including brain. NO activation of guanylate cyclase purified from bovine lung, rat liver and hu man platelets is heme-dependent [10, 27, 28], Cytosolic guanylate cyclase is a hemoprotein containing 1 mol of heme/mol ofholoenzyme dimer. The heme moiety can be easily detached to yield heme-deficient en zyme and the latter can be readily reconsti tuted with heme. Heme-deficient guanylate cyclase is unresponsive to NO, whereas the heme-containing and heme-reconstituted forms are activated up to 100-fold by NO. This is because the NO-heme complex, formed when NO reacts with heme, is re sponsible for enzyme activation. Preformed NO-heme activates heme-deficient guany late cyclase by mechanisms that are identical to those of protoporphyrin IX. Endothe lium-derived NO behaves the same as au
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68
NO and Signal Transduction
thentic NO in its mechanism of enzyme acti vation [14, 17]. NO reacts readily with other cellular hemoproteins such as hemoglobin and myoglo bin to yield the corresponding NO-heme ad ducts, both of which markedly activate guanylate cyclase via a rapid, high-affinity ex change or transfer of the NO-heme moiety to the porphyrin-binding site on guanylate cy clase [29], Thus, the endogenous NO mole cules that are available to activate guanylate cyclase may be not only immediately synthe sized from L-arginine but also derived from relatively stable NO-hemoprotein com plexes.
69
tightly bound. Thus, the portion of NOheme that the enzyme really sees is protopor phyrin IX (heme without its iron). A similar mechanism has been proposed for enzyme activation by phenylhydrazine, which forms the phenyl-heme adduct of guanylate cyclase [32]. This concept is illustrated schemati cally in figure 1. Thus, the on signal for guanylate cyclase activation is NO-induced detachment of the heme Fe2+ axial ligand from enzyme protein with continual binding of the porphyrin ring, whereas the off signal is complexation of heme Fe2+ to enzyme protein.
NO-Heme Interaction and Signal Transduction The heme group of guanylate cyclase, which contains iron in the reduced state (Fe2+), has a high binding affinity for NO and binds NO tightly to yield the nitrosylheme complex [30]. In turn, binding of NO to heme greatly increases the apparent bind ing affinity of heme for guanylate cyclase, as indicated by the low equilibrium dissocia tion constant of the guanylate cyclase-NOheme complex (K
Heme-Dependent Activation of Guanylate Cyclase by Nitric Oxide: A Novel Signal Transduction Mechanism1 Louis J. Ignarro Department o f Pharmacology, UCLA School o f Medicine, Center for the Health Sciences, Los Angeles, Calif., USA
Key Words. Cyclic GMP • Guanylate cyclase activation • Nitric oxide • Nitrogen oxides • Endothelium-dependent vasodilation • Endothelium-derived relaxing factor • Vasodilation • Endothelium Abstract. The interaction between nitric oxide (NO) synthesized in one cell and cytosolic guanylate-cyclase-bound heme located in adjacent target cells to generate the NO-heme adduct of guanylate cyclase represents a novel and widespread signal transduction mecha nism that links extracellular stimuli to the biosynthesis of cyclic GMP in target cells. A variety of chemical factors interact with selective extracellular receptors and trigger the bio synthesis of NO from L-arginine. The unique chemistry of NO endows this molecule with the capacity to diffuse rapidly into nearby cells and stimulate cyclic GMP formation. Cyclic GMP acts as a messenger in each cell type to trigger different but complementary cellular responses within a localized environment. This transcellular signaling is a form of rapid intercellular communication allowing the simultaneous local initiation of increased blood flow, inhibition of platelet-induced thrombosis and other cellular functions.
The concept of signal transduction and the involvement of G proteins and other macromolecules in linking extracellular ef fector molecules to elicitation of cellular re sponses was well appreciated long before an understanding developed of the physiologi cal importance of cyclic GMP. Neurotrans mitters, hormones, autacoids and other en dogenous substances interact with selective cell surface receptors to trigger a cascade of molecular events terminating in or constitut
ing the cellular response. In this manner, one or more extracellular effectors can turn on or turn off one or more cellular functions. Nit ric oxide (NO) is a different kind of effector molecule in that it is synthesized in one cell and diffuses into adjacent target cells to trig ger multicellular responses via a common signal transduction mechanism. The objec tive of this report is to provide an account of 1 Supported by grants HL350I4 and HL40922 from the National Institutes o f Health, and a grant from the Laubisch Fund for Cardiovascular Research.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/18/2018 4:09:32 PM
Introduction
the evidence that supports this unique signal transduction mechanism as well as an inter cellular modulatory role for NO. Activation of soluble guanylate cyclase by nitrogen oxide compounds including NO was first appreciated in the mid 1970s [1-3]. Employing crude enzyme preparations, the activation of guanylate cyclase by NO and nitroso compounds was reported to be hemedependent [3], and enzyme activation by hydroxylamine and azide was attributed to a catalase-dependent oxidation of these agents to NO [4], The knowledge that nitrovasodilators such as nitroprusside and nitroglyc erin stimulated cyclic GMP formation in tis sues [5] led to the original observations that authentic NO is a potent vascular smooth muscle relaxant [6]. Further studies revealed that organic nitrate and nitrite esters react with thiols or sulfhydryl compounds to gen erate NO via the intermediate formation of labile S-nitrosothiols [7], which themselves are potent activators of guanylate cyclase and vasodilators [8]. Soon thereafter, NO and S-nitrosothiols were found to inhibit hu man platelet aggregation [9, 10]. Six years after the discovery of endothe lium-derived relaxing factor (EDRF) [11], the proposal was made that EDRF is NO or a labile nitroso precursor [12, 13]. Our pro posal was based on the observations that the vascular responses to EDRF were virtually indistinguishable from those of authentic NO and that both substances directly acti vated purified soluble guanylate cyclase [12, 14]. Shortly thereafter, EDRF generated from arterial endothelial cells and perfused pulmonary artery and vein was identified chemically as NO [15-17], Subsequently, the formation and release of NO from other cell types or tissues such as activated macro phages [18-20], neutrophils [21, 22], hepatic
Ignarro
Kupffer cells [23] and brain tissue [24-26] were demonstrated. Thus, NO has turned out to be a fairly widespread endogenous molecule with biological actions that may include vascular smooth muscle relaxation, inhibition of platelet function, modulation of phagocytic cell function, stimulation or enhancement of cytotoxicity and modula tion of neurotransmission or other brain functions.
Activation of Guanylate Cyclase by NO NO activates cytosolic guanylate cyclase and is responsible for enzyme activation caused by nitroglycerin, nitroprusside and other nitrogen-containing substances includ ing EDRF. Guanylate cyclase activation causes cyclic GMP accumulation in vascular and nonvascular smooth muscle, endothelial cells, platelets, macrophages, neutrophils and tissues from many organs including brain. NO activation of guanylate cyclase purified from bovine lung, rat liver and hu man platelets is heme-dependent [10, 27, 28], Cytosolic guanylate cyclase is a hemoprotein containing 1 mol of heme/mol ofholoenzyme dimer. The heme moiety can be easily detached to yield heme-deficient en zyme and the latter can be readily reconsti tuted with heme. Heme-deficient guanylate cyclase is unresponsive to NO, whereas the heme-containing and heme-reconstituted forms are activated up to 100-fold by NO. This is because the NO-heme complex, formed when NO reacts with heme, is re sponsible for enzyme activation. Preformed NO-heme activates heme-deficient guany late cyclase by mechanisms that are identical to those of protoporphyrin IX. Endothe lium-derived NO behaves the same as au
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 8/18/2018 4:09:32 PM
68
NO and Signal Transduction
thentic NO in its mechanism of enzyme acti vation [14, 17]. NO reacts readily with other cellular hemoproteins such as hemoglobin and myoglo bin to yield the corresponding NO-heme ad ducts, both of which markedly activate guanylate cyclase via a rapid, high-affinity ex change or transfer of the NO-heme moiety to the porphyrin-binding site on guanylate cy clase [29], Thus, the endogenous NO mole cules that are available to activate guanylate cyclase may be not only immediately synthe sized from L-arginine but also derived from relatively stable NO-hemoprotein com plexes.
69
tightly bound. Thus, the portion of NOheme that the enzyme really sees is protopor phyrin IX (heme without its iron). A similar mechanism has been proposed for enzyme activation by phenylhydrazine, which forms the phenyl-heme adduct of guanylate cyclase [32]. This concept is illustrated schemati cally in figure 1. Thus, the on signal for guanylate cyclase activation is NO-induced detachment of the heme Fe2+ axial ligand from enzyme protein with continual binding of the porphyrin ring, whereas the off signal is complexation of heme Fe2+ to enzyme protein.
NO-Heme Interaction and Signal Transduction The heme group of guanylate cyclase, which contains iron in the reduced state (Fe2+), has a high binding affinity for NO and binds NO tightly to yield the nitrosylheme complex [30]. In turn, binding of NO to heme greatly increases the apparent bind ing affinity of heme for guanylate cyclase, as indicated by the low equilibrium dissocia tion constant of the guanylate cyclase-NOheme complex (K
