(Circulation. 1997;95:1101-1103.)
© 1997 American Heart Association, Inc.
Articles |
What Are the Molecular Mechanisms for the Antiproliferative Effects of Nitric Oxide and cGMP in Vascular Smooth Muscle?
Correspondence to Ferid Murad, MD, PhD, 1421 Lake Rd, Lake Forest, IL 60045.
Key Words: Editorials • molecular biology • muscle, smooth
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Introduction |
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 Top Introduction References |
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Although the smooth muscle antiproliferative effects of nitric oxide and cGMP in vascular preparations have been known for several years, the precise molecular mechanisms for these effects remain unknown. Presumably these effects participate in several vascular processes, including atherogenesis, reperfusion injury, angiogenesis, inflammation, tumor growth, wound healing, and tissue grafts. A molecular understanding of these effects would no doubt provide additional therapeutic interventions for these important disorders and processes. Although the information and publications in the field of nitric oxide and cGMP have grown logarithmically in the past two decades, many important questions remain to be answered and resolved. More than 4500 publications addressing these messengers were expected in 1996, probably more than for any other research topic. Nevertheless, many important experiments remain to be done. Readers are referred to several recent reviews summarizing the effects of nitric oxide and cGMP (References 1-3). Yu and colleagues4 address the vascular smooth muscle antiproliferative effects of these messengers in this issue of Circulation.
The authors find that SNP, a nitrovasodilator and nitric oxide donor or prodrug, and A-02131-1, a selective inhibitor of cGMP phosphodiesterase, decrease the proliferation of primary cultures of rat vascular smooth muscle cells that have been stimulated with one of several growth factors, such as epidermal growth factor, platelet-derived growth factor, phorbol myristate, or okadaic acid. The authors find that the effects are also mimicked by a cGMP analogue, 8-Br-cGMP, which can activate cGMP-dependent protein kinase (PKG) but is more resistant to hydrolysis by phosphodiesterase. The antiproliferative effects of SNP or 8-Br-cGMP were blocked by KT5823, presumably a selective inhibitor of cGMP-dependent protein kinase, but not by 2',5'-dideoxyadenosine (an adenylyl cyclase inhibitor) or (R)-p-adenosine 3',5'-cyclic monophosphothioate, a selective cAMP-dependent protein kinase inhibitor. The inhibitor of PKG, KT5823, reported by Yu et al4 is probably KT5822, which was reported by Kase et al,5 and probably represents a typographical error. This heterocyclic compound, a charged molecule derived from the culture broth of Nocardiopsis species, shows some partial selectivity of 
15- to 33-fold for PKG inhibition compared with protein kinase A and protein kinase C when partially purified kinase preparations are used. This competitive inhibitor with respect to the substrate ATP was not tested with other kinases or purified enzymes or in intact cells and tissues to establish its cellular permeability and specificity. Its charged nature at physiological pH would suggest rather poor cellular permeability, which is unfortunately a feature of most kinase inhibitors that markedly limits their usefulness.6
The authors go on to show that the antiproliferative effects of SNP and 8-Br-cGMP are associated with decreased activity of MAP kinase; MAP kinase kinase; and their regulatory proteins, RAS and Raf-1, which couple extracellular growth factor receptor–coupled tyrosine kinases to the MAP kinase cascade. The authors suggest that nitric oxide derived from SNP increases guanylyl cyclase activity and cGMP accumulation, which then activates PKG. They suggest that the activity of Raf-1 kinase is decreased as a result of cGMP-dependent phosphorylation of the protein, but they do not describe the precise mechanism or phosphorylation site.
These studies are of considerable interest and may relate to other effects of nitric oxide and cGMP in other cell types and model systems. The potential interrelationships of the tyrosine kinase and serine/threonine kinase cascades offer much to one's imagination, as do the potential interrelationships of cGMP and cAMP in signal transduction.
However, the study by Yu et al, although of importance, has several flaws, and additional work is certainly needed. For example, stable vascular smooth muscle cells generally are deficient in cGMP-dependent protein kinase for reasons that are unknown. Thus, primary smooth muscle cultures, which do possess cGMP-dependent protein kinase activity, are required in such studies. This invites additional problems such as cellular heterogeneity, reproducible growth conditions, and the presence of other growth factors and cytokines from the serum or contaminating cells.
The concentrations of SNP used in the experiments by Yu et al are relatively high compared with the concentrations required to elevate cGMP in vascular smooth muscle and alter endogenous smooth muscle protein phosphorylation that is cGMP dependent.7 8 9 It is possible to show marked alterations in endogenous protein phosphorylation in intact smooth muscle that is cGMP-dependent with submicromolar concentrations of SNP.
Although the effects of SNP and 8-Br-cGMP were concentration dependent, other nitrovasodilators and controls for 8-Br-cGMP were not tested. Some of the relatively long incubation times are also of concern considering the rapid effects of SNP on cGMP accumulation and vascular smooth muscle protein phosphorylation.7 8 9
Considerable progress has been made in developing some relatively selective inhibitors of some of the protein kinases, such as protein kinase A, G, and C. However, these are generally charged organic compounds or peptides with poor permeability in intact cell experiments and are more useful in cell-free incubations on permeabilized cells. The authors do not completely address the specificity of the PKG inhibitor KLT5822 and do not provide data or reference to its permeability and efficacy with intact cells. To date, dozens of protein kinases have been described, and the development of selective inhibitors is a formidable task.
With the cell-free reconstitution experiments examining PKG phosphorylation of Raf-1 (Fig 9 in the article by Yu et al), the authors use a commercial preparation of PKG and do not describe its purity and presumed lack of other kinases in their preparation. They also do not show that the Raf-1 phosphorylation in cell-free systems is indeed cGMP dependent and blocked by the PKG inhibitor KT5822. This might establish that other kinases are not participating in their effects.
It has been known for more than two decades from the work of Krebs, Greengard, and others that many proteins can serve as cyclic nucleotide–dependent protein kinase substrates in cell-free preparations. This does not provide sufficient evidence that these proteins are indeed physiological substrates in intact cells. One must proceed to demonstrate phosphorylation in intact cell preparations to establish relevance and avoid artifactual phosphorylation. Optimally, one should also show the stoichiometry of phosphorylation and, if possible, the amino acid residue of importance in the protein substrate.
Another commonly overlooked problem with commercially obtained biological reagents such as growth factors, cytokines, and enzymes is the possible presence of various preservatives, antibacterials, and stabilizers such as azide and fluoride. Azide can be converted to nitric oxide by catalase and other heme proteins and increases cGMP formation in intact cells as well as cell-free systems.10 11 12 13 14 Furthermore, azide and nitric oxide can also increase cAMP formation from ATP catalyzed by guanylyl cyclase,15 16 another reason to test the specificity of the 8-Br-cGMP effect. One should dialyze or gel filter such preparations if such additives are present to eliminate possible artifactual alterations in cGMP metabolism. Other additives and contaminants in preparations could have other effects.
Although the work of Yu et al is of considerable interest and addresses a very important topic, their work invites a number of additional experiments for confirmation of the proposed cascade and greater definition of the precise mechanism of action of nitric oxide and cGMP. For example, the rather long incubation times of some experiments (1 hour and in some cases 
1 day) do not exclude some of the effects of nitric oxide and cGMP being mediated through altered transcription or expression of some critical regulatory proteins or enzymes. Nitric oxide has been shown to decrease the transcription and expression of some proteins, such as guanylyl cyclase (A. Papapetropoulos et al, unpublished data, 1996). Demonstration of cGMP-dependent incorporation of 32P into Raf-1 in an intact cell preparation would add considerable support to their hypothesis.
In spite of the incompleteness of some of the work presented and some of the deficiencies noted, the publication presents yet another important cascade system and process that may be regulated by nitric oxide and cGMP. With some additional work and confirmation, these effects could be added to the growing list of processes regulated by these messengers and signal-transduction molecules that are summarized in the Table
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Selected Abbreviations and Acronyms |
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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1. Murad F, ed. Cyclic GMP synthesis, metabolism and function. In: Advances in Pharmacology. New York, NY: Academic Press; 1994;26:1-335.
2. Ignarro L, Murad F, eds. Nitric oxide: biochemistry, molecular biology, and therapeutic implications. In: Advances in Pharmacology. New York, NY: Academic Press; 1995;34:1-516.
3.
Murad F. Signal transduction using nitric oxide and cyclic guanosine monophosphate. JAMA. 1996;276:1189-1192.
4.
Yu S-M, Hung L-M, Lin C-C, Ou JT, Yu J-S. cGMP-Elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway. Circulation. 1997;95:1269-1277.
5. Kase H, Iwahashi K, Nakanishi S, Matsuda Y, Yamada K, Takahashi M, Murakata S, Sato A, Kaneko M. K252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide dependent protein kinase. Biochem Biophys Res Commun.. 1987;142:436-440.[Medline] [Order article via Infotrieve]
6. Francis SH, Corbin JD. Progress in understanding the mechanism and function of cyclic GMP-dependent protein kinase. In: Murad F, ed. Cyclic GMP synthesis, metabolism, and function. In: Advances in Pharmacology. New York, NY: Academic Press; 1994;26:115-170.
7. Rapoport RM, Draznin MB, Murad F. Endothelium dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature. 1983;306:274-276.[Medline] [Order article via Infotrieve]
8. Fiscus RR, Rapoport RM, Murad F. Endothelium-dependent and nitrovasodilator-induced activation of cyclic GMP-dependent protein kinase in rat aorta. J Cyclic Nucl Protein Phosphor Res. 1983;9:415-425.[Medline] [Order article via Infotrieve]
9. Rapoport RM, Murad F. Endothelium-dependent and nitrovasodilator-induced relaxation of vascular smooth muscle: role for cyclic GMP. J Cyclic Nucl Protein Phosphor Res. 1983;9:281-296.[Medline] [Order article via Infotrieve]
10.
Kimura H, Mittal CK, Murad F. Activation of guanylate cyclase from rat liver and other tissues with sodium azide. J Biol Chem. 1975;250:8016-8022.
11. Kimura H, Mittal CK, Murad F. Increases in cyclic GMP levels in brain and liver with sodium azide, an activator of guanylate cyclase. Nature. 1975;257:700-702.[Medline] [Order article via Infotrieve]
12. Mittal CK, Kimura H, Murad F. Requirement for a macromolecular factor for sodium azide activation of guanylate cyclase. J Cyclic Nucl Res. 1975;1:261-269.
13. Katsuki S, Arnold W, Mittal CK, Murad F. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cyclic Nucl Res. 1977;3:23-35.
14. Murad F, Mittal CK, Arnold WP, Katsuki S, Kimura H. Guanylate cyclase: activation by azide, nitro compounds, nitric oxide, and hydroxyl radical and inhibition by hemoglobin and myoglobin. Adv Cyclic Nucl Res. 1978;9:145-158.[Medline] [Order article via Infotrieve]
15.
Mittal CK, Murad F. Formation of adenosine 3',5'-monophosphate by preparations of guanylate cyclase from rat liver and other tissues. J Biol Chem. 1977;252:3136-3140.
16. Mittal CK, Braughler JM, Ichihara K, Murad F. Synthesis of adenosine 3',5'-monophosphate by guanylate cyclase: a new pathway for its formation. Biochim Biophys Acta. 1979;585:333-342.[Medline] [Order article via Infotrieve]
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