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(Circulation. 1995;91:248-250.)
© 1995 American Heart Association, Inc.

Articles

Regulation of Nitric Oxide Production by Tetrahydrobiopterin

Timothy Scott-Burden, PhD

From the Vascular Cell Biology Laboratory, Texas Heart Institute, Houston, Tex.

Correspondence to Dr Scott-Burden, Vascular Cell Biology Laboratory, Texas Heart Institute, PO Box 20345, MC 2-255, Houston, TX 77225-0345.

Key Words: Editorials • vasodilation • nitric oxide

	Introduction

Top Introduction References

 
The discovery of endothelium-derived relaxing factor (EDRF) by Furchgott and Zawadzki¹ in 1980 and its identification as nitric oxide (NO) by Palmer and coworkers² were key observations for our understanding of the physiology of vasodilation. Recently, the realization that tetrahydrobiopterin (BH4) may regulate the availability of EDRF has given us further insights into the regulation of vascular tone and refocused attention on the complex chemistry associated with NO synthase (EC 1.14.13.39) catalysis.^{3 4} NO synthases belong to large family of closely related enzymes that utilize a number of cofactors, including BH4, for production of NO from L-arginine. In the absence of BH4, NO synthases no longer produce NO but can catalyze reactions leading to the production of hydrogen peroxide (H₂O₂). In certain blood vessels, H₂O₂ mediates relaxation, and this may compensate for the loss of NO production due to the absence of BH4. Observations to support this have been made by Cosentino and Katui⁵ using isolated rings of canine coronary arteries and 2,4,-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of BH4 synthesis.

The importance of NO for many physiological pathways and its ubiquitous distribution throughout the body led to its nomination as the molecule of the year in 1992.⁶ An extensive family of NO synthases has been identified, and they can be subdivided into isoforms that are expressed constitutively and those that are evident only after induction. Production of NO by the latter group is often associated with cytotoxicity, and this is due in part to the very large quantities synthesized by the inducible enzyme systems.⁷ Both enzyme types have been cloned from a number of species and tissues, and they share common features related to the reactions they catalyze with L-arginine (see References 4 and 7 for reviews). The conversion of one of the guanidinonitrogens of L-arginine to NO is an oxidation process involving five electrons, and two of these are derived from BH4.⁷ Tetrahydrobiopterin is a normal cofactor for a family of enzymes called monooxygenases.⁴ The tissue distribution of BH4 is consistent with the sites of monooxygenase activity, but there is little information available on the levels of this factor in the cells (endothelial and smooth muscle) of the blood vessel wall.⁸ Tetrahydrobiopterin cannot be detected in cultured smooth muscle cells, and induction of NO production by cytokines requires de novo synthesis of the pterin.^{9 10} Guanosine triphosphate (GTP) is the precursor for the de novo biosynthesis of BH4, and the first step conversion of GTP to dihydroneopterin triphosphate is catalyzed by GTP cyclohydrolase I (EC 3.5.4.16; Reference 9). Cytokines that induce robust production of NO in several different cell types also induce the expression of GTP cyclohydrolase I.^{10 11} Rapid expression of GTP cyclohydrolase I mRNA in cultured smooth muscle cells is elicited by activation of adenylyl cyclase or by analogues of cAMP.¹² Enzyme activity is also increased by elevation of cAMP,¹³ but it is not clear if this is due to induction of enzyme synthesis or the activation of existing enzyme. Intuitively, activation of existing GTP cyclohydrolase I by elevated levels of cAMP could involve phosphorylation by cAMP-dependent protein kinase (A kinase). However, a consensus sequence for kinase A phosphorylation is absent in all the mammalian GTP cyclohydrolases that have been cloned to date.¹⁴

The induction of NO production by cultured cells in response to single, individual cytokines has yielded conflicting results suggesting that a combination of molecules is required.¹⁵ A possible explanation for this controversy may be the inability of certain cytokines to induce expression of GTP cyclohydrolase in conjunction with NO synthase. The efficacy of endotoxin induction of NO production, typically associated with sepsis, may reflect its ability to induce expression of several cytokines by target cells and thus ensure that both NO synthase and GTP cyclohydrolase I are subsequently coexpressed. The production of NO by cultured murine vascular endothelial cells is dependent on the prior expression of GTP cyclohydrolase.³ Inhibition of GTP cyclohydrolase activity by DAHP attenuates production of NO by IL-1 ß–treated smooth muscle cells.^{9 10}

These observations establish the importance of BH4 for NO synthase activity, but the mechanism of its involvement remains unresolved. Since BH4 is a cofactor for the catalysis mediated by monooxygenase, the role that it plays in these reactions may conceivably offer insight into its contribution to NO synthase catalysis. In the reaction catalyzed by the monooxygenase phenylalanine hydroxylase, BH4 contributes to hydroxylation chemistry. Also, biopterin contributes to the hydroxylation of substrates by the fatty acid monooxygenases. NO synthases exhibit a number of features in common with this group of enzymes, and in addition, they show some degree of sequence homology to the cytochrome P-450 reductase family. Purification of NO synthases from a number of sources has indicated that they possess binding sites for NAHPH, FMN, and FAD similar to the cytochrome P-450 reductases, and they contain an iron-protoporphyrin IX prosthetic group.⁴ Since NO synthases have a number of features in common with P-450 reductases and monooxygenases, BH4 may facilitate the hydroxylation of one of the amidine nitrogens of L-arginine. This is the first step catalyzed by NO synthase in the conversion of arginine to NO and citrulline. This would result in the oxidation of the pterin, which then could be recycled through the flavin nucleotides via NADPH. This attractive hypothesis remains to be proven; specific studies to address this suggest that BH4 is neither recycled in solution nor consumed during the catalysis process.¹⁶

A number of studies suggest that bound BH4 stabilizes the dimeric peptide structure of NO synthases, and this may be its mode of action. The most compelling evidence for this role comes from studies on the depression of cytokine-induced NO synthase activity by transforming growth factor-ß (TGFß). Induction of NO synthase by interferon- (INF-) in mouse peritoneal macrophages was depressed by TGFß as a consequence of elevated proteolysis of the enzyme.¹⁷ Also, this growth factor suppressed the induction of GTP cyclohydrolase mRNA in cytokine-treated smooth muscle, which presumably results in reduced BH4 production.¹⁰ NO synthase isolated in the presence of BH4 exhibited a 1:1 stoichiometry with respect to enzyme and the pterin. Addition of exogenous BH4 to such preparations did not enhance enzymatic activity. However, if enzyme was isolated in the absence of BH4, the ratio of pterin to peptide was only 0.2:1.⁴ Enzyme isolated in the absence of BH4 is less stable to freeze-thaw cycles and requires addition of exogenous cofactor to exhibit similar activity to preparations obtained in the presence of the pterin.¹²

Clearly, the exact role of BH4 in the production of NO remains to be clarified. The sequence homology between NO synthase and cytochrome P-450 reductase suggests that the presence of the pterin is key to the production of NO in contrast to superoxide anions or peroxides associated with P-450 reductase activity. Cosentino and Katui⁵ demonstrate that relaxation of canine coronary arteries, under normal conditions where presumably BH4 is not lacking, is sensitive to inhibition by N^G-nitro-L-arginine methyl ester (L-NAME), an established inhibitor of NO synthase. However, under conditions where BH4 production was attenuated by DAHP, L-NAME had no effect on the relaxations elicited by the calcium ionophore A23187, but catalase abolished such vasodilation. These data suggest that NO synthase may provide two molecules responsible for vasorelaxation of certain vascular beds. The authors also point out that under conditions where BH4 levels are suboptimal, the production of H₂O₂ by NO synthase may lead to hydroxyl radical production and oxidative tissue damage. Therefore, endothelial dysfunction may be associated with or caused by production of potential harmful oxidizing agents by endothelial cell NO synthase.

The redox state of NO is currently under close scrutiny, with different forms of the same entity having very different biological effects. EDRF is most likely the reduced form of NO (NO^·), which can interact with superoxide anion to form peroxynitrite.¹⁸ However, other forms of NO (NO⁺ and NO^-) may be responsible for vasodilation (for review, see Reference 18). Whether BH4 plays any role in the regulation of which form of NO is produced is unknown, but the complexity of the redox chemistry suggests that possibility.

Cosentino and Katui⁵ did not attempt to measure the levels of BH4 in the tissues used in their experiments, and this is not a trivial task. Their data suggest that incubation of tissues for 6 hours with DAHP (to inhibit BH4 synthesis) leads to sufficient reduction in the levels of BH4 to impair NO production, and H₂O₂ is generated instead. Since endothelial cell NO synthase is constitutively expressed and BH4 is bound to the enzyme, turnover of the synthase may be required to deplete the system of biopterin. Another possible explanation for the production of H₂O₂ may be that incubation of tissues leads to loss of L-arginine substrate, and under these conditions, NO synthase reduces O₂ to produce peroxide.¹⁸ However, the authors demonstrated that NO-dependent relaxations were restored in BH4-depleted tissues (incubation with DAHP) just by addition of 6-methyl-tetrahydrobiopterin, a liposoluble analogue of BH4. In their article, Cosentino and Katui⁵ have demonstrated that a close relation between NO synthase and GTP cyclohydrolase I exists in the endothelium. The extent of this relation and what role it plays in other tissues where NO exerts important (patho)physiological effects remain to be investigated.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

Received May 16, 1994;

	References

Top Introduction References

 

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