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EDRF: A BINDING CONTROVERSY
1376
What will these manipulations do to bioactivity of the hybrid molecule? Such is the brave new world offered by genetic engineering that one may soon have insulins that lower glucose but do not promote fat storage, or somatostatins that inhibit growth hormone but not insulin secretion. At the very least the techniques used by the Genentech group as well as helping to distinguish receptor subtypes will serve to dissect out the separate processes of receptor binding and cell activation. 1. Wharton RP, Ptashne M.
Changing the binding specificity of a repressor by redesigning an &agr;-helix. Nature 1985; 316: 601-05. 2. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from 3.
a mouse.
Nature 1986; 321: 522-25.
Cunningham BC, Henner DJ, Wells JA. Engineering human prolactin to bind to the human growth hormone receptor. Science 1990; 247:
1461-65. 4. Nicholl CS, Bern HA. On the actions of prolactin among the vertebates: is there a common demoninator? In: Wolstenholme GEW, Knight J, eds. Lactogenic hormones. Ciba Foundation Symposium. Edinburgh: Churchill Livingstone, 1972.
A further complication is that endothelium-dependent vasodilators such as acetylcholine also induce endotheliumdependent hyperpolarisation of vascular smooth muscle. However, unlike the effect of EDRF, which is sustained, this is a transient phenomenon, mediated by a diffusible hyperpolarising factor (EDHF) rather than by electrotonic spread of the hyperpolarisation which also occurs in the endothelial cells themselves-13-18 EDHF appears to be chemically distinct from L-arginine-derived EDRF since hyperpolarisation is not blocked by haemoglobin (which scavanges both NO and S-nitrosocysteine) and its action is not mimicked by authentic NO15,16,18 Moreover, different muscarinic receptors are coupled to the release of EDHF and EDRF (M1 and Mz subtypes, respectively).14 Thus EDHF is unlikely to be the putative "carrier" of NO in EDRF. Argument by correspondence cannot provide definitive evidence that EDRF is a nitrosothiol and it remains to be established whether the "NO" which is released by white cells, neurons, and other non-endothelial cell types is also "bound". We have not heard the last of this debate.
EDRF: A BINDING CONTROVERSY factor (EDRF), the endogenous vasodilator manufactured by the vascular endothelium and characterised in 1987,1 was initially thought to be the free nitric oxide (NO) radicap-4 Now Myers and colleagues5 have presented evidence that EDRF is a chemically bound form of NO, such as a nitrosothiol. The hypothesis hinges on the finding that EDRF and S-nitrosocysteine (prepared by the reaction of nitrogen dioxide with L-cysteine) are both about 80 times more potent as relaxants of vascular smooth muscle than authentic NO, while sharing the same biological half-life. Other nitrosothiols such as S-nitrosoglutathione and Snitrosomercaptoethanol were also found to be potent vasodilators but were considerably more stable than EDRF. It has been known for some years that the potency of nitrovasodilators such as glyceryl trinitrate and nitroprusside, which like EDRF relax vascular smooth muscle by stimulating soluble guanylate cyclase, is enhanced by thiol compounds and that nitrosothiols directly stimulate soluble guanylate cyclase.6 Myers et al suggest that transmembrane transport of NO could be facilitated by the presence of a carrier molecule. Variation in the biological half-life of EDRF could also be explained if the factor were a mixture of nitrosothiol compounds. However, a simpler explanation could be that there are different degrees of "destruction" by oxygen and/or the superoxide anion under different experimental conditions. Whilst it has remained clear that the "NO component" of EDRF derives from a terminal guanidino nitrogen atom of L-arginineseveral other lines of evidence have cast doubt on the identity of EDRF as simple NO-eg, EDRF is a more selective relaxant of vascular than of non-vascular smooth muscle than is authentic NO or nitroprusside,8,9 and EDRF and NO have different absorption characteristics on ion affinity exchange columns. to These differences could partly be methodological (it is technically difficult to work with compounds with half-lives of the order of seconds), but a more convincing finding is that EDRF cannot be detected as authentic NO by electroparamagnetic resonance analysisY Moreover, an EDRF which has the characteristics of a nitrovasodilator but is stabilised by low pH, and therefore cannot simply be NO, has been isolated from endothelial cells.12
Endothelium-derived
relaxing
1. Editorial. EDRF Lancet 1987; ii: 137-38. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-26. 3. Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res 1987; 61: 866-79. 4. Furchgott RF. Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte PM, ed. Vasodilatation, vol IV. New York: Raven, 1988: 401-14. 5. Myers PR, Minor RL, Guerra R, Bates JN, Harrison DG. Vasorelaxant properties of the endothelium-derived relaxin factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 1990; 345: 161-63. 6. Ignarro LJ, Gruetter CA. Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite: possible involvement of S-nitrosothiols. Biochim Biophys Acta 1980; 631: 221-31. 7. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesise nitric oxide from L-arginine. Nature 1988; 333: 664-66. 8. Shikano K, Ohlstein EH, Berkovitz BA. Differential selectivity of endothelium-derived relaxing factor and nitric oxide in smooth muscle. Br J Pharmacol 1987; 92: 483-85. 9. Shikano K, Berkowitz BA. Endothelium-derived relaxing factor is a selective relaxant of vascular smooth muscle. J Pharmacol Exp Ther
1987; 243: 55-60.
Long CJ, Shikano K, Berkowitz BA. Anion exchange resins discriminate between nitric oxide and EDRF. Eur J Pharmacol 1987; 142: 317-18. 11. Rubanyi GM, Johns A, Harrison DG, Wilcox D. Evidence that EDRF 10.
may be identical with
an
S-nitrosothiol and
not
with free nitric oxide
(NO). Circulation 1989; 80 (suppl II): 1120. 12. Chu A, Cobb FR, Hagen P-O, Murray JJ. Effects of a stabilised endothelium-derived relaxing factor on the coronary vasculature in awake dogs. Am J Physiol 1989; 257: H1895-99. 13. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarisation of canine coronary smooth muscle. Br J Pharmacol 1988; 93: 515-24. 14. Komori K, Suzuki H. Heterogeneous distribution of muscarinic receptors in the rabbit saphenous artery. Br J Pharmacol 1987; 92: 657-64. 15. Komori K, Lorenz RR, Vanhoutte PM. Nitric oxide, ACh, and electrical and mechanical properties of canine arterial smooth muscle. Am J Physiol 1988; 255: H207-12. 16. Nishiye E, Nakao K, Itoh T, Kuriyama H. Factors inducing endothelium-dependent relaxation in the guinea-pig basilar artery as estimated from the actions of haemoglobin. Br J Pharmacol 1989; 96: 645-55. 17. Beny JL. Endothelial and smooth muscle cells hyperpolarised by bradykinin are not dye coupled. Am J Physiol 1990; 258: H836-41. 18. Beny JL, Brunet PC. Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially mediated vasodilatation of pig coronary arteries. Blood Vessels 1988; 25: 308-11.
What will these manipulations do to bioactivity of the hybrid molecule? Such is the brave new world offered by genetic engineering that one may soon have insulins that lower glucose but do not promote fat storage, or somatostatins that inhibit growth hormone but not insulin secretion. At the very least the techniques used by the Genentech group as well as helping to distinguish receptor subtypes will serve to dissect out the separate processes of receptor binding and cell activation. 1. Wharton RP, Ptashne M.
Changing the binding specificity of a repressor by redesigning an &agr;-helix. Nature 1985; 316: 601-05. 2. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from 3.
a mouse.
Nature 1986; 321: 522-25.
Cunningham BC, Henner DJ, Wells JA. Engineering human prolactin to bind to the human growth hormone receptor. Science 1990; 247:
1461-65. 4. Nicholl CS, Bern HA. On the actions of prolactin among the vertebates: is there a common demoninator? In: Wolstenholme GEW, Knight J, eds. Lactogenic hormones. Ciba Foundation Symposium. Edinburgh: Churchill Livingstone, 1972.
A further complication is that endothelium-dependent vasodilators such as acetylcholine also induce endotheliumdependent hyperpolarisation of vascular smooth muscle. However, unlike the effect of EDRF, which is sustained, this is a transient phenomenon, mediated by a diffusible hyperpolarising factor (EDHF) rather than by electrotonic spread of the hyperpolarisation which also occurs in the endothelial cells themselves-13-18 EDHF appears to be chemically distinct from L-arginine-derived EDRF since hyperpolarisation is not blocked by haemoglobin (which scavanges both NO and S-nitrosocysteine) and its action is not mimicked by authentic NO15,16,18 Moreover, different muscarinic receptors are coupled to the release of EDHF and EDRF (M1 and Mz subtypes, respectively).14 Thus EDHF is unlikely to be the putative "carrier" of NO in EDRF. Argument by correspondence cannot provide definitive evidence that EDRF is a nitrosothiol and it remains to be established whether the "NO" which is released by white cells, neurons, and other non-endothelial cell types is also "bound". We have not heard the last of this debate.
EDRF: A BINDING CONTROVERSY factor (EDRF), the endogenous vasodilator manufactured by the vascular endothelium and characterised in 1987,1 was initially thought to be the free nitric oxide (NO) radicap-4 Now Myers and colleagues5 have presented evidence that EDRF is a chemically bound form of NO, such as a nitrosothiol. The hypothesis hinges on the finding that EDRF and S-nitrosocysteine (prepared by the reaction of nitrogen dioxide with L-cysteine) are both about 80 times more potent as relaxants of vascular smooth muscle than authentic NO, while sharing the same biological half-life. Other nitrosothiols such as S-nitrosoglutathione and Snitrosomercaptoethanol were also found to be potent vasodilators but were considerably more stable than EDRF. It has been known for some years that the potency of nitrovasodilators such as glyceryl trinitrate and nitroprusside, which like EDRF relax vascular smooth muscle by stimulating soluble guanylate cyclase, is enhanced by thiol compounds and that nitrosothiols directly stimulate soluble guanylate cyclase.6 Myers et al suggest that transmembrane transport of NO could be facilitated by the presence of a carrier molecule. Variation in the biological half-life of EDRF could also be explained if the factor were a mixture of nitrosothiol compounds. However, a simpler explanation could be that there are different degrees of "destruction" by oxygen and/or the superoxide anion under different experimental conditions. Whilst it has remained clear that the "NO component" of EDRF derives from a terminal guanidino nitrogen atom of L-arginineseveral other lines of evidence have cast doubt on the identity of EDRF as simple NO-eg, EDRF is a more selective relaxant of vascular than of non-vascular smooth muscle than is authentic NO or nitroprusside,8,9 and EDRF and NO have different absorption characteristics on ion affinity exchange columns. to These differences could partly be methodological (it is technically difficult to work with compounds with half-lives of the order of seconds), but a more convincing finding is that EDRF cannot be detected as authentic NO by electroparamagnetic resonance analysisY Moreover, an EDRF which has the characteristics of a nitrovasodilator but is stabilised by low pH, and therefore cannot simply be NO, has been isolated from endothelial cells.12
Endothelium-derived
relaxing
1. Editorial. EDRF Lancet 1987; ii: 137-38. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-26. 3. Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res 1987; 61: 866-79. 4. Furchgott RF. Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In: Vanhoutte PM, ed. Vasodilatation, vol IV. New York: Raven, 1988: 401-14. 5. Myers PR, Minor RL, Guerra R, Bates JN, Harrison DG. Vasorelaxant properties of the endothelium-derived relaxin factor more closely resemble S-nitrosocysteine than nitric oxide. Nature 1990; 345: 161-63. 6. Ignarro LJ, Gruetter CA. Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite: possible involvement of S-nitrosothiols. Biochim Biophys Acta 1980; 631: 221-31. 7. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesise nitric oxide from L-arginine. Nature 1988; 333: 664-66. 8. Shikano K, Ohlstein EH, Berkovitz BA. Differential selectivity of endothelium-derived relaxing factor and nitric oxide in smooth muscle. Br J Pharmacol 1987; 92: 483-85. 9. Shikano K, Berkowitz BA. Endothelium-derived relaxing factor is a selective relaxant of vascular smooth muscle. J Pharmacol Exp Ther
1987; 243: 55-60.
Long CJ, Shikano K, Berkowitz BA. Anion exchange resins discriminate between nitric oxide and EDRF. Eur J Pharmacol 1987; 142: 317-18. 11. Rubanyi GM, Johns A, Harrison DG, Wilcox D. Evidence that EDRF 10.
may be identical with
an
S-nitrosothiol and
not
with free nitric oxide
(NO). Circulation 1989; 80 (suppl II): 1120. 12. Chu A, Cobb FR, Hagen P-O, Murray JJ. Effects of a stabilised endothelium-derived relaxing factor on the coronary vasculature in awake dogs. Am J Physiol 1989; 257: H1895-99. 13. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarisation of canine coronary smooth muscle. Br J Pharmacol 1988; 93: 515-24. 14. Komori K, Suzuki H. Heterogeneous distribution of muscarinic receptors in the rabbit saphenous artery. Br J Pharmacol 1987; 92: 657-64. 15. Komori K, Lorenz RR, Vanhoutte PM. Nitric oxide, ACh, and electrical and mechanical properties of canine arterial smooth muscle. Am J Physiol 1988; 255: H207-12. 16. Nishiye E, Nakao K, Itoh T, Kuriyama H. Factors inducing endothelium-dependent relaxation in the guinea-pig basilar artery as estimated from the actions of haemoglobin. Br J Pharmacol 1989; 96: 645-55. 17. Beny JL. Endothelial and smooth muscle cells hyperpolarised by bradykinin are not dye coupled. Am J Physiol 1990; 258: H836-41. 18. Beny JL, Brunet PC. Neither nitric oxide nor nitroglycerin accounts for all the characteristics of endothelially mediated vasodilatation of pig coronary arteries. Blood Vessels 1988; 25: 308-11.