555
Invited letters to the Editor Nitric oxide and regulation of coronary vascular tone Sir, - The paper by Dr R Smith and colleagues which appears in this issue (p 508) is interesting and poses some complex but important questions which I felt were worthy of further comment. Essentially, the paper has three conceptual parts. The main section of the study addresses the question of the relative potencies of three nitric oxide synthase inhibitors on “basal” (ie, flow induced) and acetylcholine stimulated EDRF activity. The differences in the potency of the three inhibitors at reducing “basal” and stimulated EDRF activity has also been demonstrated previously. As pointed out by the authors, the reasons for these differences are not immediately apparent. The situation is even more complex since not only do differences between “basal” and stimulated EDRF activity exist, but there are also between and within species differences,’ Between and within species heterogeneity in EDRF activity in vascular tissue has been recognised for some time. The mechanisms of this are complicated, involving variations in both EDRF release and responsiveness, though the fundamental biochemical mechanisms responsible are even more obscure. Given this heterogeneity in EDRF activity it is perhaps not surprising that major differences should exist in the potency of the various EDRF synthesis inhibitors, both within and between species. Recently several isoforms of nitric oxide synthase have been described.3 In endothelial cells, the constitutive form of the enzyme has been found in both the soluble and particulate fractions of these cells. One cannot help but wonder whether different endothelial cells from different species, and even from different vessels within the same species, vary in their relative proportion of soluble and particulate isoforms. The question regarding the relative importance of the soluble and particulate enzymes in regulating “basal” and stimulated release of EDRF has also to be addressed. It is possible that the different EDRF synthesis inhibitors affect these isoforms of nitric oxide synthase in different ways especially since the inhibitors differ in their lipid solubilities. Do the ways in which the various EDRF synthesis inhibitors interact kinetically with the various isofornis of nitric oxide synthase differ? This is a possibility. Classically, four types of enzyme inhibition are described: competitive, uncompetitive, non-competitive, and irreversible. Little is known about the type of inhibition which occurs between the various EDRF synthesis inhibitors and the isoforms of nitric oxide synthase. It is usually assumed to be competitive, but this has not been vigorously demonstrated. It is clear that some endothelial cells can metabolise some EDRF synthesis inhibitors to generate L-arginine.‘ This fact alone may be of fundamental importance in determining their relative potency in different circumstances. What do we understand about the terms “basal” and “stimulated” EDRF activity‘? True basal activity implies a complete lack of any stimulus which induces EDRF release. In a purist sense this should include the absence of flow a known, very effective, stimulus for EDRF release. Caution should therefore be observed when comparing “basal” EDRF activity between static ring preparations in a tissue bath with “basal” EDRF activity in a tissue (or organ) subjected to flow, eg, perfused hearts or other vascular beds. “Stimulated’ EDRF activity implies therefore an increase over and above that seen in the “basal” state. Stimuli will therefore include flow and agonists of various sorts. But how much flow‘?
’
Which agonist? Often very little attention is paid to such considerations in much of the published work on EDRF. Unless it is, however, direct comparisons of EDRF activity between different vessels and vascular beds is virtually impossible. A further important consideration is the relative contribution of endothelium derived vasodilator substances other than nitric oxide to vascular relaxation in basal and agonist stimulated EDRF activity. The release of endothelium derived hyperpolarising factor or vasodilator prostanoids, for instance. is not inhibited by the standard inhibitors of nitric oxide. The second part of the study relates to previous work by the authors in which they showed an increased dilator effect specific to nitrovasodilators following inhibition of EDRF activity. Previously published work by other investigators has similarly showed an enhanced vascular response to nitrodilators when endothelium is removed or EDRF activity inhibited. However the situation is by no means clear. For example, previous work from our own laboratory using rabbit aorta showed that in resting ring preparations relaxation responses to glyceryl trinitrate were indeed increased in the absence of endothelium. but relaxation to isoprenaline was similarly increased, ie. was not specific to nitrodilators. When the preparations were preconstricted with 5-hydroxytryptamine, however, relaxation responses to both glyceryl trinitrate and isoprenaline were reduced in the absence of endothelium.’ Pohl and Busse, also using rabbit aorta, failed to demonstrate enhanced responses to nitrovasodilators in noradrenaline preconstricted preparations following removal of endothelium or pretreatment with EDRF inhibitors.’ Also no increased relaxation responses to glyceryl trinitrate could be seen following endothelial denudation in ring preparations of the left anterior descending coronary artery and saphenous vein of the dog preconstricted with prostaglandin F?,,.Other vessels, though, from the same species did exhibit the phenomenon.’ We have to ask the question again - why d o these differences exist? I do not have the answer, but these observations again raise the general point that we should be cautious when extrapolating the tindings seen in one vessel type or species to others. In the third part of the paper, the effectiveness of L-arginine in augmenting “basal” and acetylcholine stimulated EDRF activity and its ability to reverse the inhibition of “basal” and stimulated EDRF activity caused by L-NIO and L-NAME were examined. Many studies have been reported showing reversal by L-arginine of blockade of EDRF activity by nitric oxide synthase inhibitors. Fewer have appeared however showing augmentation of “basal” and stimulated of EDRF activity by L-arginine. It is difficult therefore to make generalised statements concerning these effects of L-arginine, particularly when no studies have addressed these questions specifically. Broadly speaking, however, the majority of studies have shown that L-arginine can reverse the effects of nitric oxide synthase inhibitors on both basal and stimulated EDRF activity. This is not universally the case. however. For example, in one study by Gardiner and co-workers investigating the inhibitory effects of L-NAME on “basal” haemodynamic responses in the rat in vivo, L-arginine was ineffective at reversing the inhibition by L-NAME in the hindquarters whereas it was effective in the renal and
556
mesenteric beds.x In another recent study by Randall and Griffith, these investigators were unable to show reversal of inhibition of EDRF activity stimulated by acetylcholine in the isolated perfused rabbit ear.9 Thus although using a perfused vascular bed from the same species as that used by Smith and colleagues, the findings show a marked contrast in the reversibility of nitric oxide synthase inhibition by L-arginine. The study by Randall and Griffith also differed from that of Smith and colleagues in that the former showed augmentation of “basal” EDRF activity by L-arginine whereas the latter did not. Randall and Griffith did not investigate augmentation of stimulated EDRF activity by L-arginine. What explanations, other than those given in the Smith paper, can be provided to resolve this complex issue? One intriguing possibility is that L-arginine acts in ways other than throu h the nitric oxide synthesis pathway. Calver and colleague)’ clearly show that “basal” vasodilator tone in human dorsal hand veins and forearm resistance bed is reduced equally by both the L- and D-enantiomers of arginine, providing evidence that the vasodilator properties of systemic infusions of L-arginine, at least in vivo in the human, cannot be ascribed to the provision of excess substrate to the L-argininehitric oxide pathway. Very few studies include D-arginine in the protocol so it is difficult to comment further on whether similar “non-specific” actions of arginine are operating in such studies. Could one such non-specific effect of L- and D-arginine result from a reduced breakdown of nitric oxide after its release from the endothelial cell? This seems unlikely since, in a recent study, L-arginine was found to increase superoxide production by endothelium, though the effect of D-arginine was not tested.” In a letter to Lancer, Paton suggests that the hypotensive effect of high doses of some basic amino acids may be due to histamine release.” The generality of this statement needs to be tested, however. The question of whether L-arginine is the only precursor of EDRF must also be asked. In the light of all the available evidence this seems unlikely. Nevertheless, two recent publications suggest this possibility.” l 3 I have tried to provide some constructive comments which I hope workers in this general area will find helpful for any future studies they undertake. The issues raised by Dr Smith and his colleagues are important and perplexing ones. I do not pretend to have the answers; as in most research, the questions raised by this study are greater than those originally posed and a great deal of further work will be necessary if they are to be resolved. MALCOLM J LEWIS
Pharmacology and Therapeutics University of Wales College of Medicine Heath Park, Cardiff CF4 4XN
Christie MI, Griffith TM, Lewis MJ. A comparison of basal and agonist-stimulated release of endothelium-derived relaxing factor from different arteries. Br J P h a m c o l 1989;98:397406. Christie MI, Lewis MJ. A comparison of endothelium-derived relaxing factor activity in the coronary and renal arteries of the pig. Eur J P harmacol 199 I ;202: 143-9. Forstermann U, Schmidt HHHW, Pollock J, ef a/. Isoforms of nitric oxide synthase. Biochem Pharmacol 199I ;42: 1849-57. Hecker M, Mitchell JA, Harris HJ, Katsura M, Thiemermann C, Vane JR. Endothelial cells metabolise N‘-monomethyl-L-arginine to L-citrulline and subsequently to L-arginine. Biochem Biophys Res Comm 1990;167:103743. White DG, Lewis MJ, Griffith TM, Edwards DH, Henderson AH. Influence of endothelium on drug-induced relaxation of the rabbit aorta. Eur J Pharmacol 1986;121:19-23.
6 Pohl U. Busse R. Endothelium-derived relaxant factor inhibits effects of nitrocompounds in isolated arterie\. An1 J Phvriol I 987;21:H307- I 3. 7 Forster C, Main JS, Armstrong PW. Endothelium modulation of the effects of nitroglycerine on blood vessels from dogs with pacing-induced heart failure. Br J Phnnnacol 1990;lOl: 109-14. 8 Gardiner SM, Compton AM, Kemq, PA, Bennett T. Regional and cardiac haemodynamic effects of N ’-nitro-L-arginine methyl ester in conscious, Long Evans rats. Br J Pharmacol 1990; 101:625-3 I , 9 Randall MD, Griffith TM. Differential effects of L-arginine on the inhibition by N”-nitro-L-arginine methyl ester of basal and agonist-stimulated EDRF activity. Br J Phtmnacd 199 I : 104: 743-9. 10 Calver A, Collier J. Vallance P. Dilator actions of arginine in human peripheral vasculature. Clin Sci 1991;81:695-700. I 1 Heim K, Thomas G , Ramwell PW. Effect of substituted arginine compounds on superoxide production in the rabbit aorta. J Phurmucol Exp Ther 1991;257:I13@5. 12 Paton WDM. L-arginine-induced hypotension. Lancer 1990; ii: 1016. 13 Thomas G, Ramwell PW. N,-Nitro-L-arginine benzyl ester, a potent irreversible inhibitor of endothelium-dependent relaxation. Biochem Biriphys Res Comm 199 1;179:1677-82.
Hypoxic preconditioning of ischaemic myocardium Sir, - In this issue of Cardiovascular Research (p 534) Shizukuda and coworkers report that five minutes of hypoxic perfusion of the dog left anterior descending coronary artery, followed by 10 minutes of normoxic reperfusion renders the subtended myocardium resistant to a subsequent prolonged coronary occlusion. In addition, these investigators have demonstrated that pretreatment with five minutes of hypoxia is similar to pretreatment with five minutes of ischaemia in terms of protection against subsequent myocardial infarction. Interestingly, these two forms of preconditioning differ in their effect on regional contractile function, with enhanced contractile function (less stunning) being observed in the hypoxic preconditioned group both before and after infarction. The separation of the phenomena of myocardial stunnin4 and ischaemic preconditioning has been noted previously.’ However in this study the interpretation of the segmental shortening data is clouded by a chance difference at the outset in coronary perfusion pressures between the hypoxic and the ischaemic preconditioned groups (table 11). Furthermore, during reperfusion following the 60 minute occlusion the percent recovery in coronary perfusion pressure is lower in the ischaemic preconditioning group, and this, by loss of the “garden hose effect”, may contribute to the greater systolic bulging seen. Despite this unfortunate disparity between the two groups there is no doubt that preconditioning with hypoxia rather than ischaemia is associated with less stunning immediately following the five minute preconditioning period (preocclusion table 11). How do the findings of this study influence our current understanding of the phenomenon of ischaemic preconditioning?’ In the first instance we must make the assumption that the protection afforded by hypoxic preconditioning has the same underlying mechanism as the protection afforded by ischaemic preconditioning. This, however, is a dangerous assumption without further information. What are the characteristics of the protection afforded by hypoxic preconditioning? Does it wear off following prolonged reperfusion?3 Is it blocked by AI antagonist^?^ Does it protect against arrhythmia?’ These questions become particularly pertinent since hypoxia has previously been noted6 to increase the heart’s resistance to subsequent ischaemia. Assuming that ischaemic and hypoxic preconditioning share the same underlying mechanisms does this study refute
’
Invited letters to the Editor Nitric oxide and regulation of coronary vascular tone Sir, - The paper by Dr R Smith and colleagues which appears in this issue (p 508) is interesting and poses some complex but important questions which I felt were worthy of further comment. Essentially, the paper has three conceptual parts. The main section of the study addresses the question of the relative potencies of three nitric oxide synthase inhibitors on “basal” (ie, flow induced) and acetylcholine stimulated EDRF activity. The differences in the potency of the three inhibitors at reducing “basal” and stimulated EDRF activity has also been demonstrated previously. As pointed out by the authors, the reasons for these differences are not immediately apparent. The situation is even more complex since not only do differences between “basal” and stimulated EDRF activity exist, but there are also between and within species differences,’ Between and within species heterogeneity in EDRF activity in vascular tissue has been recognised for some time. The mechanisms of this are complicated, involving variations in both EDRF release and responsiveness, though the fundamental biochemical mechanisms responsible are even more obscure. Given this heterogeneity in EDRF activity it is perhaps not surprising that major differences should exist in the potency of the various EDRF synthesis inhibitors, both within and between species. Recently several isoforms of nitric oxide synthase have been described.3 In endothelial cells, the constitutive form of the enzyme has been found in both the soluble and particulate fractions of these cells. One cannot help but wonder whether different endothelial cells from different species, and even from different vessels within the same species, vary in their relative proportion of soluble and particulate isoforms. The question regarding the relative importance of the soluble and particulate enzymes in regulating “basal” and stimulated release of EDRF has also to be addressed. It is possible that the different EDRF synthesis inhibitors affect these isoforms of nitric oxide synthase in different ways especially since the inhibitors differ in their lipid solubilities. Do the ways in which the various EDRF synthesis inhibitors interact kinetically with the various isofornis of nitric oxide synthase differ? This is a possibility. Classically, four types of enzyme inhibition are described: competitive, uncompetitive, non-competitive, and irreversible. Little is known about the type of inhibition which occurs between the various EDRF synthesis inhibitors and the isoforms of nitric oxide synthase. It is usually assumed to be competitive, but this has not been vigorously demonstrated. It is clear that some endothelial cells can metabolise some EDRF synthesis inhibitors to generate L-arginine.‘ This fact alone may be of fundamental importance in determining their relative potency in different circumstances. What do we understand about the terms “basal” and “stimulated” EDRF activity‘? True basal activity implies a complete lack of any stimulus which induces EDRF release. In a purist sense this should include the absence of flow a known, very effective, stimulus for EDRF release. Caution should therefore be observed when comparing “basal” EDRF activity between static ring preparations in a tissue bath with “basal” EDRF activity in a tissue (or organ) subjected to flow, eg, perfused hearts or other vascular beds. “Stimulated’ EDRF activity implies therefore an increase over and above that seen in the “basal” state. Stimuli will therefore include flow and agonists of various sorts. But how much flow‘?
’
Which agonist? Often very little attention is paid to such considerations in much of the published work on EDRF. Unless it is, however, direct comparisons of EDRF activity between different vessels and vascular beds is virtually impossible. A further important consideration is the relative contribution of endothelium derived vasodilator substances other than nitric oxide to vascular relaxation in basal and agonist stimulated EDRF activity. The release of endothelium derived hyperpolarising factor or vasodilator prostanoids, for instance. is not inhibited by the standard inhibitors of nitric oxide. The second part of the study relates to previous work by the authors in which they showed an increased dilator effect specific to nitrovasodilators following inhibition of EDRF activity. Previously published work by other investigators has similarly showed an enhanced vascular response to nitrodilators when endothelium is removed or EDRF activity inhibited. However the situation is by no means clear. For example, previous work from our own laboratory using rabbit aorta showed that in resting ring preparations relaxation responses to glyceryl trinitrate were indeed increased in the absence of endothelium. but relaxation to isoprenaline was similarly increased, ie. was not specific to nitrodilators. When the preparations were preconstricted with 5-hydroxytryptamine, however, relaxation responses to both glyceryl trinitrate and isoprenaline were reduced in the absence of endothelium.’ Pohl and Busse, also using rabbit aorta, failed to demonstrate enhanced responses to nitrovasodilators in noradrenaline preconstricted preparations following removal of endothelium or pretreatment with EDRF inhibitors.’ Also no increased relaxation responses to glyceryl trinitrate could be seen following endothelial denudation in ring preparations of the left anterior descending coronary artery and saphenous vein of the dog preconstricted with prostaglandin F?,,.Other vessels, though, from the same species did exhibit the phenomenon.’ We have to ask the question again - why d o these differences exist? I do not have the answer, but these observations again raise the general point that we should be cautious when extrapolating the tindings seen in one vessel type or species to others. In the third part of the paper, the effectiveness of L-arginine in augmenting “basal” and acetylcholine stimulated EDRF activity and its ability to reverse the inhibition of “basal” and stimulated EDRF activity caused by L-NIO and L-NAME were examined. Many studies have been reported showing reversal by L-arginine of blockade of EDRF activity by nitric oxide synthase inhibitors. Fewer have appeared however showing augmentation of “basal” and stimulated of EDRF activity by L-arginine. It is difficult therefore to make generalised statements concerning these effects of L-arginine, particularly when no studies have addressed these questions specifically. Broadly speaking, however, the majority of studies have shown that L-arginine can reverse the effects of nitric oxide synthase inhibitors on both basal and stimulated EDRF activity. This is not universally the case. however. For example, in one study by Gardiner and co-workers investigating the inhibitory effects of L-NAME on “basal” haemodynamic responses in the rat in vivo, L-arginine was ineffective at reversing the inhibition by L-NAME in the hindquarters whereas it was effective in the renal and
556
mesenteric beds.x In another recent study by Randall and Griffith, these investigators were unable to show reversal of inhibition of EDRF activity stimulated by acetylcholine in the isolated perfused rabbit ear.9 Thus although using a perfused vascular bed from the same species as that used by Smith and colleagues, the findings show a marked contrast in the reversibility of nitric oxide synthase inhibition by L-arginine. The study by Randall and Griffith also differed from that of Smith and colleagues in that the former showed augmentation of “basal” EDRF activity by L-arginine whereas the latter did not. Randall and Griffith did not investigate augmentation of stimulated EDRF activity by L-arginine. What explanations, other than those given in the Smith paper, can be provided to resolve this complex issue? One intriguing possibility is that L-arginine acts in ways other than throu h the nitric oxide synthesis pathway. Calver and colleague)’ clearly show that “basal” vasodilator tone in human dorsal hand veins and forearm resistance bed is reduced equally by both the L- and D-enantiomers of arginine, providing evidence that the vasodilator properties of systemic infusions of L-arginine, at least in vivo in the human, cannot be ascribed to the provision of excess substrate to the L-argininehitric oxide pathway. Very few studies include D-arginine in the protocol so it is difficult to comment further on whether similar “non-specific” actions of arginine are operating in such studies. Could one such non-specific effect of L- and D-arginine result from a reduced breakdown of nitric oxide after its release from the endothelial cell? This seems unlikely since, in a recent study, L-arginine was found to increase superoxide production by endothelium, though the effect of D-arginine was not tested.” In a letter to Lancer, Paton suggests that the hypotensive effect of high doses of some basic amino acids may be due to histamine release.” The generality of this statement needs to be tested, however. The question of whether L-arginine is the only precursor of EDRF must also be asked. In the light of all the available evidence this seems unlikely. Nevertheless, two recent publications suggest this possibility.” l 3 I have tried to provide some constructive comments which I hope workers in this general area will find helpful for any future studies they undertake. The issues raised by Dr Smith and his colleagues are important and perplexing ones. I do not pretend to have the answers; as in most research, the questions raised by this study are greater than those originally posed and a great deal of further work will be necessary if they are to be resolved. MALCOLM J LEWIS
Pharmacology and Therapeutics University of Wales College of Medicine Heath Park, Cardiff CF4 4XN
Christie MI, Griffith TM, Lewis MJ. A comparison of basal and agonist-stimulated release of endothelium-derived relaxing factor from different arteries. Br J P h a m c o l 1989;98:397406. Christie MI, Lewis MJ. A comparison of endothelium-derived relaxing factor activity in the coronary and renal arteries of the pig. Eur J P harmacol 199 I ;202: 143-9. Forstermann U, Schmidt HHHW, Pollock J, ef a/. Isoforms of nitric oxide synthase. Biochem Pharmacol 199I ;42: 1849-57. Hecker M, Mitchell JA, Harris HJ, Katsura M, Thiemermann C, Vane JR. Endothelial cells metabolise N‘-monomethyl-L-arginine to L-citrulline and subsequently to L-arginine. Biochem Biophys Res Comm 1990;167:103743. White DG, Lewis MJ, Griffith TM, Edwards DH, Henderson AH. Influence of endothelium on drug-induced relaxation of the rabbit aorta. Eur J Pharmacol 1986;121:19-23.
6 Pohl U. Busse R. Endothelium-derived relaxant factor inhibits effects of nitrocompounds in isolated arterie\. An1 J Phvriol I 987;21:H307- I 3. 7 Forster C, Main JS, Armstrong PW. Endothelium modulation of the effects of nitroglycerine on blood vessels from dogs with pacing-induced heart failure. Br J Phnnnacol 1990;lOl: 109-14. 8 Gardiner SM, Compton AM, Kemq, PA, Bennett T. Regional and cardiac haemodynamic effects of N ’-nitro-L-arginine methyl ester in conscious, Long Evans rats. Br J Pharmacol 1990; 101:625-3 I , 9 Randall MD, Griffith TM. Differential effects of L-arginine on the inhibition by N”-nitro-L-arginine methyl ester of basal and agonist-stimulated EDRF activity. Br J Phtmnacd 199 I : 104: 743-9. 10 Calver A, Collier J. Vallance P. Dilator actions of arginine in human peripheral vasculature. Clin Sci 1991;81:695-700. I 1 Heim K, Thomas G , Ramwell PW. Effect of substituted arginine compounds on superoxide production in the rabbit aorta. J Phurmucol Exp Ther 1991;257:I13@5. 12 Paton WDM. L-arginine-induced hypotension. Lancer 1990; ii: 1016. 13 Thomas G, Ramwell PW. N,-Nitro-L-arginine benzyl ester, a potent irreversible inhibitor of endothelium-dependent relaxation. Biochem Biriphys Res Comm 199 1;179:1677-82.
Hypoxic preconditioning of ischaemic myocardium Sir, - In this issue of Cardiovascular Research (p 534) Shizukuda and coworkers report that five minutes of hypoxic perfusion of the dog left anterior descending coronary artery, followed by 10 minutes of normoxic reperfusion renders the subtended myocardium resistant to a subsequent prolonged coronary occlusion. In addition, these investigators have demonstrated that pretreatment with five minutes of hypoxia is similar to pretreatment with five minutes of ischaemia in terms of protection against subsequent myocardial infarction. Interestingly, these two forms of preconditioning differ in their effect on regional contractile function, with enhanced contractile function (less stunning) being observed in the hypoxic preconditioned group both before and after infarction. The separation of the phenomena of myocardial stunnin4 and ischaemic preconditioning has been noted previously.’ However in this study the interpretation of the segmental shortening data is clouded by a chance difference at the outset in coronary perfusion pressures between the hypoxic and the ischaemic preconditioned groups (table 11). Furthermore, during reperfusion following the 60 minute occlusion the percent recovery in coronary perfusion pressure is lower in the ischaemic preconditioning group, and this, by loss of the “garden hose effect”, may contribute to the greater systolic bulging seen. Despite this unfortunate disparity between the two groups there is no doubt that preconditioning with hypoxia rather than ischaemia is associated with less stunning immediately following the five minute preconditioning period (preocclusion table 11). How do the findings of this study influence our current understanding of the phenomenon of ischaemic preconditioning?’ In the first instance we must make the assumption that the protection afforded by hypoxic preconditioning has the same underlying mechanism as the protection afforded by ischaemic preconditioning. This, however, is a dangerous assumption without further information. What are the characteristics of the protection afforded by hypoxic preconditioning? Does it wear off following prolonged reperfusion?3 Is it blocked by AI antagonist^?^ Does it protect against arrhythmia?’ These questions become particularly pertinent since hypoxia has previously been noted6 to increase the heart’s resistance to subsequent ischaemia. Assuming that ischaemic and hypoxic preconditioning share the same underlying mechanisms does this study refute
’
![[Regulation of vascular tone by nitric oxide (NO) derived from endothelium and nerve].](/assets/img/hold.png)
