- Email: [email protected]
Acetylcholine-mediated vasodilation in the forearm circulation of patients with heart failure: indirect evidence for the role of endothelium-derived hyperpolarizing factor
Acetylcholine-Mediated Vasodilation in the Forearm Circulation of Patients With Heart Failure: Indirect Evidence for the Role of Endothelium-Derived Hyperpolarizing Factor Stuart D. Katz,
MD,
and Henry Krum,
MD, PhD
Vasomotor responses to intraarterial administration of acetylcholine are mediated by release of nitric oxide, prostaglandins, and an unidentified hyperpolarizing factor from vascular endothelial cells. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to the vasodilatory response to acetylcholine in the skeletal muscle circulation of patients with congestive heart failure (CHF) has not been previously characterized. Accordingly, to specifically assess the role of EDHF, the regional vascular effects of sequential administration of acetylcholine and nitroglycerin in the brachial artery were determined in the forearm circulation with straingauge venous occlusion plethysmography in patients with CHF and in normal subjects during combined systemic inhibition of cyclooxygenase activity with indo-
methacin and regional inhibition of nitric oxide synthase activity with L-NG-monomethylarginine (L-NMMA). After administration of indomethacin, infusion of L-NMMA significantly decreased the forearm blood flow response to acetylcholine in normal subjects (5.4 ⴞ 1.2 to 3.5 ⴞ 0.6 ml/min/100 ml, p <0.05) but not in patients with CHF (5.7 ⴞ 1.3 to 5.7 ⴞ 1.4 ml/min/100 ml). Infusion of L-NMMA did not change forearm blood flow responses to nitroglycerin in either group. The presence of a noncyclooxygenase, non–nitric-oxide relaxing factor indicates that EDHF, rather than nitric oxide, may be the predominant endothelium-derived substance mediating vasodilation in response to acetylcholine in patients with CHF. 䊚2001 by Excerpta Medica, Inc. (Am J Cardiol 2001;87:1089 –1092)
ndothelium-dependent vasomotor effects of acetylcholine are mediated by release of nitric oxide E produced by the constitutive isozyme of nitric oxide
tric oxide and abnormal production of vasoconstricting prostaglandins.7–10 The role of EDHF in the vasodilatory response to acetylcholine in the skeletal muscle circulation of patients with CHF has not been previously characterized. The present study was undertaken to specifically determine the role of the EDHF in the vasodilatory response to administration of acetylcholine in the forearm circulation of patients with CHF. The regional vascular effects of acetylcholine were determined in patients with CHF and in normal subjects during combined systemic inhibition of cyclooxygenase activity with indomethacin and regional inhibition of nitric oxide synthase activity with G L-N -monomethylarginine (L-NMMA). Under these experimental conditions, prostaglandin- and nitric oxide-mediated effects of acetylcholine are eliminated, with residual vasodilation attributable to the actions of EDHF.1,2
synthase, prostaglandins produced by the cyclooxygenase pathway, and an unidentified endothelium-derived hyperpolarizing factor (EDHF) produced by the cytochrome P450 pathway.1,2 The relative contribution of each factor to the observed endothelium-dependent vasomotor responses to acetylcholine varies by species, vessel diameter, and regional circulation under study.2– 4 In patients with congestive heart failure (CHF), the vasodilatory response to acetylcholine in the skeletal muscle arteriolar circulation is decreased when compared with that of normal subjects.5,6 The reduced vasodilatory response in CHF is partially attributable to reduced bioavailability of niFrom Columbia Presbyterian Medical Center, Division of Circulatory Physiology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York. This study was supported by an investigatorship award from the American Heart Association, New York City Affiliate, New York, New York; by Grant NHLBI R29 HL51433 from the National Institutes of Health, Bethesda, Maryland; by the National Heart Foundation of Australia Clinical Research Program, and Grant 5 M01RR00645, Division of Research Resources, General Clinical Research Program, National Institutes of Health, Bethesda, Maryland. Manuscript received September 21, 2000; revised manuscript received and accepted November 27, 2000. Address for reprints: Stuart D. Katz, MD, Columbia Presbyterian Medical Center, Division of Circulatory Physiology, Room MHB5435, 177 Fort Washington Avenue, New York, New York 10032. E-mail address: [email protected]. ©2001 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 87 May 1, 2001
METHODS
Study group: Thirteen men and 1 woman (mean age, 50 ⫾ 12 years [range 32 to 65]) with chronic stable CHF due to nonischemic cardiomyopathy were studied. The mean left ventricular ejection fraction was 22 ⫾ 4%. Nine patients were in New York Heart Association functional class II and 5 patients were in functional class III. Cardiovascular medications, which included diuretics, angiotensin-converting enzyme inhibitors, and digoxin in all patients, were withheld 24 hours before the study. Patients with edema, serum sodium ⬍136 mEq/L, or a history of 0002-9149/01/$–see front matter PII S0002-8149(01)01466-7
1089
diabetes mellitus, hypercholesterolemia, or active smoking habits were excluded. Nine men and 1 woman (mean age, 43 ⫾ 13 years [range 27 to 66]) without a history of chronic illness participated as normal controls. The mean age of these subjects was not different from that of patients with CHF. These subjects were nonsmokers, had a normal physical examination, and were not taking chronic medications. The study was approved by the ethical review board of the Columbia Presbyterian Medical Center. All subjects gave written informed consent before the study. Venous occlusion plethysmography: Forearm blood flow (ml/min/100 ml of forearm volume) was determined with venous occlusion strain-gauge plethysmography as previously described in detail.9 Five plethysmographic measurements were averaged for each blood flow determination. Intraarterial drug administration: Acetylcholine and nitroglycerin were administered as temporally sequential 2-minute continuous infusions (1 ml/min) to achieve regional forearm blood concentrations ranging from 10⫺8 to 10⫺6 M. Infusions of acetylcholine and nitroglycerin were separated by 5 to 10 minutes to allow forearm blood flow to return to basal values. To evaluate the effects of L-NMMA at comparable levels of shear stress in patients with CHF and normal subjects, the doses of acetylcholine and nitroglycerin were individually adjusted to increase forearm blood flow to 2 to 3 times the resting value. L-NMMA, a competitive inhibitor of nitric oxide synthase, was administered as a continuous 1 ml/min infusion for 5 minutes at a concentration of 40 M. This dose of L-NMMA has previously been reported to fully inhibit nitric oxide synthase activity in isolated vascular tissue and in the intact human forearm circulation.1,11 Study protocol: To exclude the effects of prostaglandins on acetylcholine-induced vasodilation, all subjects received 50 mg of indomethacin, a cyclooxygenase inhibitor, by mouth 1 hour before the study.9,12 A 20-gauge angiocath was placed in the brachial artery under local anesthesia for regional drug administration. Forearm blood flows were measured at rest and during administration of acetylcholine and nitroglycerin before and after infusion of L-NMMA. Mean arterial pressure was recorded in the contralateral arm with an automated cuff method. Data analysis: All values are stated as mean ⫾ SEM. Forearm blood flows and mean arterial pressure in patients with CHF and normal subjects at rest and during administration of acetylcholine and nitroglycerin were compared before and after administration of L-NMMA with a repeated measures analysis of variance model. A 2-tailed p value⬍0.05 was considered statistically significant.
RESULTS Resting forearm blood flows did not differ between patients with CHF and normal subjects before infusion of L-NMMA (2.1 ⫾ 0.3 vs 2.8 ⫾ 0.3 ml/min/100 ml) or after infusion of L-NMMA (2.1 ⫾ 0.3 vs 2.1 ⫾ 0.3 ml/min/100 ml). L-NMMA significantly decreased 1090 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 1. Forearm blood flow responses to administration of acetylcholine before (open bar) and after (filled bar) infusion of L-NMMA in normal subjects and in patients with CHF. *p <0.05 versus before L-NMMA.
TABLE 1 Forearm Blood Flow Responses (ml/min/100 ml) to Administration of Nitroglycerin Before and After Infusion of L-NMMA in Normal Subjects and in Patients With CHF
Normal subjects CHF
Before L-NMMA
After L-NMMA
4.2 ⫾ 0.3 5.7 ⫾ 0.9
4.1 ⫾ 0.6 5.9 ⫾ 1.0
resting forearm blood flow in normal subjects (p ⬍0.05) but not in patients with CHF. To achieve a comparable degree of vasodilation in the 2 groups, mean doses of acetylcholine and nitroglycerin administered to patients with CHF were significantly greater than doses in normal subjects (⫺6.5 ⫾ 0.3 vs ⫺7.7 ⫾ 0.2 log units for acetylcholine and ⫺7.1 ⫾ 0.2 vs ⫺7.6 ⫾ 0.2 log units for nitroglycerin, both p ⬍0.05, CHF patients vs normal subjects). As determined by study protocol, forearm blood flows in response to administration of individually adjusted doses of acetylcholine did not differ in patients with CHF and normal subjects before infusion of L-NMMA (5.7 ⫾ 1.3 vs 5.4 ⫾ 1.2 ml/min/100 ml, Figure 1). After infusion of L-NMMA, the forearm blood flow response to acetylcholine significantly decreased to 3.5 ⫾ 0.6 ml/min/100 ml in normal subjects (p ⬍0.05 vs before L-NMMA), but did not change in patients with CHF (5.7 ⫾ 1.4 ml/min/100 ml, Figure 1). Forearm blood flows in response to administration of nitroglycerin did not differ between patients with CHF and normal subjects before infusion of L-NMMA and did not change after infusion of L-NMMA in either group (Table 1). Mean arterial pressures did not differ in patients with CHF and normal subjects before infusion of L-NMMA at rest (86 ⫾ 5 vs 91 ⫾ 4 mm Hg) and did not change during drug infusions (data not shown). MAY 1, 2001
DISCUSSION The present study demonstrates that during systemic inhibition of cyclooxygenase with indomethacin, regional inhibition of nitric oxide synthase with L-NMMA significantly decreased the vasodilatory response to acetylcholine in normal subjects but not in patients with CHF. The presence of a noncyclooxygenase, non–nitric-oxide relaxing factor indicates that EDHF, rather than nitric oxide, may be the predominant endothelium-derived substance mediating vasodilation in response to acetylcholine in the forearm circulation of patients with CHF. The presence of EDHF as an endothelium-dependent mediator of the vascular effects of acetylcholine was first described in small mesenteric arteries of the guinea pig and subsequently confirmed in other species and regional circulations by several investigators.1,2,13 Although its specific chemical identity remains elusive, pharmacologic studies suggest that EDHF is a product of the cytochrome P450 metabolic pathway, likely a member of the epoxyeicosatrienoic acid family.2 Because its specific chemical identity remains unknown, most evidence of the existence of EDHF is derived from observations of the vasomotor and membrane voltage potential effects of acetylcholine in the presence of inhibitors of nitric oxide synthase and cyclooxygenase.2 Under these experimental conditions and in endothelial nitric oxide synthase (eNOS) knockout mice, EDHF appears to play a greater role in small arterioles when compared with larger arterioles or conduit artery rings.3,14 The vasodilatory response to acetylcholine is decreased in patients with CHF when compared with normal subjects.5,6 Previous reports indicate that the decreased response to acetylcholine may be partly attributable to the presence of a vasoconstricting prostanoid.9,10 In the present study, pretreatment with indomethacin eliminated the potential contribution of cyclooxygenase metabolites to our findings. The residual vasodilation observed after combined inhibition of cyclooxygenase and nitric oxide synthase is likely attributable to EDHF.1,2 Our findings are consistent with previous studies that reported a decreased eNOS expression and metabolic activity of the L-arginine– nitric oxide pathway and a substantial residual vasodilatory response to arachidonic acid after cyclooxygenase inhibition with aspirin in experimental and clinical CHF.7,8,15 Increased EDHF effects in CHF when compared with normal subjects may be partly attributable to deficient nitric oxide production, because nitric oxide appears to tonically inhibit EDHF synthesis.2,16 Such an increase in EDHF effects may represent a compensatory adaptive vasodilatory mechanism in CHF and other disease states that are characterized by deficient nitric oxide–mediated vasodilation. Increased EDHF effects in CHF may also be related to changes in muscarinic receptor subtype distribution and/or density, or changes in the effector mechanisms that regulate membrane voltage potential in vascular smooth muscle. Our findings provide indirect evidence of the ac-
tion of EDHF. Other laboratory techniques to further characterize EDHF, such as administration of inhibitors of the calcium-activated potassium channels and depolarizing agents, are not feasible in the intact human circulation due to potential systemic toxicity.2 The doses of L-NMMA and indomethacin administered in our study have been previously reported to fully inhibit nitric oxide synthase and cyclooxygenase, respectively.1,9,11,12 Diminished inhibitory effects of L-NMMA and indomethacin in patients with CHF when compared with normal subjects do not likely account for our findings. Rather, previous reports suggest that patients with CHF are more likely to be susceptible to the inhibitory effects of L-NNMA and indomethacin than normal subjects due to downregulation of nitric oxide synthase and cyclooxygenase gene expression, and increased plasma concentrations of asymetric dimethyl arginine, an endogenous inhibitor of nitric oxide synthase.17,18 Differences in shear stress stimulus for release of nitric oxide cannot explain our findings because resting and postagonist forearm blood flows were similar in patients with CHF and in normal subjects. Similar resting blood flow values in normal subjects and patients with heart failure may be due to the selection of stable patients and/or the influence of background medications.19 Because subjects were studied at a single matched moderate degree of vasodilation, the effects of L-NMMA and cyclooxygenase in response to a wider range of acetylcholine concentrations cannot be determined from our study. Although medications were stopped before the study, residual effects of background angiotensin-converting enzyme inhibition may have influenced our findings.20 It is unlikely that digoxin contributed to our findings, because acetylcholinemediated hyperpolarization responses are not dependent on sodium–potassium pump activity.1 In conclusion, the present study demonstrates the presence of a noncyclooxygenase, non–nitric-oxide relaxing factor in response to administration of acetylcholine in patients with CHF and in normal subjects. Our findings indicate that EDHF, rather than nitric oxide, may be the predominant endotheliumderived vasoactive substance contributing to acetylcholine-mediated vasodilation in the forearm circulation of patients with CHF. The potential role of EDHF should be considered in the interpretation of studies that use acetylcholine to assess endothelium-dependent vasodilation and as a possible novel therapeutic target in patients with CHF.
1. Nagao T, Vanhoutte PM. Endothelium-derived hyperpolarizing factor and endothelium-dependent relaxations. Am J Respir Cell & Molec Biol 1993;8:1– 6. 2. Quilley J, Fulton D, McGiff JC. Hyperpolarizing factors. Biochem Pharmacol 1997;54:1059 –1070. 3. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, Takeshita A. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol 1996;28:703–711. 4. Nagao T, Illiano S, Vanhoutte PM. Heterogeneous distribution of endotheliumdependent relaxations resistant to NG-nitro-L-arginine in rats. Am J Physiol 1992;263:H1090 –H1094. 5. Kubo SH, Rector TS, Bank AJ, Williams RE, Heifetz SM. Endothelium-
CONGESTIVE HEART FAILURE/EDHF IN CONGESTIVE HEART FAILURE
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dependent vasodilation is attenuated in patients with heart failure. Circulation 1991;84:1589 –1596. 6. Katz SD, Biasucci L, Sabba C, Strom JA, Jondeau G, Galvao M, Solomon S, Nikolic SD, Forman R, LeJemtel TH. Impaired endothelium-mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol 1992;19:918 –925. 7. Agnoletti L, Curello S, Bachetti T, Malacarne F, Gaia G, Comini L, Volterrani M, Bonetti P, Parrinello G, Cadei M, Grigolato PG, Ferrari R. Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-alpha. Circulation 1999;100:1983–1991. 8. Katz SD, Khan T, Zeballos GA, Mathew L, Potharlanka P, Knecht M, Whelan J. Decreased activity of the L-arginine-nitric oxide metabolic pathway in patients with congestive heart failure. Circulation 1999;99:2113–2117. 9. Katz SD, Schwarz M, Yuen J, LeJemtel TH. Impaired acetylcholine-mediated vasodilation in patients with congestive heart failure. Role of endotheliumderived vasodilating and vasoconstricting factors. Circulation 1993;88:55– 61. 10. Kaiser L, Spickard RC, Olivier NB. Heart failure depresses endotheliumdependent responses in canine femoral artery. Am J Physiol 1989;256:H962– H967. 11. Vallance P. Use of L-arginine and its analogs to study nitric oxide pathway in humans. Methods Enzymol 1996;269:453– 459. 12. Shen TY. Indomethacin, sulindac, and their analogs. In: Rainsford KD, ed. Anti-inflammatory and Anti-rheumatic Drugs. Vol. I: Inflammation Mechanisms and Actions of Traditional Drugs. Boca Raton, FL: CRC Press, Inc., 1985:149 – 159. 13. Bolton TB, Lang RJ, Takewaki T. Mechanisms of action of noradrenaline and
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carbachol on smooth muscle of guinea-pig anterior mesenteric artery. J Physiol (Lond) 1984;351:549 –572. 14. Huang A, Sun D, Smith CJ, Connetta JA, Shesely EG, Koller A, Kaley G. In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF. Am J Physiol Heart Circ Physiol 2000;278:H762–H768. 15. Davie AP, Love MP, McMurray JJ. Even low-dose aspirin inhibits arachidonic acid-induced vasodilation in heart failure. Clin Pharmacol Ther 2000;67: 530 –537. 16. Bauersachs J, Popp R, Hecker M, Sauer E, Fleming I, Busse R. Nitric oxide attenuates the release of endothelium-derived hyperpolarizing factor. Circulation 1996;94:3341–3347. 17. Smith CJ, Sun D, Hoegler C, Roth BS, Zhang X, Zhao G, Xu XB, Kobari Y, Pritchard K Jr, Sessa WC, Hintze TH. Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure. Circ Res 1996; 78:58 – 64. 18. Usui M, Matsuoka H, Miyazaki H, Ueda S, Okuda S, Imaizumi T. Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure. Life Sci 1998;62:2425–2430. 19. Jondeau G, Katz SD, Toussaint JF, Dubourg O, Monrad ES, Bourdarias JP, LeJemtel TH. Regional specificity of peak hyperemic response in patients with congestive heart failure: correlation with peak aerobic capacity. J Am Coll Cardiol 1993;22:1399 –1402. 20. Mombouli JV, Vanhoutte PM. Endothelium-derived hyperpolarizing factor(s) and the potentiation of kinins by converting enzyme inhibitors. Am J Hypertens 1995;8:19S-27S.
MAY 1, 2001
MD,
and Henry Krum,
MD, PhD
Vasomotor responses to intraarterial administration of acetylcholine are mediated by release of nitric oxide, prostaglandins, and an unidentified hyperpolarizing factor from vascular endothelial cells. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to the vasodilatory response to acetylcholine in the skeletal muscle circulation of patients with congestive heart failure (CHF) has not been previously characterized. Accordingly, to specifically assess the role of EDHF, the regional vascular effects of sequential administration of acetylcholine and nitroglycerin in the brachial artery were determined in the forearm circulation with straingauge venous occlusion plethysmography in patients with CHF and in normal subjects during combined systemic inhibition of cyclooxygenase activity with indo-
methacin and regional inhibition of nitric oxide synthase activity with L-NG-monomethylarginine (L-NMMA). After administration of indomethacin, infusion of L-NMMA significantly decreased the forearm blood flow response to acetylcholine in normal subjects (5.4 ⴞ 1.2 to 3.5 ⴞ 0.6 ml/min/100 ml, p <0.05) but not in patients with CHF (5.7 ⴞ 1.3 to 5.7 ⴞ 1.4 ml/min/100 ml). Infusion of L-NMMA did not change forearm blood flow responses to nitroglycerin in either group. The presence of a noncyclooxygenase, non–nitric-oxide relaxing factor indicates that EDHF, rather than nitric oxide, may be the predominant endothelium-derived substance mediating vasodilation in response to acetylcholine in patients with CHF. 䊚2001 by Excerpta Medica, Inc. (Am J Cardiol 2001;87:1089 –1092)
ndothelium-dependent vasomotor effects of acetylcholine are mediated by release of nitric oxide E produced by the constitutive isozyme of nitric oxide
tric oxide and abnormal production of vasoconstricting prostaglandins.7–10 The role of EDHF in the vasodilatory response to acetylcholine in the skeletal muscle circulation of patients with CHF has not been previously characterized. The present study was undertaken to specifically determine the role of the EDHF in the vasodilatory response to administration of acetylcholine in the forearm circulation of patients with CHF. The regional vascular effects of acetylcholine were determined in patients with CHF and in normal subjects during combined systemic inhibition of cyclooxygenase activity with indomethacin and regional inhibition of nitric oxide synthase activity with G L-N -monomethylarginine (L-NMMA). Under these experimental conditions, prostaglandin- and nitric oxide-mediated effects of acetylcholine are eliminated, with residual vasodilation attributable to the actions of EDHF.1,2
synthase, prostaglandins produced by the cyclooxygenase pathway, and an unidentified endothelium-derived hyperpolarizing factor (EDHF) produced by the cytochrome P450 pathway.1,2 The relative contribution of each factor to the observed endothelium-dependent vasomotor responses to acetylcholine varies by species, vessel diameter, and regional circulation under study.2– 4 In patients with congestive heart failure (CHF), the vasodilatory response to acetylcholine in the skeletal muscle arteriolar circulation is decreased when compared with that of normal subjects.5,6 The reduced vasodilatory response in CHF is partially attributable to reduced bioavailability of niFrom Columbia Presbyterian Medical Center, Division of Circulatory Physiology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York. This study was supported by an investigatorship award from the American Heart Association, New York City Affiliate, New York, New York; by Grant NHLBI R29 HL51433 from the National Institutes of Health, Bethesda, Maryland; by the National Heart Foundation of Australia Clinical Research Program, and Grant 5 M01RR00645, Division of Research Resources, General Clinical Research Program, National Institutes of Health, Bethesda, Maryland. Manuscript received September 21, 2000; revised manuscript received and accepted November 27, 2000. Address for reprints: Stuart D. Katz, MD, Columbia Presbyterian Medical Center, Division of Circulatory Physiology, Room MHB5435, 177 Fort Washington Avenue, New York, New York 10032. E-mail address: [email protected]. ©2001 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 87 May 1, 2001
METHODS
Study group: Thirteen men and 1 woman (mean age, 50 ⫾ 12 years [range 32 to 65]) with chronic stable CHF due to nonischemic cardiomyopathy were studied. The mean left ventricular ejection fraction was 22 ⫾ 4%. Nine patients were in New York Heart Association functional class II and 5 patients were in functional class III. Cardiovascular medications, which included diuretics, angiotensin-converting enzyme inhibitors, and digoxin in all patients, were withheld 24 hours before the study. Patients with edema, serum sodium ⬍136 mEq/L, or a history of 0002-9149/01/$–see front matter PII S0002-8149(01)01466-7
1089
diabetes mellitus, hypercholesterolemia, or active smoking habits were excluded. Nine men and 1 woman (mean age, 43 ⫾ 13 years [range 27 to 66]) without a history of chronic illness participated as normal controls. The mean age of these subjects was not different from that of patients with CHF. These subjects were nonsmokers, had a normal physical examination, and were not taking chronic medications. The study was approved by the ethical review board of the Columbia Presbyterian Medical Center. All subjects gave written informed consent before the study. Venous occlusion plethysmography: Forearm blood flow (ml/min/100 ml of forearm volume) was determined with venous occlusion strain-gauge plethysmography as previously described in detail.9 Five plethysmographic measurements were averaged for each blood flow determination. Intraarterial drug administration: Acetylcholine and nitroglycerin were administered as temporally sequential 2-minute continuous infusions (1 ml/min) to achieve regional forearm blood concentrations ranging from 10⫺8 to 10⫺6 M. Infusions of acetylcholine and nitroglycerin were separated by 5 to 10 minutes to allow forearm blood flow to return to basal values. To evaluate the effects of L-NMMA at comparable levels of shear stress in patients with CHF and normal subjects, the doses of acetylcholine and nitroglycerin were individually adjusted to increase forearm blood flow to 2 to 3 times the resting value. L-NMMA, a competitive inhibitor of nitric oxide synthase, was administered as a continuous 1 ml/min infusion for 5 minutes at a concentration of 40 M. This dose of L-NMMA has previously been reported to fully inhibit nitric oxide synthase activity in isolated vascular tissue and in the intact human forearm circulation.1,11 Study protocol: To exclude the effects of prostaglandins on acetylcholine-induced vasodilation, all subjects received 50 mg of indomethacin, a cyclooxygenase inhibitor, by mouth 1 hour before the study.9,12 A 20-gauge angiocath was placed in the brachial artery under local anesthesia for regional drug administration. Forearm blood flows were measured at rest and during administration of acetylcholine and nitroglycerin before and after infusion of L-NMMA. Mean arterial pressure was recorded in the contralateral arm with an automated cuff method. Data analysis: All values are stated as mean ⫾ SEM. Forearm blood flows and mean arterial pressure in patients with CHF and normal subjects at rest and during administration of acetylcholine and nitroglycerin were compared before and after administration of L-NMMA with a repeated measures analysis of variance model. A 2-tailed p value⬍0.05 was considered statistically significant.
RESULTS Resting forearm blood flows did not differ between patients with CHF and normal subjects before infusion of L-NMMA (2.1 ⫾ 0.3 vs 2.8 ⫾ 0.3 ml/min/100 ml) or after infusion of L-NMMA (2.1 ⫾ 0.3 vs 2.1 ⫾ 0.3 ml/min/100 ml). L-NMMA significantly decreased 1090 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 1. Forearm blood flow responses to administration of acetylcholine before (open bar) and after (filled bar) infusion of L-NMMA in normal subjects and in patients with CHF. *p <0.05 versus before L-NMMA.
TABLE 1 Forearm Blood Flow Responses (ml/min/100 ml) to Administration of Nitroglycerin Before and After Infusion of L-NMMA in Normal Subjects and in Patients With CHF
Normal subjects CHF
Before L-NMMA
After L-NMMA
4.2 ⫾ 0.3 5.7 ⫾ 0.9
4.1 ⫾ 0.6 5.9 ⫾ 1.0
resting forearm blood flow in normal subjects (p ⬍0.05) but not in patients with CHF. To achieve a comparable degree of vasodilation in the 2 groups, mean doses of acetylcholine and nitroglycerin administered to patients with CHF were significantly greater than doses in normal subjects (⫺6.5 ⫾ 0.3 vs ⫺7.7 ⫾ 0.2 log units for acetylcholine and ⫺7.1 ⫾ 0.2 vs ⫺7.6 ⫾ 0.2 log units for nitroglycerin, both p ⬍0.05, CHF patients vs normal subjects). As determined by study protocol, forearm blood flows in response to administration of individually adjusted doses of acetylcholine did not differ in patients with CHF and normal subjects before infusion of L-NMMA (5.7 ⫾ 1.3 vs 5.4 ⫾ 1.2 ml/min/100 ml, Figure 1). After infusion of L-NMMA, the forearm blood flow response to acetylcholine significantly decreased to 3.5 ⫾ 0.6 ml/min/100 ml in normal subjects (p ⬍0.05 vs before L-NMMA), but did not change in patients with CHF (5.7 ⫾ 1.4 ml/min/100 ml, Figure 1). Forearm blood flows in response to administration of nitroglycerin did not differ between patients with CHF and normal subjects before infusion of L-NMMA and did not change after infusion of L-NMMA in either group (Table 1). Mean arterial pressures did not differ in patients with CHF and normal subjects before infusion of L-NMMA at rest (86 ⫾ 5 vs 91 ⫾ 4 mm Hg) and did not change during drug infusions (data not shown). MAY 1, 2001
DISCUSSION The present study demonstrates that during systemic inhibition of cyclooxygenase with indomethacin, regional inhibition of nitric oxide synthase with L-NMMA significantly decreased the vasodilatory response to acetylcholine in normal subjects but not in patients with CHF. The presence of a noncyclooxygenase, non–nitric-oxide relaxing factor indicates that EDHF, rather than nitric oxide, may be the predominant endothelium-derived substance mediating vasodilation in response to acetylcholine in the forearm circulation of patients with CHF. The presence of EDHF as an endothelium-dependent mediator of the vascular effects of acetylcholine was first described in small mesenteric arteries of the guinea pig and subsequently confirmed in other species and regional circulations by several investigators.1,2,13 Although its specific chemical identity remains elusive, pharmacologic studies suggest that EDHF is a product of the cytochrome P450 metabolic pathway, likely a member of the epoxyeicosatrienoic acid family.2 Because its specific chemical identity remains unknown, most evidence of the existence of EDHF is derived from observations of the vasomotor and membrane voltage potential effects of acetylcholine in the presence of inhibitors of nitric oxide synthase and cyclooxygenase.2 Under these experimental conditions and in endothelial nitric oxide synthase (eNOS) knockout mice, EDHF appears to play a greater role in small arterioles when compared with larger arterioles or conduit artery rings.3,14 The vasodilatory response to acetylcholine is decreased in patients with CHF when compared with normal subjects.5,6 Previous reports indicate that the decreased response to acetylcholine may be partly attributable to the presence of a vasoconstricting prostanoid.9,10 In the present study, pretreatment with indomethacin eliminated the potential contribution of cyclooxygenase metabolites to our findings. The residual vasodilation observed after combined inhibition of cyclooxygenase and nitric oxide synthase is likely attributable to EDHF.1,2 Our findings are consistent with previous studies that reported a decreased eNOS expression and metabolic activity of the L-arginine– nitric oxide pathway and a substantial residual vasodilatory response to arachidonic acid after cyclooxygenase inhibition with aspirin in experimental and clinical CHF.7,8,15 Increased EDHF effects in CHF when compared with normal subjects may be partly attributable to deficient nitric oxide production, because nitric oxide appears to tonically inhibit EDHF synthesis.2,16 Such an increase in EDHF effects may represent a compensatory adaptive vasodilatory mechanism in CHF and other disease states that are characterized by deficient nitric oxide–mediated vasodilation. Increased EDHF effects in CHF may also be related to changes in muscarinic receptor subtype distribution and/or density, or changes in the effector mechanisms that regulate membrane voltage potential in vascular smooth muscle. Our findings provide indirect evidence of the ac-
tion of EDHF. Other laboratory techniques to further characterize EDHF, such as administration of inhibitors of the calcium-activated potassium channels and depolarizing agents, are not feasible in the intact human circulation due to potential systemic toxicity.2 The doses of L-NMMA and indomethacin administered in our study have been previously reported to fully inhibit nitric oxide synthase and cyclooxygenase, respectively.1,9,11,12 Diminished inhibitory effects of L-NMMA and indomethacin in patients with CHF when compared with normal subjects do not likely account for our findings. Rather, previous reports suggest that patients with CHF are more likely to be susceptible to the inhibitory effects of L-NNMA and indomethacin than normal subjects due to downregulation of nitric oxide synthase and cyclooxygenase gene expression, and increased plasma concentrations of asymetric dimethyl arginine, an endogenous inhibitor of nitric oxide synthase.17,18 Differences in shear stress stimulus for release of nitric oxide cannot explain our findings because resting and postagonist forearm blood flows were similar in patients with CHF and in normal subjects. Similar resting blood flow values in normal subjects and patients with heart failure may be due to the selection of stable patients and/or the influence of background medications.19 Because subjects were studied at a single matched moderate degree of vasodilation, the effects of L-NMMA and cyclooxygenase in response to a wider range of acetylcholine concentrations cannot be determined from our study. Although medications were stopped before the study, residual effects of background angiotensin-converting enzyme inhibition may have influenced our findings.20 It is unlikely that digoxin contributed to our findings, because acetylcholinemediated hyperpolarization responses are not dependent on sodium–potassium pump activity.1 In conclusion, the present study demonstrates the presence of a noncyclooxygenase, non–nitric-oxide relaxing factor in response to administration of acetylcholine in patients with CHF and in normal subjects. Our findings indicate that EDHF, rather than nitric oxide, may be the predominant endotheliumderived vasoactive substance contributing to acetylcholine-mediated vasodilation in the forearm circulation of patients with CHF. The potential role of EDHF should be considered in the interpretation of studies that use acetylcholine to assess endothelium-dependent vasodilation and as a possible novel therapeutic target in patients with CHF.
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