Role of Basal and Stimulated Release of Nitric Oxide in the Regulation of Radial Artery Caliber in Humans
Abstract Although it is well established that nitric oxide contributes to the regulation of resistance arterial tone in humans, its role at the level of large arteries is less clear. Therefore, we assessed in healthy volunteers the effects of local administration of the inhibitor of nitric oxide synthesis NG-monomethyl-l-arginine (L-NMMA) on basal radial artery diameter (transcutaneous A-mode echotracking) and radial blood flow (Doppler) as well as on the radial response to acetylcholine and the nitric oxide donor sodium nitroprusside. A catheter was inserted into the brachial artery for measurement of arterial pressure and infusion of L-NMMA (2, 4, and 8 μmol/min for 5 minutes, n=11), acetylcholine (3, 30, 300, and 900 nmol/min for 3 minutes, n=8), and nitroprusside (2.5, 5, 10, and 20 nmol/min for 3 minutes, n=6). None of the treatments affected arterial blood pressure or heart rate. L-NMMA dose-dependently decreased radial blood flow (from 31±6 to 17±3 10−3 L/min after 8 μmol/min, P<.01) but did not affect radial artery diameter (from 2.93±0.11 to 2.90±0.14 mm). Acetylcholine dose-dependently increased radial blood flow (154±43% after 900 nmol/min) and radial artery diameter (16±4%), and both effects were markedly reduced after L-NMMA (increase in radial blood flow and radial artery diameter: 22±20% and 3±2%, respectively; both P<.01 versus controls). Nitroprusside also dose-dependently increased radial artery diameter (14±4% after 20 nmol/min) but only moderately affected radial blood flow (47±21%). L-NMMA markedly increased the effects of nitroprusside on radial artery diameter (26±2%, P<.01 versus controls) but not on radial blood flow (66±21%, P=NS versus controls). These experiments demonstrate for the first time that large peripheral arteries vasodilate in response to acetylcholine in vivo through the release of endogenous nitric oxide. The increased sensitivity to nitrovasodilators after L-NMMA suggests the existence of a basal release of nitric oxide at the level of large peripheral arteries. However, the lack of a decrease in diameter after L-NMMA might be explained by the presence of compensating vasodilator mechanisms occurring at the level of large arteries after inhibition of nitric oxide synthesis.
Several experiments performed in both animal models and humans have demonstrated the physiological role of endothelium-derived nitric oxide (NO) in the control of basal arteriolar tone and blood pressure.1 2 3 At the level of large arteries, however, the role of NO in the control of basal tone appears less clear. In most animal studies administration of inhibitors of NO synthesis such as NG-monomethyl-l-arginine (L-NMMA) induces a decrease in large artery diameter.4 5 In humans, however, inhibition of NO synthesis does not constantly induce vasoconstriction of large arteries.6 7 Indeed, in the human left anterior descending coronary artery (LAD), administration of L-NMMA does not induce any change in proximal LAD diameter, although it induces a small decrease in distal LAD diameter and inhibits the coronary response to acetylcholine.7 Furthermore, the role of basal and stimulated release of NO at the level of large peripheral arteries has not been clearly established.
Therefore, we designed the present study to assess the effect of local administration of L-NMMA on basal radial artery caliber and vascular resistance as well as on the radial responses to acetylcholine and the NO donor sodium nitroprusside.
Eleven normotensive subjects (6 men and 5 women; age, 24±1 years; range, 18 to 32 years) were included in the study. Subjects were nonsmokers and deemed healthy on the basis of a medical history and complete medical examination including a normal electrocardiogram and recent routine laboratory tests including glycemia and total cholesterolemia. All subjects had given written informed consent. The study was approved by the Basel Hospital Ethics Committee and followed procedures in accordance with institutional guidelines.
Radial artery internal diameter was continuously measured with a high-precision A-mode echotracking device (NIUS 02, Asulab).8 Briefly, a 10-MHz focused transducer was positioned over the radial artery. The probe was set perpendicular to the artery with the use of a stereotaxic arm with micrometric screws, while proper positioning was adjusted with the use of a stereo Doppler mode. After the switch to A-mode, the echoes from both anterior and posterior walls of the artery were visualized on a screen and tagged by electronic trackers, allowing continuous recording of the artery internal diameter.8 9 10 Briefly, the processed radio frequency line was visualized on a computer screen, and the operator selected the peaks corresponding to the interfaces, after which the exact position of each selected peak was determined with the use of an interpolation technique.11 Finally, radial blood flow velocity was continuously recorded with an 8-MHz Doppler probe (Doptek 2002, Deltex). Radial artery flow was calculated from the measurements of velocity and internal diameter. Positioning of the probes and electronic trackers was always performed by the same investigator, after which calculation of radial parameters was performed automatically without any further intervention by the investigator. The reproducibility of the measurements of internal diameter and blood flow velocity was calculated from two consecutive measurements separated by 30 minutes after the device had been repositioned. These parameters were determined after acquisition in a blind manner by postprocessing of the data in a random order with no information on the subject. The coefficients of variation were 6±1% and 2±1% for radial blood flow velocity and diameter, respectively.
Preparation of Drugs
L-NMMA (Clinalfa), acetylcholine (Ciba Vision AG), and sodium nitroprusside (Hoffmann–La Roche SA) were dissolved in sterile physiological saline (0.9% NaCl) and infused at a constant rate (0.8 mL/min) with an infusion pump (Sage Instruments Inc). The forearm volume of each subject was measured by the water displacement method, and drug doses were adjusted accordingly. However, as the forearm volume was close to 1 L in each subject, the values expressed in moles per minute per liter were close to those expressed in moles per minute; therefore, all doses are expressed in moles per minute.
The study was performed while subjects were supine in a quiet air-conditioned room maintained at a constant temperature (20° to 22°C). With subjects under local anesthesia (lidocaine 1%) an 18-gauge catheter was inserted into the left brachial artery for regional infusion of L-NMMA, acetylcholine, and sodium nitroprusside and for recording of arterial pressure (Statham P23 Pb pressure transducer, Gould Instruments).12 Subjects were allowed to rest for 30 minutes after instrumentation. After control measurements (physiological saline) had been obtained, increasing doses of acetylcholine (3, 30, 300, and 900 nmol/min for 3 minutes) and sodium nitroprusside (2.5, 5, 10, and 20 nmol/min for 3 minutes) were infused through the same catheter. Thirty minutes after sodium nitroprusside the radial response to increasing doses of L-NMMA (2, 4, and 8 μmol/min for 5 minutes) was assessed. Sixty minutes after completion of the dose-response curve to L-NMMA, the effects of the same doses of acetylcholine and sodium nitroprusside were again assessed after the administration of a single dose of L-NMMA (8 μmol/min for 5 minutes). The doses of acetylcholine and sodium nitroprusside were chosen on the basis of previous studies as those producing maximal regional vasodilation without systemic hemodynamic changes.12 13 Hemodynamic parameters were measured continuously from baseline to the end of each infusion period. From this continuous recording mean values were calculated from 10 consecutive cardiac cycles during the last minute of the infusion of each dose. When an oscillatory pattern was noted, we extended the calculations to 30 to 40 consecutive cardiac cycles to cover the half period of oscillation and obtain stable mean values.14 During the control period the coefficients of variation calculated from four repeated measurements within the 15-minute period were 1.2±0.4% and 12.6±1.9% for internal diameter and radial blood flow, respectively (P=NS).
Results are expressed as mean±SEM. The effect of L-NMMA on baseline hemodynamic parameters as well as on the responses to acetylcholine or sodium nitroprusside was assessed with ANOVA for repeated measurements followed by Tukey’s test when applicable. A value of P≤.05 was considered statistically significant.
Forearm infusion of L-NMMA, acetylcholine, or sodium nitroprusside did not modify arterial pressure or heart rate (Table 1⇓).
L-NMMA dose-dependently decreased radial blood flow (Fig 1⇓; from 31±6 to 17±3 10−3 L/min after 8 μmol/min L-NMMA; n=11; P<.01) but did not affect radial artery internal diameter (Fig 1⇓; from 2.93±0.11 to 2.90±0.14 mm; P<NS).
Before L-NMMA, acetylcholine induced dose-dependent increases in radial blood flow and internal diameter (Fig 2⇓ and Table 2⇓; increase from baseline after 900 nmol/min: 154±43% and 16±4%, respectively; n=8; both P<.01). After L-NMMA, the increases in radial blood flow and internal diameter observed after acetylcholine were significantly reduced (Fig 2⇓ and Table 2⇓; increase from baseline after 900 nmol/min: 22±20%, P<.02; and 3±2%, P<.01, respectively).
Before L-NMMA, sodium nitroprusside induced a dose-dependent increase in radial artery internal diameter (Fig 2⇑ and Table 2⇑; increase from baseline after 20 nmol/min: 14±4%; n=6; P<.01) but only moderately affected radial blood flow (47±21%; P<.05). The increase in radial diameter was enhanced by previous infusion of L-NMMA (Fig 2⇑ and Table 2⇑; from 14±4% to 26±2%; P<.01). In contrast, the increase in radial blood flow was not modified by L-NMMA.
The present experiments performed at the level of the radial artery in humans demonstrate that local administration of L-NMMA (1) induced a dose-dependent decrease in radial blood flow without affecting radial artery diameter, (2) markedly reduced the effect of acetylcholine on radial blood flow and radial artery diameter, and (3) augmented the large artery response to the NO donor sodium nitroprusside but did not affect the nitroprusside-induced increase in flow.
The high-resolution echotracking method coupled to Doppler enabled us to obtain continuous and stable measurements of internal diameter and blood flow velocity and thus to study simultaneously the effects of drugs on both conduit and resistance arteries. Our basal values and estimates of reproducibility were similar to those previously reported by others15 16 and us.9 10 The spontaneous fluctuations of internal diameter and flow noted during the control period were minimal, and the return to basal values at the end of each rest period demonstrated the stability of the signal measured and the reversibility of the changes observed after infusion of the different drugs.
The decrease in radial blood flow after L-NMMA, which is in agreement with previous results, illustrates the major role of the basal release of NO at the level of peripheral resistance arteries in humans.2 3 Furthermore, our observation that the increase in radial blood flow induced by acetylcholine was markedly reduced by L-NMMA confirms previous studies2 3 and suggests that acetylcholine dilates peripheral resistance arteries mostly through the release of NO. In addition, the inhibitory effect of L-NMMA on basal radial blood flow and on the flow response to acetylcholine suggests that the present experiments were performed with an L-NMMA dose that was effective in blocking NO synthesis in this bed.
In this context, our experiments demonstrate for the first time in humans that acetylcholine increases the internal diameter of large peripheral arteries through NO release. After L-NMMA the effect on radial artery diameter was markedly depressed. This finding is in agreement with a recent study performed in the human coronary circulation in which L-NMMA inhibited the response of the distal LAD to acetylcholine.7 Numerous in vitro studies have suggested that the relaxing response of the isolated large arteries to acetylcholine is dependent on endothelium and mediated by NO.17 18 Our experiments would suggest that the same phenomenon occurs in vivo in humans. However, it is well established that large arteries accommodate changes in blood flow by increasing their diameter, and we have shown recently that such “flow-dependent vasodilation” is mediated by NO.10 Since in the present experiments the acetylcholine-induced increase in diameter was accompanied by a significant increase in flow, the respective contribution of direct and indirect (ie, flow-mediated) release of NO to the response of the radial artery to acetylcholine cannot be easily evaluated. However, animal experiments in which the effect of acetylcholine was studied in either the absence or presence of a flow-limiting stenosis suggested that the dilating effect of acetylcholine on large arteries was partly a direct, flow-independent effect.19 20
The NO synthase inhibitor L-NMMA decreased radial blood flow but did not affect the diameter of the radial artery. This effect has been already reported in humans at the level of the pulmonary artery6 and the proximal site of coronary arteries7 and contrasts with previous studies performed in dogs in which administration of inhibitors of NO synthesis was associated with significant decreases in epicardial coronary4 or femoral5 diameter. It must be noted, however, that the vasoconstrictor effects observed in those studies were accompanied by significant increases in blood pressure. Thus, it is possible that the vasoconstriction observed in animal studies at the level of the large arteries may be attributed to confounding systemic effects.
Another possibility to explain the lack of effect of L-NMMA on radial diameter would be that the doses used were insufficient to induce full blockade of NO synthesis. This is unlikely, however, because we show that the same dose of L-NMMA induced a marked decrease in radial blood flow and virtually abolished the radial response to acetylcholine.
In our experiments administration of nitroprusside after L-NMMA occurred in a setting of reduced baseline values for radial diameter. This reduced baseline value is probably not the consequence of a delayed L-NMMA–induced vasoconstrictor effect because we did not observe a similar delayed vasoconstriction during the dose-response curve to the l-arginine analogue, despite the fact that the hemodynamic parameters were recorded for 60 minutes after the highest dose. Thus, we believe that the decreased baseline values observed before nitroprusside are an unspecific time effect or an unmasked delayed vasoconstrictor response to acetylcholine after L-NMMA. Indeed, in our experiments the order of drug administration was not randomized, and nitroprusside was always administered after acetylcholine because we were concerned that possible prolonged effects of nitroprusside (especially on large artery diameter, an effect common to many nitrates or NO donors) might have influenced the response to acetylcholine if acetylcholine had been infused after nitroprusside.
Our results show that the vasodilator response to nitroprusside at the level of the radial artery was significantly increased after prior treatment with L-NMMA. Although we cannot totally exclude the possibility that the lower baseline values of radial diameter before nitroprusside may contribute to this increased response, it must be noted that the absolute values of radial diameter obtained after the highest dose of nitroprusside were higher after than before L-NMMA. Furthermore, it was suggested previously that nonspecific vasoconstriction through either infusion of norepinephrine or lower body negative pressure does not affect the maximal vasodilatation observed after sodium nitroprusside in healthy volunteers.21 Finally, it must be noted that a similar potentiation has already been observed both in vitro5 22 23 and in vivo5 23 and has generally been thought to reflect a supersensitivity to nitrovasodilators after inhibition of basal NO synthesis. Thus, the fact that we observed a similar potentiation in the present experiments suggests the existence of a basal release of NO at the level of the radial artery. In this context, it is possible that the lack of effect of L-NMMA on radial diameter is the consequence of compensating vasodilator mechanisms occurring after inhibition of NO synthesis.
In contrast to the effect at the level of large arteries, the increase in radial blood flow induced by sodium nitroprusside was not affected by L-NMMA. This confirms previous human studies performed in the same vascular bed2 and could be related to the weak vasodilator effect observed with this NO donor at the level of resistance arteries.
Our study performed in vivo in humans demonstrates for the first time that acetylcholine vasodilates peripheral conduit arteries through NO release. In addition, we observed an increased sensitivity to nitrovasodilators after NO synthase inhibition, which suggests in vivo the existence of a basal release of NO at the level of large peripheral arteries. The absence of a decrease in radial diameter noted after L-NMMA might be explained by the presence of compensating vasodilator mechanisms occurring at the level of large arteries on inhibition of NO synthesis.
These studies were supported by the Association Charles Nicolle, Rouen, France, and the Swiss National Science Foundation (grant No. 32-36575.92), the Scheizerische Rentenanstalt, and the Freiwillige Akademische Gesellschaft Basel, Switzerland.
Rees DD, Palmer RMJ, Moncada S. The role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A. 1989;86:3375-3378.
Panza JA, Casino PR, Crescence MK, Quyyumi AA. Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation. 1993;87:1468-1474.
Chu A, Chambers DE, Lin C-C, Kuehl WD, Palmer RMJ, Moncada S, Cobb FR. Effects of inhibition of nitric oxide formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs. J Clin Invest. 1991;87:1964-1968.
Celermajer DS, Dollery C, Burch M, Deanfield JE. Role of endothelium in the maintenance of low pulmonary vascular tone in normal children. Circulation. 1994;89:2041-2044.
Leffroy DC, Crake T, Uren NG, Davies GJ, Maseri A. Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation. 1993;88:43-54.
Joannides R, Richard V, Moore N, Godin M, Thuillez C. Influence of sympathetic tone on mechanical properties of muscular arteries in humans. Am J Physiol. 1995;268:H794-H801.
Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, Lüscher TF. Nitric oxide is responsible for flow-dependent dilation of human peripheral conduit arteries in vivo. Circulation. 1995;91:1314-1319.
Tardy Y, Hayoz D, Mignot JP, Richard PH, Brunner HR, Meister JJ. Dynamic non-invasive measurements of arterial diameter and wall thickness. J Hypertens. 1992;10(suppl 6):S105-S109.
Linder L, Kiowski W, Bühler FR, Lüscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo: blunted response in essential hypertension. Circulation. 1990;8:1762-1767.
Bruning TA, Hendriks MGC, Chang PC, Kuypers EAP, van Zwieten PA. In vivo characterization of vasodilating muscarinic-receptor subtypes in humans. Circ Res. 1994;74:912-919.
Hayoz D, Tardy Y, Rutschmann B, Mignot JP, Achakri H, Feihl F, Meister JJ, Waeber B, Brunner HR. Spontaneous diameter oscillations of the radial artery in humans. Am J Physiol. 1993;264:H2080-H2084.
Perret F, Mooser V, Hayoz D, Tardy Y, Meister J-J, Etienne J-D, Farine P-A, Marrazzi A, Burnier M, Nussberger J, Waeber B, Brunner H-R. Evaluation of arterial compliance-pressure curves: effect of antihypertensive drugs. Hypertension. 1991;18(suppl II):II-77-II-83.
Young MA, Vatner SF. Blood flow- and endothelium-mediated vasomotion of iliac arteries in conscious dogs. Circ Res. 1987;61(suppl II):II-88-II-93.
Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension. 1986;8:37-44.
Lüscher TF, Richard V, Yang Z. Interaction between endothelium-derived nitric oxide and SIN-1 in human and porcine coronary arteries. J Cardiovasc Pharmacol. 1989;14(suppl 11):S76-S80.
Moncada S, Rees DD, Schulz R, Palmer RMJ. Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo. Proc Natl Acad Sci U S A. 1991;88:2166-2170.