Eur J Clin Pharmacol (2003) 59: 499–505 DOI 10.1007/s00228-003-0662-7

PHARMACODYNAMICS

Lisbeth Hjorth Lassen Æ Helle Klingenberg Iversen Jes Olesen

A dose–response study of nitric oxide synthase inhibition in different vascular beds in man

Received: 29 March 2003 / Accepted: 11 July 2003 / Published online: 6 September 2003
Springer-Verlag 2003

Abstract Background and objective: Nitric oxide (NO) is an almost ubiquitous messenger molecule and is implicated in several disorders. NG monomethyl L-arginine (L-NMMA:546C88) is an inhibitor of all three NO synthases (NOS), the enzymes that catalyse the production of NO. The present study was performed to evaluate the dose-response relation of L-NMMA to improve the design and interpretation of studies in migraine sufferers and other diseases. Methods: In a double-blind, placebo-controlled, crossover design, six healthy volunteers were randomised to receive three different doses of L-NMMA (0.3 mg/kg, 1 mg/kg, 3 mg/kg) or placebo (5% dextrose) intravenously (iv) over 5 min on four different days. On a fifth study day, in an open design, the same subjects received L-NMMA in the dose 6 mg/kg iv over 15 min. The effect of L-NMMA on the maximal mean blood velocity (Vmean) in the middle cerebral artery (MCA) (transcranial Doppler), the luminal diameter of the radial artery (high-frequency ultrasound), mean arterial blood pressure (MAP), heart rate and electrocardiogram were repeatedly followed every 5 min until 60 min after start of the infusion, then every 15 min during the following hour, and at 3 h and 4 h. Results: Inhibition of NOS had no effect on Vmean in MCA or on the diameter of the radial artery, but MAP increased and heart rate decreased dose dependently. With a dose of 6-mg/kg L-NMMA infused over a 15-min period, the maximum MAP increase was 20% 20 min after the start of L-NMMA infusion. The maximum decrease of heart rate was 24% 15 min after start of the L-NMMA infusion.

L. Hjorth Lassen (&) Æ H. Klingenberg Iversen J. Olesen Danish Headache Center, Department of neurology, Glostrup University Hospital, 2600 Glostrup, Denmark E-mail: [email protected] Tel.: +45-39-650042 L. Hjorth Lassen Skraenten 8, DK-2820 Gentofte, Denmark

Conclusion: L-NMMA in a dose that caused marked changes in systemic blood pressure and heart rate had no effect on cerebral and radial arteries in man. Keywords NOS Æ L-NMMA Æ TCD

Introduction Since the first publications about endothelium derived relaxing factor [1] and nitric oxide (NO) [2], the interest in NO has exploded, and it seems to be implicated in several disorders, such as migraine, diabetes and stroke [3, 4, 5, 6]. One way to study the effects of NO is to block its production using an inhibitor of NO synthase (NOS). Many such compounds have been studied in animals, but only few have been toxicologically tested to allow human experiments. NG monomethyl L-arginine (L-NMMA: 546C88, Glaxo Smith Kline) is a non-selective NOS antagonist that has been extensively tested for safety in man [7]. Previous studies have established that L-NMMA markedly increases blood pressure and constricts peripheral arterioles [8, 9, 10, 11, 12], whereas the effect on the larger peripheral arteries has not been thoroughly investigated. In the same way, the effect of L-NMMA on blood velocity in the middle cerebral artery and on regional cerebral blood flow has not received much attention, and the results are in dispute [13, 14, 15, 16, 17]. The present study was performed to examine the effect of four different doses of L-NMMA on blood velocity in the middle cerebral artery, diameter of the radial artery, blood pressure, heart rate and electrocardiogram (ECG) in healthy volunteers. We tested the hypotheses that NOS inhibition and therefore, NO, is more important in arterioles than in larger arteries both in the cerebral and the extracerebral circulations.

Materials and methods Six non-smoking healthy subjects (five males, one female, mean age 25 years, range 22–28 years, mean bodyweight 79 kg, range

500 65–105 kg) were included. No daily medications except contraceptives were allowed. Before inclusion, a physical examination including ECG and a routine chemistry was performed and had to be within normal limits. The ethics committee of the County of Copenhagen approved the study (ka 95002 s) and it was conducted according to the Helsinki II declaration of 1964 with amendments in 1989. All subjects gave their informed consent and were free to withdraw at any time during the study. In a double-blind, placebo-controlled, cross-over design, each subject was randomised to receive either A: placebo (5% dextrose) or L-NMMA (B: 0.3 mg/kg, C: 1 mg/kg, D: 3 mg/kg) intravenously (i.v.) over 5 min on four different days separated by at least 1 week. The randomisation was conducted by the statistical program (Med Stat) and the randomisation sequence (ADCB, DACB, ABCD, ABDC, DCAB and DBCA) was not known by investigators before the study. Blinding was ascertained by letting a medical doctor not participating in the study prepare the infusions. Time-averaged mean of the maximal blood velocity (Vmean) in the middle cerebral artery (MCA) was measured bilaterally with transcranial Doppler ultrasonography (2 MHz, Multidop X Doppler; DWL, Sipplingen, Germany) [18]. The luminal diameter of the left radial artery was measured with high-frequency ultrasound (Dermascan C; Cortex Technology, Hadsund, Denmark) [19]. Blood pressure and heart rate were measured on the right arm with an automatic inflatable arm cuff (Tonoprint, Jungingen, Germany), and end-tidal pCO2 was followed using a capnograph (Poet, Criticare Systems) connected to an anaesthetic mask. L-NMMA was kindly delivered from GSK (UK), and immediately before use diluted to the required strength with 5% dextrose. On the study days, no medication, coffee, tea, cola, alcohol or tobacco were allowed 12 h prior the study. The subjects arrived at the laboratory at 0800 hours and were placed in a quiet room in the supine position. A cannula (Viggo Venflon) was placed in the left cubital vein for L-NMMA/placebo infusion; another cannula was placed in the ante-brachial vein on the right arm for blood sampling. Baseline values of blood velocity (Vmean) in MCA, the luminal diameter of the radial artery, blood pressure, heart rate and pCO2 were recorded after at least 30 min of rest in the supine position. L-NMMA (0.3 mg/kg or 1 mg/kg or 3 mg/kg) or placebo (5% dextrose) was then infused i.v. over 5 min. Vmean in MCA, the luminal diameter of the radial artery, blood pressure, heart rate and end tidal pCO2 were recorded every 5 min until 60 min after start of the L-NMMA/placebo infusion. During the subsequent 60 min, the parameters were recorded every 15 min and then at 3 h and 4 h; furthermore, blood pressure and heart rate were measured at 6, 8 and 10 h. ECG was monitored continuously during the first 2 h and then at time 3, 4, 6, 8 and 10 h. Subjects were placed in the supine position until 2 h after infusion. They were then allowed to walk around until 30 min before the next examination. Blood samples for measuring pL-NMMA to a pharmacokinetic component of the study were drawn at given time points up to 10 h after start of infusion (in preparation, GSK). Since the effect of L-NMMA in the doses up to 3 mg/kg was less than expected, we added a fifth study day, with an open study design where the same six subjects were treated with L-NMMA at the dose 6 mg/kg i.v. over 15 min. The infusion time was increased to 15 min and no blood samples for plasma kinetics were taken. In all other aspects, the procedure was the same during the first 2 h after starting the infusion. The subjects were then sent home.

Statistics Values are expressed as mean and standard deviation (±SD). All measurements are expressed as absolute values and percentage change from baseline. Differences among the sum of percentage changes from baseline in the first 60 min from start of infusion in blood velocity, arterial

diameters, blood pressure and heart rate at each different dose of L-NMMA were compared with each other and to the placebo response using multivariate analysis of variance (MANOVA, Statgraphics 7.0)[20]. Changes were then located with a multiple range test (confidence intervals, Statgraphics 7.0). The differences among peak values in blood velocity, arterial diameters, blood pressure and heart rate among study days were tested in the same way. MANOVA was also used to test for differences in blood velocity, blood pressure, heart rate and pCO2 for each dose over time and a multiple range test was used to locate changes. Statistical significance was defined as P<0.05.

Results The mean values (±SD) of Vmean, the diameter of the radial artery, blood pressure and heart rate at baseline and at time for peak value during the first 60 min after start of infusion and at 120 min are given in Table 1. Table 1 Mean blood velocity (Vmean) in the middle cerebral artery. Diameter of the radial artery. Blood pressure and heart-rate at placebo and different doses of NGmonomethyl L-arginine (L-NMMA:546C88). Mean values (SD) Baseline Vmean (cm/s) Placebo 72.7 0.3 mg/kg 78.7 1 mg/kg 76.0 3 mg/kg 75.8 6 mg/kg 71.5

(11.4) (14.2) (14.1) (11.3) (17.5)

Diameter of the radial Placebo 2.42 (0.44) 0.3 mg/kg 2.33 (0.43) 1 mg/kg 2.32 (0.38) 3 mg/kg 2.35 (0.37) 6 mg/kg 2.41 (0.46)

Peak

60 min

120 min

(9.3) (13.4) (15.5) (13.8) (16.5)

72.2 72.3 70.0 72.8 71.8

(13.3) (12.4) (11.7) (13.1) (16.2)

73.7 70.3 68.5 73.3 75.3

(12.6) (11.8) (14.3) (17.2) (17.2)

artery (mm) 2.59 (0.44) 2.58 (0.33) 2.47 (0.40) 2.58 (0.45) 2.60 (0.42)

2.66 2.50 2.46 2.51 2.44

(0.46) (0.32) (0.40) (0.42) (0.39)

2.55 2.54 2.43 2.40 2.47

(0.40) (0.28) (0.37) (0.41) (0.44)

(5.3) (5.6) (5.7) (7.1) (7.7)

87.8 88.6 93.3 88.3 91.7

(10.2) (10.4) (10.3) (10.1) (8.6)

74.5 78.2 75.7 77.2 73.0

Mean arterial blood pressure (mmHg) Placebo 84.6 (7.5) 86.6 (7.6) 87.6 0.3 mg/kg 79.3 (4.9) 84.6 (6.3)* 83.6 1 mg/kg 80.8 (5.3) 87.4 (7.3)* 82.9 3 mg/kg 81.4 (6.5) 91.2 (7.6)* 88.3 6 mg/kg 80.2 (7.1) 95.8 (8.8)* 91.2 Systolic blood pressure Placebo 115.7 (9.9) 0.3 mg/kg 112.8 (10.4) 1 mg/kg 111.8 (8.5) 3 mg/kg 111.5 (5.3) 6 mg/kg 112.5 (7.3) Diastolic blood Placebo 69.0 0.3 mg/kg 62.5 1 mg/kg 65.3 3 mg/kg 66.3 6 mg/kg 63.8 Heart rate Placebo 0.3 mg/kg 1 mg/kg 3 mg/kg 6 mg/kg

(mmHg) 115.6 (11.3) 118.5 (7.3) 118.5 (10.4) 116.3 (5.4) 121.5 (13.4)

pressure (mmHg) (7.6) 72.3 (7.2) (3.0) 69.5 (6.4) (4.1) 72.3 (9.5) (9.0) 79.5 (9.6)* (7.8) 85.2 (9.4)*

(beats/min) 61.2 (8.8) 62.0 (5.3) 64.7 (6.8) 61.0 (6.5) 61.8 (3.7)

58.2 59.5 54.2 53.3 46.7

(6.1) (3.3) (6.1)* (8.3)* (5.8)*

115.3 114.8 115.0 117.2 117.0

(7.4) (13.1) (8.0) (7.1) (8.0)

116.0 114.7 120.5 115.8 121.3

(12.7) (7.1) (9.9) (5.8) (8.4)

73.7 68.0 66.8 73.8 78.2

(5.6) (5.2) (4.9) (8.6) (9.8)

73.7 75.5 79.7 74.5 77.0

(9.8) (12.5) (11.8) (12.7) (10.8)

60.5 64.0 64.8 59.0 69.5

(6.7) (10.2) (6.4) (8.0) (5.5)

63.7 60.8 66.3 64.5 59.5

(7.6) (7.2) (3.8) (10.0) (5.4)

*Indicates when peak value is significant different from placebo P<0.05

501 Fig. 1 Vmean in the middle cerebral artery. There were no significant differences in Vmean between doses (P=0.27) and none of the doses changed Vmean significantly over time. n:6 mg/kg NG monomethyl L-arginine (L-NMMA:546C88) over 15 min, ¤: 3 mg/kg L-NMMA over 5 min, m: 1 mg/kg L-NMMA over 5 min, d: 0.3 mg/kg L-NMMA over 5 min, ;: 5% dextrose over 5 min

Cerebral haemodynamic

The diameter of the radial artery

Since no systematic side-to-side difference was found (paired t-test), the blood velocity data is presented as the results from the right MCA. During the 60 min after the start of infusion, there was no significant difference in Vmean between the different doses, including placebo (P=0.27). None of the doses changed Vmean significantly from baseline over time, (Fig. 1). During the same time period, pCO2 did not differ between doses (P=0.53), and pCO2 did not change significantly over time on any of the doses. Therefore, no pCO2 corrections of Vmean have been made.

During the first 60 min after start of the infusion, the diameter of the radial artery did not differ among the different doses or placebo (P=0.50), but the radial artery diameter changed significantly over time with placebo and with all doses (except 1 mg/kg, P=0.16), (Fig. 2).

Fig. 2 Changes in the radial artery diameter. There were no significant differences in the diameter of the radial artery between doses (P=0.50). With all doses, inclusive placebo (except 1 mg/kg, P=0.16), the radial diameter changed significantly over time. This is most likely caused by an increase in room temperature (approximately 2C) at all study days. An open symbol denotes a statistical difference from the baseline value; filled symbols, no statistical difference from baseline. h, n: 6 mg/kg NG monomethyl L-arginine (L-NMMA:546C88) over 15 min, e, r: 3 mg/kg L-NMMA over 5 min, D, m: 1 mg/kg L-NMMA over 5 min, s, d: 0.3 mg/kg L-NMMA over 5 min, x, ;: 5% dextrose over 5 min

Blood pressure The mean arterial blood pressure (MAP) at 3 mg/kg was significantly higher than after placebo, and MAP at 6 mg/kg was significantly higher than after all other doses except the 3-mg/kg dose (P<0.001). During the first 60 min after start of the infusion, the peak value of MAP on all doses was significantly higher than the peak value for placebo, and peak MAP at 6 mg/kg also differed significantly from peak MAP at all lower doses (P<0.0001). The maximal increase of MAP (20%) occurred 20 min after the start of the infusion of 6 mg/kg, and MAP did not reach baseline in the 2-h observation period (Table 1; Fig. 3).

502 Fig. 3 Mean arterial blood pressure (MAP) after 6 mg/kg NG monomethyl L-arginine (L-NMMA:546C88) was significantly different from all other doses except the 3 mg/kg dose. MAP at the 3 mg/kg dose differed significantly from MAP after placebo (P<0.001). An open symbol denotes a statistical difference from the baseline value; filled symbols, no statistical difference from baseline. h, n: 6 mg/kg L-NMMA over 15 min, e, ¤: 3 mg/kg L-NMMA over 5 min, D, m: 1 mg/kg L-NMMA over 5 min, s, d: 0.3 mg/kg L-NMMA over 5 min, x, ;: 5% dextrose over 5 min

Systolic blood pressure Systolic blood pressure was not significantly affected by any of the doses compared with placebo (P=0.12) and peak systolic blood pressure during the first 60 min from start of infusion did not vary significantly among doses (P=0.15). With all four doses, but not with placebo (P=0.35), the systolic blood pressure changed significantly over time (Table 1).

the three highest doses, but not when placebo and 0.3 mg/kg were given (Table 1; Fig. 4). Adverse effects No adverse effects were observed in any subject during or after the infusion of L-NMMA. Apart from the decrease in heart rate, there were no changes in the ECG (no signs of ishaemia or significant changes in PR intervals).

Diastolic blood pressure The diastolic blood pressure at 3 mg/kg was significantly higher than placebo, and at 6 mg/kg it was significantly increased compared with all lower doses (P<0.001). The peak diastolic blood pressure at 6 mg/kg was significantly higher than all other doses, and the value at 3 mg/kg was significantly higher than placebo (P<0.001). Diastolic blood pressure at 6 mg/kg did not reach baseline during the 2-h observation period. For all doses, including placebo, the diastolic blood pressure changed significantly over time (Table 1). Heart rate Heart rate at 6 mg/kg was significantly different (decreased) from the 0.3-mg/kg dose and placebo. Furthermore, heart rate at the 1-mg/kg and 3-mg/kg doses was significantly lower than at the 0.3-mg/kg dose, but not at placebo (P<0.05). The peak value of the heart rate during the first 60 min from the start of infusion at the doses 1, 3 and 6-mg/kg rate was significantly lower than at placebo and 0.3 mg/kg (P<0.001). The maximal decrease in heart rate (24%) occurred 15 min after the start of infusion of 6 mg/kg and reached baseline after 45 min. Heart rate changed significantly over time with

Discussion The three NOS—endothelial, neuronal and inducible NOS—are all inhibited by L-NMMA, a guanidinosubstituted analogue of L-arginine [7]. L-NMMA was, in the present study, administered in increasing doses to healthy human volunteers during the resting condition, and we found that inhibition of NOS had no effect on: (1) the mean maximal blood velocity in the middle cerebral artery, (2) the basal diameter of the radial artery and (3) the end tidal pCO2. However, the MAP increased dose dependently, with a maximal increase of 20% 20 min after start of infusion (6 mg/kg L-NMMA over 15 min), and heart rate decreased dose dependently with a maximum decrease of 24% 15 min after the start of the infusion (same dose and infusion time). NO in the regulation of the basal tone of the MCA Kiss et al. found an 11% decrease in MCA velocity using 6 mg L-NMMA/kg given over 5 min followed by 60 lg/kg min for 95 min [13], but, in another study from the same group using a slightly smaller dose, no such change was found [14]. We found no effect of any of the four

503 Fig. 4 Heart rate after 6 mg/kg NG monomethyl L-arginine (L-NMMA:546C88) was significantly decreased compared with heart rate at 0.3 mg/kg and placebo (P<0.001). An open symbol denotes a statistical difference from the baseline value; filled symbols, no statistical difference from baseline. h, n: 6 mg/kg L-NMMA over 15 min, e, ¤: 3 mg/kg L-NMMA over 5 min, D, m: 1 mg/kg L-NMMA over 5 min, s, d: 0.3 mg/kg LNMMA over 5 min, x, ;: 5% dextrose over 5 min

studied doses of L-NMMA ranging from 0.3 mg/kg to 6 mg/kg on MCA velocity. The SD of Vmean is quite high among subjects; however, the intra-subject variation was much less from a methodological study (the inter-subject coefficient of variability was 26 cm/s whereas the intrasubject coefficient of variability was 7 cm/s) [18]. When following the same changes in the same subject, as done in the present crossover study, the intra-subject SD can be estimated to 5 cm/s. Using this SD, we would not overlook a difference of 10 cm/s with a type-1 error of 5% and type-2 error of 10% using a number of five subjects (Med Stat). We did not examine the diameter of MCA directly, but studied the blood velocity, Vmean, in MCA. Vmean is related to the cerebral blood flow (CBF) and radius of the MCA by the following equation: regional cerebral blood flow (rCBF)=Vmean ·(2 · phi · r2) [21]. The effect of 3 mg L-NMMA/kg over 5 min on resting CBF has recently been studied in the basilar artery and in both internal carotid arteries by the use of a gradient-echo phase contrast MRI technique. At this small dose, there was no significant change in CBF [15]. When a bolus of 10 mg L-NMMA/kg followed by 10 mg/kg/h was used in a positron emission tomography (PET) study, a 20% decrease of resting global CBF was found [16]. No recordings of MCA blood velocity were performed, but the PET group had previously studied blood velocity in the middle cerebral artery with simultaneous colour velocity ultrasonic imaging of volume flow in the common (CCA) and internal carotid arteries (ICA). At 10 mg L-NMMA/kg (bolus), there was no significant reduction in MCA velocity, but there was a 29.5% decrease in CCA flow and 14.9% decrease in ICA flow, suggesting a constriction of MCA [17]. Based on those results and the fact that the NO-donor glyceryl trinitrate (GTN) dilates MCA without affecting CBF [22], it was proposed that MCA diameter alters with NOS inhibition [17]. Since L-NMMA does not affect

MCA velocity, it is most likely that the proposed MCA constriction represents a physiological adaptation to the reduction of CBF rather than that MCA has a basal NO tonus. Nitric oxide in the regulation of the basal tone of the radial artery We found no significant effect of iv L-NMMA on the resting tone of the radial artery at any of the doses. This is in agreement with studies of intra-brachial artery L-NMMA infusions [23, 24]. Radial artery flow was reduced but there was no change in the internal diameter of the radial artery. Neither in the human pulmonary artery [25] nor in the proximal part of the human coronary artery [26] was an effect of NOS inhibition on the basal diameter found. It has been suggested that the absence of a decrease in radial diameter on inhibition of NO synthesis may be explained by the presence of compensating vasodilator mechanisms occurring in the large arteries [23]. However, it seems more reasonable to assume that the basal tone of the conduit arteries is not under influence of NO. The production of NO and, thereby, the effect of NOS inhibition may be completely different between basal circumstances and during activation. Vasodilatation induced by different autacoids such as acetylcholine [27], bradykinin [28], substance P [27] and possibly also calcitonin gene-related protein (CGRP) [29] and histamine [30] plus vasodilatation induced by mechanical factors such as shear stress and pulsatile flow [31] is caused by increased intracellular production of NO. In our study, the diameter of the radial artery increased significantly over time for all doses, including placebo. This was probably caused by a rising temperature in the laboratory of approximately 2C at the end of the experiments on all study days.

504

Effect of NOS inhibition on resting mean arterial blood pressure and heart rate It is well established that mean arterial blood pressure increase is dose dependent in response to i.v. L-NMMA [8, 9, 10, 11, 12, 32]. L-NMMA 3 mg/kg over 3–5 min causes a 2–16% increase in MAP [8, 9, 10, 11]. In the present study this dose caused a 12% increase in MAP. After 6 mg/kg over 5–15 min, MAP increased 9–17% [10, 12, 32]. In the present study, it caused a 20% increase in MAP. When L-NMMA 10 mg/kg was given as a bolus, a 16% increase in MAP was observed [17], but when the bolus was followed by infusion of 10 mg/kg/h, MAP increased 24% [16, 33]. In most studies, including our study, the MAP increase has been due to an increase in diastolic blood pressure, whereas the systolic blood pressure has only contributed to a minor degree, or not at all. Mice lacking the gene for eNOS are hypertensive, and mice lacking nNOS are normotensive [34]. Therefore, the blockade of eNOS probably causes the L-NMMAinduced increase in blood pressure [35]. A contribution of nNOS blockade with a sympatho-excitatory effect has, however, been proposed as an explanation for the continued MAP elevation after NOS-inhibitor administration [12, 36]. L-NMMA causes a dose-dependent decrease in heart rate [9, 10, 11, 13, 32]. Given as 3 mg/kg over 3–5 min, it causes a heart rate decrease ranging between 6% and 19% [9, 10, 11, 13]. In the present study, we found a 12% decrease. When 6 mg/kg has been given over 5–15 min, a 16–21% heart rate decrease has been reported [10, 13, 32]. In the present study, we found 24% decrease. Recently 10 mg/kg L-NMMA has been reported to induce a 23–26% decrease in heart rate, both when given as a bolus and when followed by continued infusion (10 mg/kg/h) [16, 17, 33]. The time from end of infusion to the time for recordings may vary between the studies. Whether baroreceptor reflexes cause this decrease in heart rate is discussed. Thereby, the L-NMMA-induced increase in systemic blood pressure should induce a reflex-mediated withdrawal of sympathetic nervous system activity with subsequent decrease in heart rate. In the early studies of intra-arterial infusion of L-NMMA, the vasoconstrictor effect on the human forearm suggested that systemic doses of this inhibitor would lead to a near doubling of mean arterial blood pressure, if without the effect of reflex compensatory changes [37]. When phenylephedrine is used to elevate systemic blood pressure to the same extent as with L-NMMA, heart rate also decreases to the same degree as with L-NMMA [33]. Furthermore, when blood pressure experimentally is held constant during L-NMMA infusion to eliminate a confounding influence of a baroreflex activation, heart rate is unchanged from baseline [35]. When L-NMMA has been infused directly into a coronary artery in humans, no change in heart rate or systolic blood pressure was observed [26].

However, in a recent study of the direct role of NOS inhibition on pacemaker activity in human cardiac transplant recipients who posses a denervated donor heart, it was found that NO exerts a direct facilitating influence on pacemaker activity in the sino-atrial node (chronotropic action); as injection of the non-NOdependent vasoconstrictor, phenylephedrine, archived a similar rise in MAP as L-NMMA, but failed to change the R-R interval [38]. The decline in heart rate after L-NMMA may, however, be due to other mechanisms. A direct effect of L-NMMA on cardiac pump function seems unlikely since NO itself reduces contractility of cardiac myocytes [39, 40]. A direct toxic effect of L-NMMA on myocytes seems also unlikely since no changes in ECG have been observed during the administration of L-NMMA in the present or any other study. In conclusion, our study confirms that tonic endothelial NO production is an important determinant of resting blood pressure and suggests that the basal tone of the middle cerebral artery and of the radial artery in humans is not under influence of NO. Furthermore, it was concluded that the dose 6 mg/kg L-NMMA given over 15 min was well tolerated in young healthy volunteers. The use of a higher dose might have provided us with further information, but the increase in MAP and decrease in heart rate observed at the 6-mg/kg dose was considered the maximal acceptable changes in healthy subjects; therefore, this dose was chosen for further studies. Acknowledgements The skilful assistance of the technicians Mrs. Kirsten Enghave and Mrs. Bente Leisner and the medical student Pernille A. Haderslev is gratefully acknowledged. The study was supported financially by The Wellcome Foundation, Novo-Nordisk Foundation and by The Danish Research Academy. This experiment complies with the current laws of Denmark.

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