Journal of Human Hypertension (2014) 28, 494–499 & 2014 Macmillan Publishers Limited All rights reserved 0950-9240/14 www.nature.com/jhh
ORIGINAL ARTICLE
Lack of changes in carotid artery compliance with systemic nitric oxide synthase inhibition J Sugawara1,2, Y Saito3, S Maeda3, M Yoshizawa1,3, H Komine1, M Nakamura3, R Ajisaka3 and H Tanaka2 Proximal large elastic arteries (ascending aorta and carotid artery) have an important role in buffering the pulsatile pressure generated from the left ventricle, which forwards continuous peripheral blood flow and protects the brain microcirculation from end-organ damage. Although compliance of distal conduit arteries (extremities’ arteries) is attenuated by the nitric oxide synthase (NOS) inhibition, it is yet unknown whether compliance of proximal elastic arteries changes by the systemic NOS inhibition. To address this question, we measured central artery compliance in 17 young adults (26±1 years) who underwent intravenous infusions of NG-monomethyl-L-arginine (L-NMMA) or saline (placebo) on separate days. Following the systemic NOS inhibition, the mean arterial pressure (MAP), total peripheral resistance and aortic augmentation index were significantly increased. However, carotid artery compliance was not affected significantly (from 0.10±0.01 to 0.11±0.01 mm2 per mmHg) and the b-stiffness index (an index of arterial compliance adjusted for the distending pressure) tended to decrease (from 6.63±0.35 to 6.06±0.42 a.u., P ¼ 0.07). These parameters were not altered with saline infusion. Changes in the b-stiffness index tended to correlate negatively with the corresponding changes in MAP (r ¼ 0.31, P ¼ 0.07). These results suggest that carotid artery compliance remains unchanged during the systemic NOS inhibition in spite of systemic vasoconstriction. Journal of Human Hypertension (2014) 28, 494–499; doi:10.1038/jhh.2013.137; published online 9 January 2014 Keywords: endothelial function; vascular tone; arterial stiffness
INTRODUCTION In the arterial tree, it is the central elastic arteries in the cardiothoracic region that dampen the pulsatile pressure and flow generated from the left ventricle.1 However, such function is deteriorated with advancing age.2,3 The decreased compliance of large elastic arteries (for example, aorta and carotid artery) is associated with the greater incidence of future cardio- and cerebrovascular diseases.4–7 Central arterial stiffening is associated with an increased susceptibility to white matter rarefaction or cerebral leukoaraiosis.8,9 Hence, physiological factors contributing to large arterial compliance have been a topic of intense investigation in the field of cardiovascular and cerebral vascular research. Compliance of artery is determined by the contractile state of the vascular smooth muscle cells as well as structural factors (for example, elastin and collagen) in the arterial wall.1,10 In particular, nitric oxide (NO) bioavailability, which has emerged as an important regulatory mechanism of vasomotor tone, could regulate arterial compliance.11,12 In animal experiments,11 intraarterial infusion of NG-monomethyl-L-arginine (L-NMMA), a NO synthase (NOS) inhibitor, significantly increased iliac artery pulse wave velocity (PWV) independent of changes in the blood pressure. These results indicated that basal NO bioavailability influences compliance of ‘peripheral muscular’ arteries. On the other hand, Stewart et al.13 determined the relation between changes in carotid-femoral PWV and mean arterial pressure (MAP) with systemic infusions of vasoactive drugs. Changes in PWV caused by three pressor agents, including L-NMMA, were closely correlated with changes in MAP. On the basis of these results,
the systemic NOS inhibition-induced increase in aortic PWV was attributed to the corresponding increases in blood pressure rather than any specific effect of NOS inhibition on the large arterial wall. As carotid-femoral PWV includes stiffness of the distal part of aorta (that is, the thoracic and abdominal descending aorta), the influence of inhibition of basal NO release on compliance of proximal vessels (that is, the ascending aorta and carotid artery) is not known. We reason that the elucidation of physiological mechanisms responsible for carotid artery compliance might give us distinct target for prevention of future cardio- and cerebrovascular diseases. Accordingly, the primary aim of this study was to determine the impact of the basal NO bioavailability inhibition on carotid artery compliance as a measure of large elastic artery stiffness. In the present study, we studied young healthy adults as their high basal NO bioavailability could facilitate distinguishing role of NO when NOS inhibitors were applied. MATERIALS AND METHODS Subjects Seventeen sedentary or recreationally active young adults (26±1 years; nine men and eight women) were studied. All the subjects were non-obese (height 167.2±2.3 cm, body mass 58.6±2.0 kg and body mass index 21.0±0.3 kg m 2), normotensive and free of overt cardiovascular disease as assessed by medical history. All the subjects had not been taking any medications including oral contraceptives. Subjects who were current smokers or smoked within the past 2 years were excluded. Before the study, all the subjects were explained about potential risks and procedures of the study and they gave their written informed consent to participate in
1 Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan; 2Department of Kinesiology and Health Education, The University of Texas at Austin, Austin, TX, USA and 3Graduate School of Comprehensive Human Sciences, The University of Tsukuba, Tsukuba, Japan. Correspondence: Dr J Sugawara, Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan. E-mail: [email protected] Received 31 July 2013; revised 4 November 2013; accepted 21 November 2013; published online 9 January 2014
Arterial compliance and NO bioavailability J Sugawara et al
495 the study. The study conformed with the principles outlined in the Helsinki Declaration and was reviewed and approved by the Institutional Review Board.
Experimental protocol After overnight fast, each subject underwent two experiments (NOS inhibition and saline control conditions) with a randomized counterbalanced design on separate days. All subjected were blinded to the order of sessions (saline or L-NMMA) (for example, single-blind manner); however, the investigators were aware of which drugs were infused for the safety reasons. Female subjects were studied at the same menstrual cycle phase for both experiments. The subjects abstained from alcohol, caffeine and vigorous exercise for at least 24 h before the experiments. All measurements were conducted in a quiet temperature-controlled room (25 1C). The timeline of the experiment was illustrated in Figure 1. After 10 min of supine rest, the baseline measurements were performed. Then, the subjects were administrated with intravenous NOS inhibitor (L-NMMA, 3 mg kg 1 bolus over 5 min and a subsequent continuous (0.05 mg kg 1 min 1) infusion) or the same amount of saline (control condition). The procedure of infusions and dose of the systemic drug infusion were consistent with previous studies.14 The second measurements were made 5–10 min after the commencement of continuous intravenous infusions. The total volume of the infusion throughout each experiment was 17.5 ml.
Hemodynamic measurements Blood pressure and beat-to-beat arterial blood pressure waveforms were recorded by using photoplethysmography (Portapres Model 2, TNO TPD Biomedical Instrumentation, Amsterdam, The Netherlands) on the finger. Heart rate was obtained with a standard electrocardiogram (ML132 Bio Amp, AD Instruments, Colorado Springs, CO, USA). Systolic and diastolic blood pressures (SBP and DBP, respectively) and MAP of brachial artery were estimated from the reconstructed pressure waveform from finger arterial pressure waveform. Stroke volume and cardiac output were calculated from the finger blood pressure waveform using the validated Model flow method incorporating age, sex, height and weight (BeatScope 1.0; TNO TPD; Biomedical Instrumentation).15 This methodology has been shown to reliably estimate rapid changes in cardiac output during a variety of experimental protocols.16–19 Total peripheral resistance (TPR) was calculated as MAP/cardiac output.
Arterial compliance measures The combination of ultrasound imaging of the common carotid artery diameter with applanation tonometry recording of blood pressure on the contralateral carotid artery was applied to obtain arterial compliance as previously described.20,21 A longitudinal image of the common carotid artery was measured with an ultrasound machine equipped with a highresolution multifrequency linear-array transducer (EnVisor; Koninklijke Philips Electronics, Eindhoven, The Netherlands). Successive 10 beats of common carotid artery longitudinal image were acquired at maximal (systolic) and minimal (diastolic) diameters. Arterial lumen diameter (from the media/adventitia interface of the near wall to the lumen/intima interface of the far wall of the vessel) was measured offline using the commercially available software (Carotid Analyzer, Medical Imaging Applications, Coralville, IA, USA). Arterial pressure waveforms were recorded noninvasively on the common carotid artery with arterial applanation tonometry incorporating an array of 15 micropiezoresistive transducers (VP-2000, Colin Medical Instrument, San Antonio, TX, USA) and sampled at 1 kHz using an analog-to-digital converter (PowerLab 8/30, AD Instruments, Bella Vista, New South Wales, Australia) interfaced with a
personal computer equipped with data acquisition software (LabChart 6, AD Instruments). Carotid arterial pressures were calibrated by brachial MAP and DBP.22 Arterial compliance, Young’s elastic modulus and the b-stiffness index were calculated using the equations: [(D1 D0)/D0]/[2(P1–P0)] p D20, ([Ps Pd] Dd)/([Ds Dd]/IMT) and ln(P1/P0)/[(D1 D0)/D0], where D1 and D0 are the maximal and minimum arterial diameters, P1 and P0 are the highest and lowest carotid arterial pressures and IMT is carotid artery intima-media thickness.23–25 Carotid artery diameter and IMT were analyzed using image analysis software (Carotid Analyzer, Medical Imaging Applications). All image analyses were performed by the same investigator who was blinded to the sessions (saline or L-NMMA). The day-to-day coefficients of variation for carotid artery diameter and pulse pressure are 2±1% and 7±3%, respectively. The carotid arterial waveform data were also fed into the SphygmoCor software (AtCor Medical, Sydney, New South Wales, Australia), and a validated transfer function was applied to estimate aortic blood pressure.26 Aortic augmentation index (AIx) and the reflected wave transit time (RWTT) were also computed.27 AIx is a measure of the contribution of wave reflection to the aortic waveform, and depends on PWV and the magnitude and site of the reflected pressure wave.28 To account for the influence of heart rate on AIx, the index of AIx normalized for heart rate of 75 bpm (AIx75) was also obtained. The SphygmoCor software could not calculate AIx75 of one subject because of low heart rate (o45 bpm). RWTT was measured from the foot of the carotid pressure waveform to the first inflection point, which corresponds to the foot of the global reflected pressure wave.27
Statistical analyses Repeated measures analysis of variance (general linear model) was used to determine the effects of systemic NOS blockades on carotid artery compliance and other hemodynamic measures. Analysis of covariance was performed to compare responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for. In the case of a significant F-value, a post hoc test using the Newman–Keuls test identified significant differences among the mean values. For all three arterial compliance measures, normality of the distribution was confirmed in all parameters by Kolmogorov–Smirnov test. Owing to a relatively small sample size used in the present study, nonparametric test was also performed. The association between the variables of interest was evaluated by parametric and non-parametric simple correlation analyses (Pearson’s r and Spearman’s r). All data are reported as the mean±s.e.m. Statistical significance was set a priori at Po0.05.
RESULTS There was no significant difference in baseline hemodynamic variable between the saline control and the NOS inhibition conditions (Table 1). Brachial SBP, DBP and, mean blood pressure increased significantly with the L-NMMA administration but did not change with the saline infusion. Brachial pulse pressure did not change significantly with either L-NMMA or saline infusion. Similar trend of changes were observed in carotid and aortic blood pressures (Table 2). Stroke volume did not change with either saline or L-NMMA infusion. Heart rate and cardiac output decreased significantly with the L-NMMA administration, and mild but significant reductions in these measures were also observed
Before infusion (baseline)
During infusion
Measurement
Commencement of resting
Bolus infusion (3mg/kg)
Measurement
Continuous infusion (0.05mg/kg/min)
L-NMMA or saline 0
5
10
15
0
5
10
15
(min)
Figure 1.
Timeline of experimental protocol.
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Journal of Human Hypertension (2014) 494 – 499
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496 Table 1.
Hemodynamic responses to saline/L-NMMA infusions
Variables
Saline
Brachial systolic BP, mmHg Brachial diastolic BP, mmHg Brachial mean BP, mmHg Brachial pulse pressure, mmHg Heart rate, bpm Stroke volume, ml Cardiac output, l min 1
L-NMMA
Baseline
Infusion
Baseline
Infusion
120±3 59±2 78±2 60±2 64±2 94±4 6.0±0.3
119±4 61±2 79±3 58±2 61±2a 90±4 5.5±0.3a
123±2 62±2 81±2 61±2 65±2 94±4 6.1±0.3
134±3a,b 74±2a,b 93±3a,b 60±1 56±1a,b 91±4 5.1±0.3a,b
Abbreviations: BP, blood pressure; L-NMMA, NG-monomethyl-L-arginine. Data are means±s.e.m. Brachial arterial pressure was obtained from the reconstructed finger arterial pressure waveform. aPo0.05 versus baseline. bPo0.05 versus saline infusion.
Table 2.
Responses of carotid arterial and aortic characteristics to saline/L-NMMA infusions
Variables
Carotid systolic BP, mmHg Carotid diastolic BP, mmHg Carotid pulse pressure, mmHg Carotid arterial systolic diameter, mm Carotid arterial diastolic diameter, mm Carotid arterial distention, mm Aortic systolic BP, mmHg Aortic diastolic BP, mmHg Aortic pulse pressure, mmHg Aortic AIx, %
Saline
L-NMMA
Baseline
Infusion
Baseline
Infusion
104±2 59±2 45±1 6.3±0.1 5.8±0.1 0.52±0.03 100±2 59±2 41±1 5±4
102±3 61±2 42±1 6.5±0.1a 6.0±0.1a 0.5±0.03 99±3 61±2 39±1 4±4
107±2 62±2 45±1 6.3±0.1 5.8±0.1 0.5±0.02 104±2 62±2 42±1 0±3
118±3a,b 74±2a,b 44±1 6.5±0.1a 6.0±0.1a 0.5±0.03 117±3a,b 74±2a,b 43±1 15±3a,b
Abbreviations: AIx, augmentation index; BP, blood pressure; L-NMMA, NG-monomethyl-L-arginine. Data are means±s.e.m. aPo0.05 versus baseline. b Po0.05 versus saline infusion.
with the saline infusion. Aortic AIx and AIx75 increased and RWTT decreased significantly with the L-NMMA administration but did not change with the saline infusion (Figure 2). At baseline, there were no significant differences in carotid artery diastolic or systolic diameter between the two conditions (Table 2). Carotid artery diastolic and systolic diameters increased significantly with both the saline and L-NMMA infusions. Baseline carotid artery compliance and the b-stiffness index were comparable between the two conditions (Figure 3). Carotid artery compliance and Young’s elastic modulus were not changed significantly with either the saline (from 0.11±0.01 to 0.11 ±0.01 mm2 per mmHg and from 244±20 to 245±21 mmHg*mm, respectively) or L-NMMA infusion (from 0.10±0.01 to 0.11 ±0.01 mm2 per mmHg and from 249±14 to 260±19 mmHg*mm, respectively). The b-stiffness index was not affected by the saline infusion (from 6.63±0.38 to 6.66±0.42 a.u.) and tended to decrease with the L-NMMA infusion (from 6.63±0.35 to 6.06±0.42 a.u., P ¼ 0.07). Additionally, there were no significant differences in responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for in ANCOVA. Changes in the b-stiffness index induced by the drug infusions tended to correlate negatively with the corresponding changes in MAP (r ¼ 0.31, P ¼ 0.07, Figure 4). Non-parametric correlational analysis (Spearman’s ranks order correlation) identified a significant correlation (r ¼ 0.343, Po0.05). The saline/L-NMMA administration-induced changes in RWTT, aortic AIx and AIx75 were significantly correlated with the changes in MAP (r ¼ 0.49, 0.41 and 0.43, respectively). Journal of Human Hypertension (2014) 494 – 499
DISCUSSION As NO is a potent vasodilator, it is reasonable to speculate that NOS inhibition would increase vascular tone and decrease arterial compliance. However, the present findings do not support such notion. We found that the systemic L-NMMA infusion did not alter measures of the carotid artery compliance. There are a number of possible explanations for a lack of effects of NO inhibition on arterial stiffness. One interpretation is that, as the amount of smooth muscle cells surrounding the vascular wall decreases from the peripheral arteries to central arteries in the arterial tree, NO has smaller or negligible roles in determining the distensibility of proximal elastic arteries (that is, ascending aorta and carotid artery). Stewart et al.13 suggested that effects of inhibition of basal NO release on carotid-femoral PWV could be explained by the elevation of MAP rather than any specific effect of NO inhibition within the aorta. The study by Stewart et al.13 is consistent with the findings of the current study. In contrast, Wilkinson et al.29 suggested that large artery stiffness is regulated by NO because of the significant increase in aortic AIx via intravenous infusion of L-NMMA in healthy young adults. However, AIx is no longer been considered as a measure of arterial stiffness as it is an index of arterial wave reflection from the periphery.1 Similar to that study,29 L-NMMA induced significant increases in aortic AIx and AIx normalized for HR (AIx75) in the present study. Additionally, changes in aortic AIx and AIx75 induced by the saline/L-NMMA administrations were significantly correlated with the corresponding changes in MAP. Therefore, the systemic NOS inhibitioninduced increase in AIx may not be because of the alterations in the elastic properties of central arteries but may rather be explained by the epiphenomenon of MAP increase and the & 2014 Macmillan Publishers Limited
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497 Saline L-NMMA 0.14
*
90 80 70
0.10 0.08
60
0.06
40
350
35 TPR (unit)
0.12
*†
30 25
*
20
Young’s elastic modulus (mmHg*mm)
MAP (mmHg)
100
Arterial compliance (mm2/mmHg)
110
Saline L-NMMA
300 250 200 150
15 8
Aortic AIx75 (%)
5 0
*
-5 -10
β-stiffness index (au)
10 7 6 5
-15
4 Baseline
L-NMMA /saline
-20
Figure 3. Changes in carotid arterial compliance, Young’s elastic modulus and b-stiffness index in response to systemic nitric oxide synthase blockade (L-NMMA) and saline administrations.
190 6 180 * 170 160 Baseline
L-NMMA /saline
Figure 2. Changes in MAP, TPR, aortic augmentation index normalized for heart rate of 75 bpm (AIx75) and RWTT in response to systemic nitric oxide synthase blockade (L-NMMA) and saline administrations. *Po0.05 versus baseline; wPo0.05 versus saline infusion.
elevations in peripheral vasoconstriction augmenting the reflected waves. An alternative interpretation is that the effect of L-NMMA would have been masked by the subsequent reductions in sympathetic vasoconstrictor tone that occurs as a counter-regulatory effect. In other word, NO may be likely involved in modulating arterial compliance but the reflex reductions in sympathetic vasoconstrictor tone may have masked such contribution, resulting in unchanged carotid artery compliance. The inverse relation between responses of MAP and the b-stiffness index, an index & 2014 Macmillan Publishers Limited
Changes in β-stiffness index (au)
RWTT (msec)
200
Saline L-NMMA
4 2 0 -2 -4 r= -0.31, P=0.07 -6 -20
-10
0
10
20
30
Changes in MAP (mmHg)
Figure 4. Relation between changes in MAP and changes in b-stiffness index in response to infusions of saline and L-NMMA.
adjusted for the distending pressure, supports this hypothesis. In this context, we have previously investigated the effect of NOS inhibition on carotid artery compliance under systemic a-adrenergic receptor blockade, phentolamine.30 The increase in arterial stiffness was observed after the systemic administration of L-NMMA preceded by phentolamine infusions. Although the prior a-adrenergic receptor blockade is a reasonable approach to Journal of Human Hypertension (2014) 494 – 499
Arterial compliance and NO bioavailability J Sugawara et al
498 isolate the effect of NO on arterial compliance, there may be secondary effects that confound the interpretation. Under conditions of a-adrenergic receptor blockade, the facilitation of noradrenaline release by phentolamine (because of blockade of presynaptic a2 receptors) can stimulate b-adrenergic receptors.31,32 Additionally, NOS inhibition augments b-adrenoceptor-mediated vasorelaxation33 and a loss of a1-adrenergic receptors leads to enhancement of vascular responsiveness to vasoconstrictors.34 Moreover, our previous study involving prior a-adrenergic receptor blockade30 was conducted in middle-aged and older adults, making the comparison of these studies with the present study involving young adults difficult. Clearly, more studies are needed to fully answer the question regarding the role of NO on the distensibility of the central elastic arteries in humans. There is a growing recognition that central arterial pressure differs from peripheral arterial pressure. Reflections of the forward propagating pressure wave occur at multiple sites of the arterial bed because of changes in arterial properties (for example, elasticity/stiffness gradient and vasomotor tone) or in the architecture of the arterial tree (for example, branching points and calcifications).3,35,36 The multiple reflected waves are integrated as a single combined reflected wave, which sums with the forward ejected wave and thus forms the local pressure pulse wave. In young healthy populations, a large elasticity/ stiffness gradient introduces a greater peripheral pulse amplification and disparity between central and peripheral pulse pressures. As such, it is important to evaluate vascular wall responses matching to local pressure changes. We determined the influence of acute systemic NOS inhibition on carotid artery compliance in young healthy adults. Carotid artery compliance was determined by simultaneously measuring diameter changes and pressure changes both in the common carotid artery. The systemic L-NMMA administration did not affect measures of carotid artery compliance in spite of a greater vasoconstriction as seen in elevated MAP and TPR. The compliance of large elastic arteries (for example, aorta and carotid artery) buffers the repeated pulsatile pressure generated from the left ventricle and fosters smooth continuous blood flow to the frail brain tissue, and its decline is associated with the greater incidence of future cerebrovascular diseases.7,9 Thus, the absence of change in carotid artery compliance might be a protective mechanism that some other redundant factors (for example, reductions in local sympathetic vasoconstrictor tone) may have contributed to preserve arterial compliance in order to prevent potential cerebrovascular disease. We identified significant changes in heart rate, cardiac output and carotid artery diameter under the saline control condition. Although these changes are fairly small, they are statistically significant. It is possible that blood volume expansion induced by infusions might have lowered heart rate and cardiac output via cardiopulmonary baroreflex. The most important issue to note here is that all of these changes were observed also under the experimental conditions (for example, L-NMMA infusion). Furthermore, there were no significant differences in responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for in ANCOVA. As in any other studies, there are some drawbacks of our study that need to be emphasized. First, the sample size for this study was relatively small. This was primarily because of the fact that the study was highly invasive as well as risky as it involved the infusion of systemic pharmacological blockade. In order to determine the minimal sample size, the power calculations were performed prior to the study using the mean acute changes in arterial compliance that were expected with the pharmacological administration studies.13,20,29 The sample size was based on a power calculation that showed that 16 subjects were required to achieve 80% power Journal of Human Hypertension (2014) 494 – 499
to detect a difference of 15% in arterial compliance, with a ¼ 0.05. Second, the completeness of NOS blockade was not confirmed, although significant vasoconstriction was identified with the systemic administration of L-NMMA in the present study. Third, this study was conducted in the single-blind manner. For the safety reasons, the investigators were aware of which drugs were being infused. However, all subjected were blinded to the order of sessions (saline or L-NMMA), and image analyses were performed by the same investigator who was blinded to the sessions. Fourth, the measurement of peripheral artery stiffness was omitted from the present study because it has been fairly well established that the stiffness/compliance of peripheral arteries (brachial or femoral arteries) do not change with aging and disease state and are not considered to be clinically important. As the safety of the subjects was the number one priority in this highly risky protocol that involves systemic drug infusion, one extra measurement that did not provide much clinical insight was dropped from the measurement battery. Fifth, even though we attributed no significant change in carotid artery compliance with systemic NOS inhibition to the contribution of the reflex reductions in sympathetic vasoconstrictor tone, we did not directly measure sympathetic nervous activity. In this context, there are currently no experimental approaches in humans to measure sympathetic nervous system activity directed to the central elastic arteries. Finally, systemic NOS inhibition may not be the ideal procedure to address this issue. Arguably, the most effective procedure to investigate this in humans is a ‘local’ infusion of L-NMMA applied in the human aorta and/or other central elastic arteries (for example, carotid arteries). However, such procedure is extremely risky as well as difficult to interpret, as the associated elevation in blood pressure and/or reflex reductions in sympathetic vasoconstrictor tone would confound the interpretation. In conclusion, we found that carotid artery compliance remained unchanged with the systemic NOS inhibition, despite systemic vasoconstriction as seen in the elevated MAP and TPR. Taken together, with our previous study involving prior systemic a-adrenergic receptor blockade, it is likely that NO may likely be involved in regulating arterial compliance but the complex compensation/redundant mechanisms may have masked such effects in the present study. Further studies are clearly needed to address this issue.
‘What is known about the topic?’ Central artery compliance buffers the repeated pulsatile pressure generated from the left ventricle and fosters smooth continuous blood flow to the frail brain tissue and has an important role in the pathogenesis of cerebrovascular disease. The basal NO bioavailability influences compliance of ‘peripheral muscular’ arteries. It is unknown whether the inhibition of basal NO release affects compliance of central elastic artery. ‘What this study adds?’ Carotid artery compliance remained unchanged during the systemic NO synthase inhibition despite a clear sign of systemic vasoconstriction.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18300215 and
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Arterial compliance and NO bioavailability J Sugawara et al
499 18650186), JSPS Postdoctoral Fellowships for Research Abroad and NIH grant AG20966.
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19 Sugawara J, Tanabe T, Miyachi M, Yamamoto K, Takahashi K, Iemitsu M et al. Non-invasive assessment of cardiac output during exercise in healthy young humans: comparison between Modelflow method and Doppler echocardiography method. Acta Physiol Scand 2003; 179(4): 361–366. 20 Sugawara J, Komine H, Hayashi K, Yoshizawa M, Otsuki T, Shimojo N et al. Reduction in alpha-adrenergic receptor-mediated vascular tone contributes to improved arterial compliance with endurance training. Int J Cardiol 2009; 135(3): 346–352. 21 Tanaka H, Dinenno FA, Monahan KD, Clevenger CM, DeSouza CA, Seals DR. Aging, habitual exercise, and dynamic arterial compliance. Circulation 2000; 102: 1270–1275. 22 Armentano R, Megnien JL, Simon A, Bellenfant F, Barra J, Levenson J. Effects of hypertension on viscoelasticity of carotid and femoral arteries in humans. Hypertension 1995; 26(1): 48–54. 23 Hirai T, Sasayama S, Kawasaki T, Yagi S. Stiffness of systemic arteries in patients with myocardial infarction. A noninvasive method to predict severity of coronary atherosclerosis. Circulation 1989; 80(1): 78–86. 24 Reneman RS, van Merode T, Hick P, Muytjens AM, Hoeks AP. Age-related changes in carotid artery wall properties in men. Ultrasound Med Biol 1986; 12(6): 465–471. 25 Roman MJ, Pini R, Pickering TG, Devereux RB. Non-invasive measurements of arterial compliance in hypertensive compared with normotensive adults. J Hypertens Suppl 1992; 10(6): S115–S118. 26 Sugawara J, Hayashi K, Tanaka H. Distal shift of arterial pressure wave reflection sites with aging. Hypertension 2010; 56(5): 920–925. 27 Mitchell GF, Parise H, Benjamin EJ, Larson MG, Keyes MJ, Vita JA et al. Changes in arterial stiffness and wave reflection with advancing age in healthy men and women: the Framingham Heart Study. Hypertension 2004; 43(6): 1239–1245. 28 Murgo JP, Westerhof N, Giolma JP, Altobelli SA. Aortic input impedance in normal man: relationship to pressure wave forms. Circulation 1980; 62(1): 105–116. 29 Wilkinson IB, MacCallum H, Cockcroft JR, Webb DJ. Inhibition of basal nitric oxide synthesis increases aortic augmentation index and pulse wave velocity in vivo. Br J Clin Pharmacol 2002; 53(2): 189–192. 30 Sugawara J, Komine H, Hayashi K, Yoshizawa M, Yokoi T, Otsuki T et al. Effect of systemic nitric oxide synthase inhibition on arterial stiffness in humans. Hypertens Res 2007; 30(5): 411–415. 31 Frewin DB, Whelan RF. The mechanism of action of tyramine on the blood vessels of the forearm in man. Br J Pharmacol Chemother 1968; 33(1): 105–116. 32 Saeed M, Sommer O, Holtz J, Bassenge E. Alpha-adrenoceptor blockade by phentolamine causes beta-adrenergic vasodilation by increased catecholamine release due to presynaptic alpha-blockade. J Cardiovasc Pharmacol 1982; 4(1): 44–52. 33 Kang KB, van der Zypp A, Majewski H. Endogenous nitric oxide attenuates beta-adrenoceptor-mediated relaxation in rat aorta. Clin Exp Pharmacol Physiol 2007; 34(1-2): 95–101. 34 Sanbe A, Tanaka Y, Fujiwara Y, Miyauchi N, Mizutani R, Yamauchi J et al. Enhanced vascular contractility in alpha1-adrenergic receptor-deficient mice. Life Sci 2009; 84(21-22): 713–718. 35 Avolio AP, Van Bortel LM, Boutouyrie P, Cockcroft JR, McEniery CM, Protogerou AD et al. Role of pulse pressure amplification in arterial hypertension: experts’ opinion and review of the data. Hypertension 2009; 54(2): 375–383. 36 Safar ME, Protogerou AD, Blacher J. Statins central blood pressure, and blood pressure amplification. Circulation 2009; 119(1): 9–12.
Journal of Human Hypertension (2014) 494 – 499
ORIGINAL ARTICLE
Lack of changes in carotid artery compliance with systemic nitric oxide synthase inhibition J Sugawara1,2, Y Saito3, S Maeda3, M Yoshizawa1,3, H Komine1, M Nakamura3, R Ajisaka3 and H Tanaka2 Proximal large elastic arteries (ascending aorta and carotid artery) have an important role in buffering the pulsatile pressure generated from the left ventricle, which forwards continuous peripheral blood flow and protects the brain microcirculation from end-organ damage. Although compliance of distal conduit arteries (extremities’ arteries) is attenuated by the nitric oxide synthase (NOS) inhibition, it is yet unknown whether compliance of proximal elastic arteries changes by the systemic NOS inhibition. To address this question, we measured central artery compliance in 17 young adults (26±1 years) who underwent intravenous infusions of NG-monomethyl-L-arginine (L-NMMA) or saline (placebo) on separate days. Following the systemic NOS inhibition, the mean arterial pressure (MAP), total peripheral resistance and aortic augmentation index were significantly increased. However, carotid artery compliance was not affected significantly (from 0.10±0.01 to 0.11±0.01 mm2 per mmHg) and the b-stiffness index (an index of arterial compliance adjusted for the distending pressure) tended to decrease (from 6.63±0.35 to 6.06±0.42 a.u., P ¼ 0.07). These parameters were not altered with saline infusion. Changes in the b-stiffness index tended to correlate negatively with the corresponding changes in MAP (r ¼ 0.31, P ¼ 0.07). These results suggest that carotid artery compliance remains unchanged during the systemic NOS inhibition in spite of systemic vasoconstriction. Journal of Human Hypertension (2014) 28, 494–499; doi:10.1038/jhh.2013.137; published online 9 January 2014 Keywords: endothelial function; vascular tone; arterial stiffness
INTRODUCTION In the arterial tree, it is the central elastic arteries in the cardiothoracic region that dampen the pulsatile pressure and flow generated from the left ventricle.1 However, such function is deteriorated with advancing age.2,3 The decreased compliance of large elastic arteries (for example, aorta and carotid artery) is associated with the greater incidence of future cardio- and cerebrovascular diseases.4–7 Central arterial stiffening is associated with an increased susceptibility to white matter rarefaction or cerebral leukoaraiosis.8,9 Hence, physiological factors contributing to large arterial compliance have been a topic of intense investigation in the field of cardiovascular and cerebral vascular research. Compliance of artery is determined by the contractile state of the vascular smooth muscle cells as well as structural factors (for example, elastin and collagen) in the arterial wall.1,10 In particular, nitric oxide (NO) bioavailability, which has emerged as an important regulatory mechanism of vasomotor tone, could regulate arterial compliance.11,12 In animal experiments,11 intraarterial infusion of NG-monomethyl-L-arginine (L-NMMA), a NO synthase (NOS) inhibitor, significantly increased iliac artery pulse wave velocity (PWV) independent of changes in the blood pressure. These results indicated that basal NO bioavailability influences compliance of ‘peripheral muscular’ arteries. On the other hand, Stewart et al.13 determined the relation between changes in carotid-femoral PWV and mean arterial pressure (MAP) with systemic infusions of vasoactive drugs. Changes in PWV caused by three pressor agents, including L-NMMA, were closely correlated with changes in MAP. On the basis of these results,
the systemic NOS inhibition-induced increase in aortic PWV was attributed to the corresponding increases in blood pressure rather than any specific effect of NOS inhibition on the large arterial wall. As carotid-femoral PWV includes stiffness of the distal part of aorta (that is, the thoracic and abdominal descending aorta), the influence of inhibition of basal NO release on compliance of proximal vessels (that is, the ascending aorta and carotid artery) is not known. We reason that the elucidation of physiological mechanisms responsible for carotid artery compliance might give us distinct target for prevention of future cardio- and cerebrovascular diseases. Accordingly, the primary aim of this study was to determine the impact of the basal NO bioavailability inhibition on carotid artery compliance as a measure of large elastic artery stiffness. In the present study, we studied young healthy adults as their high basal NO bioavailability could facilitate distinguishing role of NO when NOS inhibitors were applied. MATERIALS AND METHODS Subjects Seventeen sedentary or recreationally active young adults (26±1 years; nine men and eight women) were studied. All the subjects were non-obese (height 167.2±2.3 cm, body mass 58.6±2.0 kg and body mass index 21.0±0.3 kg m 2), normotensive and free of overt cardiovascular disease as assessed by medical history. All the subjects had not been taking any medications including oral contraceptives. Subjects who were current smokers or smoked within the past 2 years were excluded. Before the study, all the subjects were explained about potential risks and procedures of the study and they gave their written informed consent to participate in
1 Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan; 2Department of Kinesiology and Health Education, The University of Texas at Austin, Austin, TX, USA and 3Graduate School of Comprehensive Human Sciences, The University of Tsukuba, Tsukuba, Japan. Correspondence: Dr J Sugawara, Human Technology Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8566, Japan. E-mail: [email protected] Received 31 July 2013; revised 4 November 2013; accepted 21 November 2013; published online 9 January 2014
Arterial compliance and NO bioavailability J Sugawara et al
495 the study. The study conformed with the principles outlined in the Helsinki Declaration and was reviewed and approved by the Institutional Review Board.
Experimental protocol After overnight fast, each subject underwent two experiments (NOS inhibition and saline control conditions) with a randomized counterbalanced design on separate days. All subjected were blinded to the order of sessions (saline or L-NMMA) (for example, single-blind manner); however, the investigators were aware of which drugs were infused for the safety reasons. Female subjects were studied at the same menstrual cycle phase for both experiments. The subjects abstained from alcohol, caffeine and vigorous exercise for at least 24 h before the experiments. All measurements were conducted in a quiet temperature-controlled room (25 1C). The timeline of the experiment was illustrated in Figure 1. After 10 min of supine rest, the baseline measurements were performed. Then, the subjects were administrated with intravenous NOS inhibitor (L-NMMA, 3 mg kg 1 bolus over 5 min and a subsequent continuous (0.05 mg kg 1 min 1) infusion) or the same amount of saline (control condition). The procedure of infusions and dose of the systemic drug infusion were consistent with previous studies.14 The second measurements were made 5–10 min after the commencement of continuous intravenous infusions. The total volume of the infusion throughout each experiment was 17.5 ml.
Hemodynamic measurements Blood pressure and beat-to-beat arterial blood pressure waveforms were recorded by using photoplethysmography (Portapres Model 2, TNO TPD Biomedical Instrumentation, Amsterdam, The Netherlands) on the finger. Heart rate was obtained with a standard electrocardiogram (ML132 Bio Amp, AD Instruments, Colorado Springs, CO, USA). Systolic and diastolic blood pressures (SBP and DBP, respectively) and MAP of brachial artery were estimated from the reconstructed pressure waveform from finger arterial pressure waveform. Stroke volume and cardiac output were calculated from the finger blood pressure waveform using the validated Model flow method incorporating age, sex, height and weight (BeatScope 1.0; TNO TPD; Biomedical Instrumentation).15 This methodology has been shown to reliably estimate rapid changes in cardiac output during a variety of experimental protocols.16–19 Total peripheral resistance (TPR) was calculated as MAP/cardiac output.
Arterial compliance measures The combination of ultrasound imaging of the common carotid artery diameter with applanation tonometry recording of blood pressure on the contralateral carotid artery was applied to obtain arterial compliance as previously described.20,21 A longitudinal image of the common carotid artery was measured with an ultrasound machine equipped with a highresolution multifrequency linear-array transducer (EnVisor; Koninklijke Philips Electronics, Eindhoven, The Netherlands). Successive 10 beats of common carotid artery longitudinal image were acquired at maximal (systolic) and minimal (diastolic) diameters. Arterial lumen diameter (from the media/adventitia interface of the near wall to the lumen/intima interface of the far wall of the vessel) was measured offline using the commercially available software (Carotid Analyzer, Medical Imaging Applications, Coralville, IA, USA). Arterial pressure waveforms were recorded noninvasively on the common carotid artery with arterial applanation tonometry incorporating an array of 15 micropiezoresistive transducers (VP-2000, Colin Medical Instrument, San Antonio, TX, USA) and sampled at 1 kHz using an analog-to-digital converter (PowerLab 8/30, AD Instruments, Bella Vista, New South Wales, Australia) interfaced with a
personal computer equipped with data acquisition software (LabChart 6, AD Instruments). Carotid arterial pressures were calibrated by brachial MAP and DBP.22 Arterial compliance, Young’s elastic modulus and the b-stiffness index were calculated using the equations: [(D1 D0)/D0]/[2(P1–P0)] p D20, ([Ps Pd] Dd)/([Ds Dd]/IMT) and ln(P1/P0)/[(D1 D0)/D0], where D1 and D0 are the maximal and minimum arterial diameters, P1 and P0 are the highest and lowest carotid arterial pressures and IMT is carotid artery intima-media thickness.23–25 Carotid artery diameter and IMT were analyzed using image analysis software (Carotid Analyzer, Medical Imaging Applications). All image analyses were performed by the same investigator who was blinded to the sessions (saline or L-NMMA). The day-to-day coefficients of variation for carotid artery diameter and pulse pressure are 2±1% and 7±3%, respectively. The carotid arterial waveform data were also fed into the SphygmoCor software (AtCor Medical, Sydney, New South Wales, Australia), and a validated transfer function was applied to estimate aortic blood pressure.26 Aortic augmentation index (AIx) and the reflected wave transit time (RWTT) were also computed.27 AIx is a measure of the contribution of wave reflection to the aortic waveform, and depends on PWV and the magnitude and site of the reflected pressure wave.28 To account for the influence of heart rate on AIx, the index of AIx normalized for heart rate of 75 bpm (AIx75) was also obtained. The SphygmoCor software could not calculate AIx75 of one subject because of low heart rate (o45 bpm). RWTT was measured from the foot of the carotid pressure waveform to the first inflection point, which corresponds to the foot of the global reflected pressure wave.27
Statistical analyses Repeated measures analysis of variance (general linear model) was used to determine the effects of systemic NOS blockades on carotid artery compliance and other hemodynamic measures. Analysis of covariance was performed to compare responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for. In the case of a significant F-value, a post hoc test using the Newman–Keuls test identified significant differences among the mean values. For all three arterial compliance measures, normality of the distribution was confirmed in all parameters by Kolmogorov–Smirnov test. Owing to a relatively small sample size used in the present study, nonparametric test was also performed. The association between the variables of interest was evaluated by parametric and non-parametric simple correlation analyses (Pearson’s r and Spearman’s r). All data are reported as the mean±s.e.m. Statistical significance was set a priori at Po0.05.
RESULTS There was no significant difference in baseline hemodynamic variable between the saline control and the NOS inhibition conditions (Table 1). Brachial SBP, DBP and, mean blood pressure increased significantly with the L-NMMA administration but did not change with the saline infusion. Brachial pulse pressure did not change significantly with either L-NMMA or saline infusion. Similar trend of changes were observed in carotid and aortic blood pressures (Table 2). Stroke volume did not change with either saline or L-NMMA infusion. Heart rate and cardiac output decreased significantly with the L-NMMA administration, and mild but significant reductions in these measures were also observed
Before infusion (baseline)
During infusion
Measurement
Commencement of resting
Bolus infusion (3mg/kg)
Measurement
Continuous infusion (0.05mg/kg/min)
L-NMMA or saline 0
5
10
15
0
5
10
15
(min)
Figure 1.
Timeline of experimental protocol.
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496 Table 1.
Hemodynamic responses to saline/L-NMMA infusions
Variables
Saline
Brachial systolic BP, mmHg Brachial diastolic BP, mmHg Brachial mean BP, mmHg Brachial pulse pressure, mmHg Heart rate, bpm Stroke volume, ml Cardiac output, l min 1
L-NMMA
Baseline
Infusion
Baseline
Infusion
120±3 59±2 78±2 60±2 64±2 94±4 6.0±0.3
119±4 61±2 79±3 58±2 61±2a 90±4 5.5±0.3a
123±2 62±2 81±2 61±2 65±2 94±4 6.1±0.3
134±3a,b 74±2a,b 93±3a,b 60±1 56±1a,b 91±4 5.1±0.3a,b
Abbreviations: BP, blood pressure; L-NMMA, NG-monomethyl-L-arginine. Data are means±s.e.m. Brachial arterial pressure was obtained from the reconstructed finger arterial pressure waveform. aPo0.05 versus baseline. bPo0.05 versus saline infusion.
Table 2.
Responses of carotid arterial and aortic characteristics to saline/L-NMMA infusions
Variables
Carotid systolic BP, mmHg Carotid diastolic BP, mmHg Carotid pulse pressure, mmHg Carotid arterial systolic diameter, mm Carotid arterial diastolic diameter, mm Carotid arterial distention, mm Aortic systolic BP, mmHg Aortic diastolic BP, mmHg Aortic pulse pressure, mmHg Aortic AIx, %
Saline
L-NMMA
Baseline
Infusion
Baseline
Infusion
104±2 59±2 45±1 6.3±0.1 5.8±0.1 0.52±0.03 100±2 59±2 41±1 5±4
102±3 61±2 42±1 6.5±0.1a 6.0±0.1a 0.5±0.03 99±3 61±2 39±1 4±4
107±2 62±2 45±1 6.3±0.1 5.8±0.1 0.5±0.02 104±2 62±2 42±1 0±3
118±3a,b 74±2a,b 44±1 6.5±0.1a 6.0±0.1a 0.5±0.03 117±3a,b 74±2a,b 43±1 15±3a,b
Abbreviations: AIx, augmentation index; BP, blood pressure; L-NMMA, NG-monomethyl-L-arginine. Data are means±s.e.m. aPo0.05 versus baseline. b Po0.05 versus saline infusion.
with the saline infusion. Aortic AIx and AIx75 increased and RWTT decreased significantly with the L-NMMA administration but did not change with the saline infusion (Figure 2). At baseline, there were no significant differences in carotid artery diastolic or systolic diameter between the two conditions (Table 2). Carotid artery diastolic and systolic diameters increased significantly with both the saline and L-NMMA infusions. Baseline carotid artery compliance and the b-stiffness index were comparable between the two conditions (Figure 3). Carotid artery compliance and Young’s elastic modulus were not changed significantly with either the saline (from 0.11±0.01 to 0.11 ±0.01 mm2 per mmHg and from 244±20 to 245±21 mmHg*mm, respectively) or L-NMMA infusion (from 0.10±0.01 to 0.11 ±0.01 mm2 per mmHg and from 249±14 to 260±19 mmHg*mm, respectively). The b-stiffness index was not affected by the saline infusion (from 6.63±0.38 to 6.66±0.42 a.u.) and tended to decrease with the L-NMMA infusion (from 6.63±0.35 to 6.06±0.42 a.u., P ¼ 0.07). Additionally, there were no significant differences in responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for in ANCOVA. Changes in the b-stiffness index induced by the drug infusions tended to correlate negatively with the corresponding changes in MAP (r ¼ 0.31, P ¼ 0.07, Figure 4). Non-parametric correlational analysis (Spearman’s ranks order correlation) identified a significant correlation (r ¼ 0.343, Po0.05). The saline/L-NMMA administration-induced changes in RWTT, aortic AIx and AIx75 were significantly correlated with the changes in MAP (r ¼ 0.49, 0.41 and 0.43, respectively). Journal of Human Hypertension (2014) 494 – 499
DISCUSSION As NO is a potent vasodilator, it is reasonable to speculate that NOS inhibition would increase vascular tone and decrease arterial compliance. However, the present findings do not support such notion. We found that the systemic L-NMMA infusion did not alter measures of the carotid artery compliance. There are a number of possible explanations for a lack of effects of NO inhibition on arterial stiffness. One interpretation is that, as the amount of smooth muscle cells surrounding the vascular wall decreases from the peripheral arteries to central arteries in the arterial tree, NO has smaller or negligible roles in determining the distensibility of proximal elastic arteries (that is, ascending aorta and carotid artery). Stewart et al.13 suggested that effects of inhibition of basal NO release on carotid-femoral PWV could be explained by the elevation of MAP rather than any specific effect of NO inhibition within the aorta. The study by Stewart et al.13 is consistent with the findings of the current study. In contrast, Wilkinson et al.29 suggested that large artery stiffness is regulated by NO because of the significant increase in aortic AIx via intravenous infusion of L-NMMA in healthy young adults. However, AIx is no longer been considered as a measure of arterial stiffness as it is an index of arterial wave reflection from the periphery.1 Similar to that study,29 L-NMMA induced significant increases in aortic AIx and AIx normalized for HR (AIx75) in the present study. Additionally, changes in aortic AIx and AIx75 induced by the saline/L-NMMA administrations were significantly correlated with the corresponding changes in MAP. Therefore, the systemic NOS inhibitioninduced increase in AIx may not be because of the alterations in the elastic properties of central arteries but may rather be explained by the epiphenomenon of MAP increase and the & 2014 Macmillan Publishers Limited
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497 Saline L-NMMA 0.14
*
90 80 70
0.10 0.08
60
0.06
40
350
35 TPR (unit)
0.12
*†
30 25
*
20
Young’s elastic modulus (mmHg*mm)
MAP (mmHg)
100
Arterial compliance (mm2/mmHg)
110
Saline L-NMMA
300 250 200 150
15 8
Aortic AIx75 (%)
5 0
*
-5 -10
β-stiffness index (au)
10 7 6 5
-15
4 Baseline
L-NMMA /saline
-20
Figure 3. Changes in carotid arterial compliance, Young’s elastic modulus and b-stiffness index in response to systemic nitric oxide synthase blockade (L-NMMA) and saline administrations.
190 6 180 * 170 160 Baseline
L-NMMA /saline
Figure 2. Changes in MAP, TPR, aortic augmentation index normalized for heart rate of 75 bpm (AIx75) and RWTT in response to systemic nitric oxide synthase blockade (L-NMMA) and saline administrations. *Po0.05 versus baseline; wPo0.05 versus saline infusion.
elevations in peripheral vasoconstriction augmenting the reflected waves. An alternative interpretation is that the effect of L-NMMA would have been masked by the subsequent reductions in sympathetic vasoconstrictor tone that occurs as a counter-regulatory effect. In other word, NO may be likely involved in modulating arterial compliance but the reflex reductions in sympathetic vasoconstrictor tone may have masked such contribution, resulting in unchanged carotid artery compliance. The inverse relation between responses of MAP and the b-stiffness index, an index & 2014 Macmillan Publishers Limited
Changes in β-stiffness index (au)
RWTT (msec)
200
Saline L-NMMA
4 2 0 -2 -4 r= -0.31, P=0.07 -6 -20
-10
0
10
20
30
Changes in MAP (mmHg)
Figure 4. Relation between changes in MAP and changes in b-stiffness index in response to infusions of saline and L-NMMA.
adjusted for the distending pressure, supports this hypothesis. In this context, we have previously investigated the effect of NOS inhibition on carotid artery compliance under systemic a-adrenergic receptor blockade, phentolamine.30 The increase in arterial stiffness was observed after the systemic administration of L-NMMA preceded by phentolamine infusions. Although the prior a-adrenergic receptor blockade is a reasonable approach to Journal of Human Hypertension (2014) 494 – 499
Arterial compliance and NO bioavailability J Sugawara et al
498 isolate the effect of NO on arterial compliance, there may be secondary effects that confound the interpretation. Under conditions of a-adrenergic receptor blockade, the facilitation of noradrenaline release by phentolamine (because of blockade of presynaptic a2 receptors) can stimulate b-adrenergic receptors.31,32 Additionally, NOS inhibition augments b-adrenoceptor-mediated vasorelaxation33 and a loss of a1-adrenergic receptors leads to enhancement of vascular responsiveness to vasoconstrictors.34 Moreover, our previous study involving prior a-adrenergic receptor blockade30 was conducted in middle-aged and older adults, making the comparison of these studies with the present study involving young adults difficult. Clearly, more studies are needed to fully answer the question regarding the role of NO on the distensibility of the central elastic arteries in humans. There is a growing recognition that central arterial pressure differs from peripheral arterial pressure. Reflections of the forward propagating pressure wave occur at multiple sites of the arterial bed because of changes in arterial properties (for example, elasticity/stiffness gradient and vasomotor tone) or in the architecture of the arterial tree (for example, branching points and calcifications).3,35,36 The multiple reflected waves are integrated as a single combined reflected wave, which sums with the forward ejected wave and thus forms the local pressure pulse wave. In young healthy populations, a large elasticity/ stiffness gradient introduces a greater peripheral pulse amplification and disparity between central and peripheral pulse pressures. As such, it is important to evaluate vascular wall responses matching to local pressure changes. We determined the influence of acute systemic NOS inhibition on carotid artery compliance in young healthy adults. Carotid artery compliance was determined by simultaneously measuring diameter changes and pressure changes both in the common carotid artery. The systemic L-NMMA administration did not affect measures of carotid artery compliance in spite of a greater vasoconstriction as seen in elevated MAP and TPR. The compliance of large elastic arteries (for example, aorta and carotid artery) buffers the repeated pulsatile pressure generated from the left ventricle and fosters smooth continuous blood flow to the frail brain tissue, and its decline is associated with the greater incidence of future cerebrovascular diseases.7,9 Thus, the absence of change in carotid artery compliance might be a protective mechanism that some other redundant factors (for example, reductions in local sympathetic vasoconstrictor tone) may have contributed to preserve arterial compliance in order to prevent potential cerebrovascular disease. We identified significant changes in heart rate, cardiac output and carotid artery diameter under the saline control condition. Although these changes are fairly small, they are statistically significant. It is possible that blood volume expansion induced by infusions might have lowered heart rate and cardiac output via cardiopulmonary baroreflex. The most important issue to note here is that all of these changes were observed also under the experimental conditions (for example, L-NMMA infusion). Furthermore, there were no significant differences in responses of carotid artery compliance measures between two conditions when the influences of confounding factors (that is, changes in blood pressure, heart rate and stroke volume) were accounted for in ANCOVA. As in any other studies, there are some drawbacks of our study that need to be emphasized. First, the sample size for this study was relatively small. This was primarily because of the fact that the study was highly invasive as well as risky as it involved the infusion of systemic pharmacological blockade. In order to determine the minimal sample size, the power calculations were performed prior to the study using the mean acute changes in arterial compliance that were expected with the pharmacological administration studies.13,20,29 The sample size was based on a power calculation that showed that 16 subjects were required to achieve 80% power Journal of Human Hypertension (2014) 494 – 499
to detect a difference of 15% in arterial compliance, with a ¼ 0.05. Second, the completeness of NOS blockade was not confirmed, although significant vasoconstriction was identified with the systemic administration of L-NMMA in the present study. Third, this study was conducted in the single-blind manner. For the safety reasons, the investigators were aware of which drugs were being infused. However, all subjected were blinded to the order of sessions (saline or L-NMMA), and image analyses were performed by the same investigator who was blinded to the sessions. Fourth, the measurement of peripheral artery stiffness was omitted from the present study because it has been fairly well established that the stiffness/compliance of peripheral arteries (brachial or femoral arteries) do not change with aging and disease state and are not considered to be clinically important. As the safety of the subjects was the number one priority in this highly risky protocol that involves systemic drug infusion, one extra measurement that did not provide much clinical insight was dropped from the measurement battery. Fifth, even though we attributed no significant change in carotid artery compliance with systemic NOS inhibition to the contribution of the reflex reductions in sympathetic vasoconstrictor tone, we did not directly measure sympathetic nervous activity. In this context, there are currently no experimental approaches in humans to measure sympathetic nervous system activity directed to the central elastic arteries. Finally, systemic NOS inhibition may not be the ideal procedure to address this issue. Arguably, the most effective procedure to investigate this in humans is a ‘local’ infusion of L-NMMA applied in the human aorta and/or other central elastic arteries (for example, carotid arteries). However, such procedure is extremely risky as well as difficult to interpret, as the associated elevation in blood pressure and/or reflex reductions in sympathetic vasoconstrictor tone would confound the interpretation. In conclusion, we found that carotid artery compliance remained unchanged with the systemic NOS inhibition, despite systemic vasoconstriction as seen in the elevated MAP and TPR. Taken together, with our previous study involving prior systemic a-adrenergic receptor blockade, it is likely that NO may likely be involved in regulating arterial compliance but the complex compensation/redundant mechanisms may have masked such effects in the present study. Further studies are clearly needed to address this issue.
‘What is known about the topic?’ Central artery compliance buffers the repeated pulsatile pressure generated from the left ventricle and fosters smooth continuous blood flow to the frail brain tissue and has an important role in the pathogenesis of cerebrovascular disease. The basal NO bioavailability influences compliance of ‘peripheral muscular’ arteries. It is unknown whether the inhibition of basal NO release affects compliance of central elastic artery. ‘What this study adds?’ Carotid artery compliance remained unchanged during the systemic NO synthase inhibition despite a clear sign of systemic vasoconstriction.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18300215 and
& 2014 Macmillan Publishers Limited
Arterial compliance and NO bioavailability J Sugawara et al
499 18650186), JSPS Postdoctoral Fellowships for Research Abroad and NIH grant AG20966.
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