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Systemic nitric oxide synthase inhibition improves coronary flowreserve to adenosine in patients with significant stenoses

Kaufmann, P A; Rimoldi, O E; Gnecchi-Ruscone, T; Luscher, T F; Camici, P G

Kaufmann, P A; Rimoldi, O E; Gnecchi-Ruscone, T; Luscher, T F; Camici, P G (2007). Systemic nitric oxidesynthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses. AmericanJournal of Physiology. Heart and Circulatory Physiology, 293(4):H2178-H2182.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology. Heart and Circulatory Physiology 2007, 293(4):H2178-H2182.

Kaufmann, P A; Rimoldi, O E; Gnecchi-Ruscone, T; Luscher, T F; Camici, P G (2007). Systemic nitric oxidesynthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses. AmericanJournal of Physiology. Heart and Circulatory Physiology, 293(4):H2178-H2182.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:American Journal of Physiology. Heart and Circulatory Physiology 2007, 293(4):H2178-H2182.

Systemic nitric oxide synthase inhibition improves coronary flowreserve to adenosine in patients with significant stenoses

Abstract

We studied the impact of systemic infusion of the nitric oxide synthase (NOS) inhibitorN(G)-monomethyl-L-arginine (L-NMMA) on coronary flow reserve (CFR) in patients with coronaryartery disease (CAD). We have previously demonstrated that CFR to adenosine was significantlyincreased after systemic infusion of L-NMMA in normal volunteers but not in recently transplanteddenervated hearts. At baseline, myocardial blood flow (MBF; ml x min(-1) x g(-1)) was measured at restand during intravenous administration of adenosine (140 microg x kg(-1) x min(-1)) in 10 controls (47+/- 5 yr) and 10 CAD patients (58 +/- 8 yr; P < 0.01 vs. controls) using positron emission tomographyand (15)O-labeled water. Both MBF measurements were repeated during intravenous infusion of 10mg/kg L-NMMA. CFR was calculated as the ratio of MBF during adenosine to MBF at rest. CFR wassignificantly higher in healthy volunteers than in CAD patients and increased significantly afterL-NMMA in controls (4.00 +/- 1.10 to 6.15 +/- 1.35; P < 0.0001) and in patients, both in territoriessubtended by stenotic coronary arteries (>70% luminal diameter; 2.06 +/- 1.13 to 3.21 +/- 1.07; P <0.01) and in remote segments (3.20 +/- 1.23 to 3.92 +/- 1.62; P < 0.05). In conclusion, CFR can besignificantly increased in CAD by a systemic infusion of L-NMMA. Similarly to our previous findingsin normal volunteers, this suggests that adenosine-induced hyperemia in CAD patients is constrained bya mechanism that can be relieved by systemic NOS inhibition with L-NMMA.

Systemic nitric oxide synthase inhibition improves coronary flow reserve toadenosine in patients with significant stenoses

Philipp A. Kaufmann,1,2,3 Ornella E. Rimoldi,1 Tomaso Gnecchi-Ruscone,1 Thomas F. Luscher,2,3

and Paolo G. Camici11Medical Research Council Clinical Sciences Center, Imperial College, Hammersmith Hospital, London, United Kingdom;2Cardiovascular Center, Nuclear Cardiology, University Hospital, Zurich; and 3Zurich Center for Integrative HumanPhysiology, University of Zurich, Switzerland

Submitted 26 November 2006; accepted in final form 27 July 2007

Kaufmann PA, Rimoldi OE, Gnecchi-Ruscone T, Luscher TF,Camici PG. Systemic nitric oxide synthase inhibition improves cor-onary flow reserve to adenosine in patients with significant stenoses.Am J Physiol Heart Circ Physiol 293: H2178–H2182, 2007. Firstpublished July 27, 2007; doi:10.1152/ajpheart.01292.2006.—Westudied the impact of systemic infusion of the nitric oxide synthase(NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) on coronaryflow reserve (CFR) in patients with coronary artery disease (CAD).We have previously demonstrated that CFR to adenosine was signif-icantly increased after systemic infusion of L-NMMA in normalvolunteers but not in recently transplanted denervated hearts. Atbaseline, myocardial blood flow (MBF; ml �min�1 �g�1) was mea-sured at rest and during intravenous administration of adenosine (140�g �kg�1 �min�1) in 10 controls (47 � 5 yr) and 10 CAD patients(58 � 8 yr; P � 0.01 vs. controls) using positron emission tomogra-phy and 15O-labeled water. Both MBF measurements were repeatedduring intravenous infusion of 10 mg/kg L-NMMA. CFR was calcu-lated as the ratio of MBF during adenosine to MBF at rest. CFR wassignificantly higher in healthy volunteers than in CAD patients andincreased significantly after L-NMMA in controls (4.00 � 1.10 to6.15 � 1.35; P � 0.0001) and in patients, both in territories subtendedby stenotic coronary arteries (�70% luminal diameter; 2.06 � 1.13 to3.21 � 1.07; P � 0.01) and in remote segments (3.20 � 1.23 to3.92 � 1.62; P � 0.05). In conclusion, CFR can be significantlyincreased in CAD by a systemic infusion of L-NMMA. Similarly toour previous findings in normal volunteers, this suggests that ade-nosine-induced hyperemia in CAD patients is constrained by a mech-anism that can be relieved by systemic NOS inhibition withL-NMMA.

coronary circulation; autonomic nervous system; ischemic heart dis-ease; cardiac imaging; positron emission tomography

IT IS WELL ESTABLISHED THAT myocardial ischemia is a powerfulstimulus for vasodilatation of coronary resistive vessels. Thevasodilator response is reported to be near maximal for ische-mic times of up to 15–20 s, while no further changes areobserved for longer ischemic times (16).

Experimental studies in animals have proven that intracoro-nary or intravenous infusion of adenosine achieves a degree ofcoronary vasodilatation comparable to that obtained after aperiod of ischemia of 15–20 s. In normal humans, intravenousinfusion of 140 �g/kg adenosine produces an increase inmyocardial blood flow (MBF) approximately fourfold abovethe resting value with no further increase at higher adenosinedoses (12, 29).

In a recent study in normal volunteers (15), we demonstratedthat hyperemic MBF measured during the combined intravenousinfusion of adenosine (140 �g/kg) and the nitric oxide synthase(NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA; 10 mg/kg) was 53% higher than that during infusion of adenosinealone. This effect seems to necessitate an intact cardiac inner-vation, since no increase in MBF during the combined intra-venous infusion of adenosine and L-NMMA was observed intransplant recipients (6.5 � 2 mo after transplant) whosehearts remain denervated for more than a year after thegrafting (1).

The purpose of the present study was to assess whether alimitation of adenosine-induced hyperemia, similar to thatpreviously demonstrated in normal volunteers, is present inpatients with coronary artery disease (CAD).

METHODS

Study population. Ten patients (1 female) age 58 � 8 yr withsingle-vessel CAD were studied. Mean total cholesterol was 6.1 � 0.8mM. All patients described at least a 3-mo history of chronic stableangina pectoris and had electrocardiographic (ECG) evidence ofmyocardial ischemia, defined as �0.1 mV of horizontal or downslop-ing ST segment depression, during exercise stress. All patients had asignificant stenosis (�70% of luminal diameter) in a major coronarybranch. The coronary angiograms were analyzed by two independent,experienced operators. The luminal diameter of the stenosed arteryand adjacent reference lumen was measured at end diastole in theprojection that demonstrated the most severe stenosis, and quantitativeanalysis was performed using an automated edge-contour detectionsystem (Centricity QCA; General Electric Medical Systems, Milwau-kee, WI). A significant stenosis was found in the left anterior descend-ing artery in five patients, in the left circumflex in one patient, and inthe right coronary artery in four patients. All patients were receivingtreatment with aspirin and �-blocking agents. However, in all patients�-blockers were withdrawn 48–72 h before the study day. Exclusioncriteria were a recent history (�3 mo) of myocardial infarction orunstable angina, an inability to undergo exercise tolerance testing, orresting ECG patterns that would interfere with interpretation of STchanges during exercise.

A group of 10 healthy male volunteers age 47 � 5 yr (P � 0.01 vs.patients) served as controls. The lipid profile was assessed in allvolunteers, and those with total cholesterol �6.4 mM (250 mg/100ml) were excluded from the study. Their total cholesterol was 5.2 �1.0 mM. None of them had a history of cardiovascular disease,smoking, or any other cardiovascular risk factor. None of the volun-teers was receiving any form of drug treatment. Enrollment criteriaincluded normal heart rate, blood pressure, ECG, two-dimensional

Address for reprint requests and other correspondence: P. G. Camici, MRCClinical Sciences Centre, Hammersmith Hospital, London W12 ONN, UK(e-mail: paolo.camici@csc.mrc.ac.uk).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Heart Circ Physiol 293: H2178–H2182, 2007.First published July 27, 2007; doi:10.1152/ajpheart.01292.2006.

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echocardiogram, and low clinical probability for CAD (6). Some ofthe healthy volunteers’ data have been published previously (15).

In addition, all study subjects were carefully instructed to refrainfrom intake of caffeine-containing beverages or food during the 24 hpreceding the study. A screening test for caffeine was performed in ablood sample taken immediately before the positron emission tomog-raphy (PET) scan from each subject. Caffeine was not detectable inany of the blood samples.

PET measurement of MBF. MBF was measured using 15O-labeledwater (H2

15O) and an ECAT 931-08/12 15-slice PET scanner (CTI/Siemens, Knoxville, TX). H2

15O (700–900 MBq) was injected as anintravenous bolus over 20 s at an infusion rate of 10 ml/min, anddynamic scanning was acquired over a period of 5.5 min (13, 14, 29).The sinograms obtained were corrected for attenuation and recon-structed on a MicroVax II computer (Digital Equipment, Marlboro,MA) employing dedicated array processors and standard reconstruc-tion algorithms. On factor images, generated by iterative reconstruc-tion, regions of interest were drawn within the left atrium and on leftventricular myocardium on consecutive image planes. These wereprojected onto the dynamic H2

15O images to generate blood and tissuetime activity curves, which were fitted to a single-tissue compartmenttracer kinetic model to give values of MBF (ml �min�1 �g�1) (12, 14,26, 28–30).

Coronary flow reserve (CFR) was calculated as the ratio of MBFduring adenosine to resting MBF. To account for the variability ofcoronary driving pressure, we calculated resting and minimal (i.e.,during adenosine infusion) coronary resistance (mmHg �ml�1 �min �g)as the ratio of mean systemic arterial pressure to MBF (10, 26).

Study protocol. Under baseline conditions, MBF was measuredboth at rest and during intravenous administration of adenosine (140�g �kg�1 �min�1), as previously reported (14, 28). Arterial bloodpressure was recorded by an automatic cuff sphygmomanometer at1-min intervals, and the ECG was monitored continuously throughoutthe procedure. A 12-lead ECG was recorded at baseline and everyminute during adenosine administration. Thereafter, repeat measure-ments of MBF both at rest and during intravenous adenosine (140�g �kg�1 �min�1) were carried out following a 30-min intravenousinfusion of 10 mg/kg L-NMMA (Clinalfa, Laufelfingen, Switzerland)dissolved in 50 ml of isotonic saline.

The study protocol was approved by the Research Ethics commit-tees of Hammersmith Hospital. Radiation exposure was licensed bythe UK Administration of Radioactive Substances Advisory Commit-tee. All patients gave informed and written consent before the study.

Statistical analysis. Data are means � SD. Statistical comparisonof hemodynamic data, MBF, CFR, and coronary resistance during thedifferent study conditions was carried out using analysis of variancefor repeated measurements and post hoc Fisher’s protected leastsignificant difference test. A P value �0.05 was considered signifi-cant.

RESULTS

Baseline study. The main hemodynamic parameters in nor-mal volunteers and patients during the different study phases

are reported in Table 1. No significant difference was detect-able between the two groups except for a higher heart rate-systolic pressure product (RPP) in CAD patients at rest. In bothgroups, RPP increased significantly with adenosine.

In patients, resting MBF in territories subtended by a ste-notic artery was not statistically different from MBF in normalvolunteers, whereas MBF in remote myocardium subtended bya nonstenotic artery tended to be higher (Table 2 and Fig. 1).During adenosine infusion, the MBF increase in normal vol-unteers was greater than that observed in patients in territoriessubtended by a stenotic artery, whereas it was comparable tothat in remote myocardium subtended by a nonstenotic artery(Table 2 and Fig. 1). CFR was significantly higher in volunteersthan in CAD patients (P � 0.01 vs. remote myocardium and P �0.001 vs. myocardium subtended by a stenotic artery) (see Table2). Minimal coronary resistance (during intravenous adenosine)was 28.5 � 9.9 mmHg �ml�1 �min �g in normal volunteers,64.1 � 26.0 mmHg �ml�1 �min �g in territories subtended by astenotic artery (P � 0.0005 vs. healthy volunteers), and 32.2 �11.2 mmHg �ml�1 �min �g in remote myocardium [P � nonsig-nificant (NS) vs. healthy volunteers and P � 0.005 vs. stenoticterritories].

L-NMMA infusion. Mean arterial pressure both at rest andduring adenosine increased significantly after L-NMMA infusion,whereas corresponding heart rates were reduced (Table 1). Rest-ing MBF was substantially unchanged in normal volunteersand patients both in territories subtended by a stenotic arteryand in remote myocardium (Table 2 and Fig. 1). Duringadenosine infusion, MBF increased significantly comparedwith the respective baseline data both in normal volunteers andin patients. In the latter, there was a significant increase both interritories subtended by stenotic arteries and in remote myo-cardium. Similarly, CFR increased significantly in both groups(Table 2). Minimal coronary resistance decreased to 17.4 � 3.1mmHg �ml�1 �min �g in normal volunteers (P � 0.0005 vs.baseline) and to 47.1 � 18.8 mmHg �ml�1 �min �g in territoriessubtended by a stenotic artery (P � 0.05 vs. baseline and P �0.0001 vs. normal volunteers) and tended to decrease in remotemyocardium (29.4 � 15.3 mmHg �ml�1 �min �g, P � NS vs.baseline and P � 0.01 vs. normal volunteers).

DISCUSSION

The main finding of the present study is that the systemicinfusion of L-NMMA in patients with CAD significantly in-creases the MBF response to adenosine in territories subtendedby stenotic (�70% luminal diameter) coronary arteries. Theresults of the present study extend our previous observations inhealthy volunteers and demonstrate that the maximum MBF

Table 1. Hemodynamics

Group

Baseline L-NMMA

Rest Adenosine Rest Adenosine

MAP HR RPP MAP HR RPP MAP HR RPP MAP HR RPP

1 86�9 59�9 6,714�1,239 90�9 93�15b 11,574�2,239b 95�7d 52�9d 6,592�1,181 90�11a 78�13b,d 9,596�1,800b,d

2 95�10 63�11 8,308�1,957 99�15 94�17b 13,306�3,572b 113�13d 59�11 9,574�2,701 107�13 83�21b,c 13,057�4,408a

P ns ns �0.05 ns ns ns ns ns �0.01 ns ns �0.05

Values are heart rate (HR; beats/min), mean arterial pressure (MAP; mmHg), and rate-pressure product (RPP; systolic blood pressure � HR) at baseline andafter NG-monomethyl-L-arginine (L-NMMA) infusion in controls (group 1) and coronary artery disease (CAD) patients (group 2). P values shown are forbetween-group comparison; ns, no significant difference between groups. aP � 0.05; bP � 0.005 vs. rest. cP � 0.05; dP � 0.005 vs. respective baseline.

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response to adenosine in humans is constrained by what couldto be a neurally mediated vasoconstriction of resistance ves-sels. This latter is relieved by systemic infusion of L-NMMApotentially via central neuronal NOS (nNOS) inhibition, assuggested by the lack of flow increase after L-NMMA observedin transplant recipients that was demonstrated in our group’sprevious study (15).

The notion that the MBF response to adenosine and/ordipyridamole does not represent the maximum flow achievablein the coronary system has been previously demonstrated inboth animals and humans. In open-chest anesthetized dogs,L’Abbate et al. (16) demonstrated that prolonged intracoronaryinfusion of adenosine provoked a biphasic flow response: atfirst, within 15–30 s, coronary flow increased to a level similarto that observed during reactive hyperemia after a 30-s coro-nary occlusion; subsequently, continuing the infusion led tofurther coronary vasodilatation that within 20–45 min from thestart of the infusion reached a plateau at a value that was twiceas much as that observed during reactive hyperemia andremained constant up to 2 h.

-Adrenoceptor-mediated coronary vasoconstriction cancompete with local metabolic vasodilation, as has been shownin a number of studies in dogs, limiting the coronary vascularresponse during sympathetic activation, e.g., exercise (4, 7, 9,

19), norepinephrine infusion, or carotid sinus reflex (18). Therole of -adrenoceptor-mediated coronary vasoconstriction hasalso been investigated during maximal vasodilation to over-come the confounding influence of autoregulation. In twodifferent studies, during adenosine infusion, Vlahakes et al.(27) found higher flow after phentolamine and lower flow afterphenylephrine, and Johannsen et al. (11) observed lower flowduring cardiac sympathetic nerve stimulation and followinginfusion of phenylephrine or norepinephrine. However, al-though sympathetic discharge to the heart has been shown toresult in both -adrenergic constriction in the upstream vesselsand �-adrenoceptor-mediated dilation in the coronary micro-circulation, the latter has been found to be the dominant effectin pigs and dogs (24). This may not necessarily apply tohumans. In fact, in a study in normal human volunteers inwhom MBF was measured noninvasively by means of PET,Lorenzoni et al. (17) showed that the MBF response to dipy-ridamole was increased by 40% when the study was repeatedduring pharmacological blockade of 1-adrenoceptors.

Our data suggest that the reflex sympathetic activation elic-ited following the systemic administration of vasodilators suchas adenosine and dipyridamole should result in a further fall inminimal coronary resistance. The latter, however, is blunted byan NO-modulated suppression of sympathetic outflow in the

Fig. 1. Linear graphs showing individual values of rest and hyperemic (Ado) myocardial blood flow (MBF) at baseline and after NG-monomethyl-L-arginine(L-NMMA) infusion in controls and in patients with coronary artery disease (CAD) in remote segments and in segments subtended by a stenotic artery (stenosis).

Table 2. Myocardial blood flow and coronary flow reserve

Group

MBF, ml � min�1 � g�1

CFR (Relative Values)Baseline L-NMMA

Rest Adenosine Rest Adenosine Baseline L-NMMA

Controls 0.88�0.13 3.48�0.99 0.88�0.12 5.26�0.65c 4.00�1.10 6.15�1.35c

CADRemote 1.06�0.22d 3.27�0.96 1.14�0.22d 4.34�1.59a 3.20�1.23 3.92�1.62a,e

Stenosis 0.96�0.36 1.86�0.94e 0.84�0.29 2.69�1.24a,e 2.06�1.13e 3.21�1.07b,e

P ns �0.0001 �0.05 �0.005 �0.01 ns

Values are myocardial blood flow (MBF) at baseline and after adenosine infusion and coronary flow reserve (CFR) at baseline and after L-NMMA infusionin controls and in territories subtended by a stenotic artery and in remote myocardium in CAD patients. P values shown are for within-group comparison (CAD).aP � 0.05; bP � 0.01; cP � 0.0001 vs. baseline. dP � 0.05; eP � 0.005 vs. controls.

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central nervous system, where neuronal NO modulates neuro-transmitter release (20). This is in line with our previous study,which showed that an increase in the standard adenosineinfusion rate of 140 �g �kg�1 �min�1 (12) does not furtherdecrease coronary resistance, whereas systemic coinfusion ofadenosine with L-NMMA further decreased coronary resis-tance in healthy volunteers (15).

In the present study, the increase in MBF was significant inboth ischemic and remote territories of CAD patients. Never-theless, CFR remained significantly lower in remote segmentsthan in controls after L-NMMA. This is in line with previousobservations in CAD patients reporting an impaired hyperemicresponse in remote territories supplied by an angiographicallynormal coronary artery (5, 23).

It remains controversial whether inhibition of nNOS elicitssympathoexcitatory effects (21) or not (8), since it may exertopposing effects at different sites (22). The present results inCAD patients indicate that when the vasodilator is adminis-tered during systemic inhibition of NOS, this constraint isremoved and the overall effect is a further dilatation and ahigher hyperemic flow.

Limitations of the study. Although our observations supportthe above suggestion that neurally mediated vasoconstriction isrelieved by systemic NOS inhibition with L-NMMA, this mustremain a hypothesis. Similarly, we cannot comment on apossible involvement of a parasympathetic component. Toprovide final proof, a future study would need to demonstrate,first, that an -adrenergic blockade causes augmentation ofcoronary flow rates during adenosine similar to that producedby L-NMMA [which has been reported by Lorenzoni et al. (17)as mentioned above], and second, that after L-NMMA admin-istration, -adrenergic blockade would not further increaseMBF response to adenosine. This, however, was beyond thescope of the present study.

An additional limitation may be that CAD patients wereolder than controls, and this difference may potentially havehampered the comparability of the two groups’ CFR, since thelatter has been shown to decrease after the age of 60 yr (25),although mainly because of an increase in basal flow (3). Bycontrast, maximal hyperemic response decreases only after theage of 70 yr (2), which would be irrelevant to our study sincenone of our patients was older than 68 yr. Furthermore, theexperimental design using every subject as his own controlbefore and after L-NMMA administration and comparing ste-notic versus remote segments within each subject furtherstrengthens our results.

L-NMMA induced a significant increase in mean arterialpressure, which could theoretically have contributed to theincrease in hyperemic response after L-NMMA. This, however,was been ruled out in our group’s previous study, in whichphenylephrine was used to increase blood pressure but did notaffect hyperemic response and CFR (4).

In conclusion, the present study provides evidence thatadenosine-induced hyperemic flow response in CAD patients isconstrained, potentially by neurally mediated vasoconstriction,and that this can be relieved by systemic NOS inhibition withL-NMMA. Further studies are needed to ascertain whether thefindings of this investigation offer the possibility of devisingsome new form of treatment to improve myocardial perfusionin patients with chronic CAD.

GRANTS

P. A. Kaufmann was supported by Swiss National Science FoundationProfessorship Grants PP00A-68835 and PP00A-114706.

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