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Journal of the American College of Cardiology Vol. 36, No. 1, 2000 © 2000 by the American College of Cardiology ISSN 0735-1097/00/$20.00 Published by Elsevier Science Inc. PII S0735-1097(00)00674-4 Cardiac Physiology Dysfunction Induced by Nitric Oxide Synthase Inhibition in Humans Lukas E. Spieker, MD, Roberto Corti, MD, Christian Binggeli, MD, Thomas F. Lu¨scher, MD, FACC, FRCP, FESC, Georg Noll, MD, FESC Zurich, Switzerland

OBJECTIVES We sought to investigate baroreceptor regulation of sympathetic nerve activity and hemody- namics after inhibition of nitric oxide (NO) synthesis. BACKGROUND Both the sympathetic nervous system and endothelium-derived substances play essential roles in cardiovascular and diseases. Little is known about their interactions. METHODS In healthy volunteers, we recorded muscle sympathetic nerve activity (MSA) with microneu- rography and central measured at different levels of induced by lower body negative pressure. G RESULTS After administration of the NO synthase inhibitor N -monomethyl-L-arginine (L-NMMA, 1 mg/kg/min), systolic increased by 24 mm Hg (p ϭ 0.01) and diastolic blood pressure by 12 mm Hg (p ϭ 0.009), while index (measured by thermodilution) fell from 53 to 38 mL/min/m2 (p Ͻ 0.002). Administration of L-NMMA prevented the compensatory increase of rate, but not MSA, to orthostatic stress. The altered response of was not due to higher blood pressure, because heart rate responses were not altered during infusion of the alpha-1-adrenoceptor agonist phenylephrine (titrated to an equal increase of systolic blood pressure). In the presence of equal systolic blood pressure and central venous pressure, we found no difference in MSA during phenylephrine and L-NMMA infusion. CONCLUSIONS This study demonstrates a highly specific alteration of baroreceptor regulation of heart rate but not muscle sympathetic activity after inhibition of NO synthesis in healthy volunteers. This suggests an important role of NO in reflex-mediated heart rate regulation in humans. (J Am Coll Cardiol 2000;36:213–8) © 2000 by the American College of Cardiology

The vascular endothelium synthesizes and releases nitric abnormal baroreflex contributes to sympathetic activation, oxide (NO) and plays a fundamental role in the basal and which is an unfavorable prognostic factor (15–19). Alter- dynamic regulation of the circulation (1–4). Apart from the ations in endothelial function, with a resulting imbalance of tone of peripheral resistance vessels, NO also regulates endothelium-derived relaxing and contracting factors, may and is synthesized in neuronal cells affect baroreceptor function because endothelial factors may (5–8). However, little is known about the interactions of act on baroreceptor nerve endings in the vessel wall. Various NO and other cardiovascular regulating systems, especially endogenous substances (e.g., NO, endothelin-1, prostacy- with the sympathetic nervous system. These interactions are clin, norepinephrine) have been shown to influence barore- of particular interest in congestive heart failure, arterial ceptor function in animals (12,20–24). It is not clear if these , atherosclerosis and septic shock, where sym- mediators also affect human baroreceptor function. pathetic nerves innervating and constricting blood vessels The aim of our study was to investigate the effects of NO act as important counterpart of the endothelium-derived synthase inhibition and alpha-1-adrenergic stimulation on vasodilator NO (5,9–11). baroreceptor-mediated regulation of sympathetic nerve ac- Sympathetic activity is mainly regulated by tivity, heart rate and hemodynamics in healthy humans. located in the cardiopulmonary and carotid vessel walls, which centrally inhibit sympathetic outflow. These stretch- METHODS activated not only regulate short-term changes of blood pressure but also react to chronic elevation Subjects. Nine healthy normotensive volunteers (118 Ϯ of blood pressure by resetting sensitivity to a lower level 2/65 Ϯ 2 mm Hg, seven men and two women) with a mean (12,13). In hypertension, altered baroreflex-mediated regu- age of 26 years (range 21 to 35 years) participated in this study. lation of muscle sympathetic nerve activity (MSA) has been Right heart catheterization was performed in seven subjects described (14). Similarly, in congestive heart failure, an (five men and two women), of whom three (one man and two women) were simultaneously studied by microneurography. From the Department of Cardiology, University Hospital, Zu¨rich, Switzerland. Microneurographic measurements of sympathetic activity and This study was supported by the Swiss National Foundation (Nos. 32-42560.94 and noninvasive hemodynamic measurements were additionally 32-5106997) and the Italian Society of Hypertension. Manuscript received August 5, 1999; revised manuscript received December 30, performed in two male volunteers (without right heart cathe- 1999, accepted February 28, 2000. terization). Each volunteer gave written, informed consent. 214 Spieker et al. JACC Vol. 36, No. 1, 2000 NO and Baroreceptor Regulation July 2000:213–8

ilance Monitor Model VGS1; Baxter Healthcare Corporation, Abbreviations and Acronyms Irvine, California) with a Swan-Ganz catheter (Model 746H- LBNP ϭ lower body negative pressure 8F; Baxter Healthcare Corporation) inserted through a sheath G L-NMMA ϭ N -monomethyl-L-arginine introducer system (Cordis, Langenthal, Switzerland) in a ϭ MSA muscle sympathetic nerve activity cubital and propagated to the under NO ϭ nitric oxide fluoroscopic guidance. Arterial blood pressure was measured noninvasively (relative changes: Finapres; Ohmeda, Sweden; absolute baseline values: Dinamap; Critikon, Tampa, Florida) This study was approved by the local ethical committee. and a one-lead electrocardiogram was recorded and digitized Smokers and offspring of hypertensive parents were excluded for computation of heart rate (29). (25). None of the subjects was taking any medications. Experimental protocol. After recording stable baseline val- Microneurographic measurements. Microneurography ues for 15 min, phenylephrine was administered intravenously was performed as described previously (25–27). Subjects through the distal lumen of the Swan-Ganz catheter (or a were studied in standardized fashion, that is, at 2 PM in a peripheral intravenous line in three volunteers) at two dosages quiet, temperature-controlled room, after micturition to (75 and 100 ␮g/min, each over 3 min at 0.75 and 1 ml/min). avoid any increase of sympathetic nerve activity through was measured by thermodilution after each bladder distension (10). Multifiber recordings of MSA were dose of the drugs. During the last dose, LBNP was performed. obtained from the peroneal nerve. A reference electrode was G After reestablishing baseline conditions, N -monomethyl-L- inserted subcutaneously 1 to 2 cm from the recording arginine (L-NMMA) (Clinalfa, La¨ufelfingen, Switzerland) electrode. Signals were amplified, filtered (700 to was administered in two doses (0.3 and 1 mg/kg/min, each for 2,000 Hz), integrated (time constant, 0.1 s) and digitized 3 min at 1 to 3 ml/min) and cardiac output was determined using an analog-digital board (MIO-16L; National Instru- after each dose. Lower body negative pressure was performed ments, Austin, TX) and a modified commercial software again. The drug infusion order was not alternated because of (LabView; National Instruments). Data recorded on a the long elimination half life of L-NMMA. computer (Macintosh Power PC 7100; Apple) were ana- Statistical analysis. Results are presented as means Ϯ lyzed offline (MatLab; MathWorks, Natick, Massachu- SEM. Differences between drugs were evaluated using setts). The results were expressed as bursts per minute and paired Student t tests with Bonferroni adjusted p values for cumulative sum of the amplitude in volts per minute. multiple comparisons. The effects of LBNP were evaluated Lower body negative pressure. Lower body negative pres- by repeated-measures analysis of variance (StatView 4.5; sure (LBNP) decreases central venous pressure by restricting Abacus Concepts, Berkeley, California) (30). Statistical venous return of blood from the lower extremities to the significance was accepted at p Ͻ 0.05. heart (28). We used an air-tight plexiglass chamber in which the subjects were enclosed up to the waist. Negative RESULTS pressure was applied with a commercial vacuum cleaner (15 and 30 mm Hg, each for 2 min), monitored with a Hemodynamic effects. Changes in hemodynamic param- manometer connected to the interior of the chamber. eters and sympathetic nerve activity after administration of Hemodynamic measurements. Cardiac output (average of drugs are shown in Table 1. L-NMMA and phenylephrine three measurements) was determined by thermodilution (Vig- caused dose-related increases in arterial blood pressure.

Table 1. Hemodynamic and Sympathetic Nervous Effects of L-NMMA and Phenylephrine L-NMMA Phenylephrine (mg/kg/min) (␮g/min) Baseline 0.3 1.0 75 100 SBP 134 Ϯ 7 152 Ϯ 5 150 Ϯ 3* 155 Ϯ 7 153 Ϯ 8* DBP 73 Ϯ 487Ϯ 585Ϯ 5† 88 Ϯ 690Ϯ 5† SVR 1,114 Ϯ 124 1,413 Ϯ 145* 1,973 Ϯ 203‡ 1,602 Ϯ 205† 1,743 Ϯ 209† HR 67 Ϯ 470Ϯ 663Ϯ 657Ϯ 5‡ 52 Ϯ 3‡ CI 3.5 Ϯ 0.3 3.1 Ϯ 0.2* 2.3 Ϯ 0.2§ 2.7 Ϯ 0.3† 2.6 Ϯ 0.2† SVI 53 Ϯ 648Ϯ 638Ϯ 5† 51 Ϯ 851Ϯ 4 CVP 4 Ϯ 0.7 3.6 Ϯ 0.4 4.3 Ϯ 0.05 6.3 Ϯ 0.5* 9.5 Ϯ 0.6† B/min 23.1 Ϯ 3.5 14 Ϯ 4.5* 18.6 Ϯ 5.5 12.8 Ϯ 5.8 17.6 Ϯ 5.7 B/100 HB 35.6 Ϯ 5.8 21.3 Ϯ 7.6 32.8 Ϯ 10.8 21.7 Ϯ 9.4 33.8 Ϯ 10 ACS 100 53 Ϯ 27 67 Ϯ 21 26 Ϯ 16* 78 Ϯ 17

Values represent means Ϯ SEM (n ϭ 9 for noninvasive, n ϭ 7 for invasive hemodynamic, and n ϭ 5 for microneurographic measurements). *p Ͻ 0.05, †p Յ 0.01, ‡p Յ 0.001, §p Ͻ 0.0001, for each data point compared with baseline values. G L-NMMA ϭ N -monomethyl-L-arginine; SBP ϭ systolic blood pressure (mm Hg); DBP ϭ diastolic blood pressure (mm Hg); SVR ϭ systemic (dyne/s/cm5); HR ϭ heart rate (beats/min); CI ϭ (L/min/m2); SVI ϭ stroke volume index (ml/min/m2); CVP ϭ central venous pressure (mm Hg); B/min, sympathetic bursts per minute; B/100 HB ϭ sympathetic bursts per 100 heartbeats; ACS ϭ cumulative amplitude sum of sympathetic bursts (% change from baseline). JACC Vol. 36, No. 1, 2000 Spieker et al. 215 July 2000:213–8 NO and Baroreceptor Regulation

Comparison of L-NMMA and phenylephrine. Muscle sympathetic nerve activity was not significantly different after L-NMMA and phenylephrine infusions, in the pres- ence of equal systolic blood pressure and comparable central venous pressure (Fig. 2). Stroke volume index was signifi- cantly decreased by L-NMMA, and systemic peripheral resistance was significantly higher during inhibition of NO synthesis with L-NMMA than during infusion of phenyl- ephrine (p Ͻ 0.05). There were no adverse events during the study. Specifi- cally, no clinical or electrocardiographic signs of ischemia were observed.

DISCUSSION This study shows that baroreflex regulation of heart rate but not MSA in response to orthostatic stress is altered after inhibition of NO synthesis in healthy subjects. Further- more, inhibition of NO synthesis caused a decrease in cardiac contractility. Figure 1. Relative changes of central venous pressure (CVP), systolic blood pressure, muscle sympathetic nerve activity (MSA, bursts per minute and Baroreceptor regulation in orthostatic stress. After in- volts per minute) and heart rate during lower body negative pressure. hibition of NO synthesis, heart rate failed to increase as a Ͻ Central venous pressure similarly decreased in both drugs (*p 0.05 vs. counterregulatory mechanism to falling central venous pres- baseline, intergroup differences NS). Also, there was no difference in systolic blood pressure, which remained constant during lower body sure induced by LBNP. Lower body negative pressure negative pressure in both drugs. During infusion of L-NMMA simulates the cardiovascular effects of orthostatic stress or (1 mg/kg/min), heart rate failed to increase in response to decreased central hemorrhage by venous pooling of blood in the legs and venous pressure. This was in contrast to phenylephrine (100 ␮g/min), where heart rate increased (†p Ͻ 0.05 vs. phenylephrine; *p Ͻ 0.01 by decreasing central venous pressure (31,32). Lower body repeated-measures analysis of variance). Open circles ϭ control; black negative pressure Ͻ20 mm Hg unloads the cardiopulmo- ϭ ϭ circles L-NMMA; black boxes phenylephrine. nary mechanoreceptors; suction Ն20 mm Hg additionally deactivates arterial baroreceptors and increases heart rate After 0.3 mg/kg/min of L-NMMA, blood pressure increased (33). Both maneuvers result in an increase in sympathetic Ͻ Ͻ by 16.3% (p 0.01); and by 18.6% after 1 mg/kg/min (p nerve traffic to blood vessels of arm and leg muscles (28). ␮ 0.01). Similar increases were achieved after 75 g/min of The observed altered baroreceptor response to orthostatic ϩ Ͻ ␮ phenylephrine ( 18.6%, p 0.05) and after 100 g/min stress is not due to an increased blood pressure level after ϩ Ͻ ( 19.4%, p 0.01; intergroup differences not significant). inhibition of NO synthesis, because heart rate increased ϩ Systemic vascular resistance changed accordingly by 26.8% similarly during titrated infusions of phenylephrine. Addi- Ͻ ϩ Ͻ (p 0.05) and 77.1% (p 0.001) after L-NMMA and by tionally, it has been previously shown that different central ϩ Ͻ ϩ Ͻ 43.8% (p 0.01) and 56.5% (p 0.01) after phenyleph- venous pressure levels do not influence sinus node responses rine (intergroup differences not significant). At the higher to arterial baroreceptor responses (34). doses (1.0 mg/kg/min and 100 ␮g/min, respectively), increases Potential explanations. The paradoxical bradycardia dur- in arterial blood pressure were equal. ing NO synthase inhibition in response to falling central Heart rate was not affected by L-NMMA infusion venous pressure and decreased stroke volume suggests the (ϩ4.5% and Ϫ6.0%, NS), in contrast to phenylephrine requirement of some basal NO for baroreceptor regulation where heart rate decreased (Ϫ14.9% and Ϫ22.4%, p Ͻ of heart rate. This may take place at various anatomical 0.001). Stroke volume index declined after the higher dose levels from alterations in: 1) baroreceptor sensitivity of L-NMMA (Ϫ28.3%, p ϭ 0.01) but not after phenyl- (20,21,35–41); 2) central nervous coupling (8,35,42,43); 3) ephrine. After phenylephrine, the decreased cardiac index pre- and postsynaptic autonomic nerve traffic (44,45); and was related to a dose- and blood pressure-dependent fall in 4) transmission of autonomic nerve activity to end-organ heart rate. Central venous pressure was increased by phen- cells (41,46–48). Our experiments do not allow us to ylephrine (ϩ43.6% and ϩ123%, p Ͻ 0.01), but was not differentiate between these possibilities. It could be hypoth- changed by L-NMMA. esized that NO is required as a parasympathetic messenger LBNP. During administration of L-NMMA, heart rate, for transmission of the effects of acetylcholine to cardiac but not MSA, failed to show a compensatory increase in pacemaker cells. It was reported that alkyl esters of arginine response to decreased central venous pressure induced by are muscarinic receptor antagonists; however, this is not the LBNP (Fig. 1). This was in contrast to control conditions case for L-NMMA (49). Because the response of peripheral and phenylephrine infusion. sympathetic nerve activity to orthostatic stress was not 216 Spieker et al. JACC Vol. 36, No. 1, 2000 NO and Baroreceptor Regulation July 2000:213–8

Figure 2. Hemodynamics and muscle sympathetic nervous activity (MSA) at similar levels of systemic arterial (sBP) and central venous (CVP) blood pressure after infusion of L-NMMA (1 mg/kg/min), and phenylephrine (PE, 100 ␮g/min) in combination with lower body negative pressure (LBNP Ϫ30 mm Hg). Stroke volume index (SVI) was significantly lower during L-NMMA infusion than during phenylephrine, whereas systemic vascular resistance (SVR) was significantly higher. No significant difference in sympathetic nerve activity between L-NMMA and phenylephrine could be observed (*p Ͻ 0.05). affected by L-NMMA, differential regulation of muscle and hemodynamic abnormalities, have been demonstrated cardiac sympathetic activity could be postulated (50,51). In (15,17,53,54). Alterations of baroreflex function may con- fact, a dissociation of MSA in arm and leg has been tribute to elevated sympathetic nerve activity in heart failure described (52). (18), which is an unfavorable prognostic factor Endothelial and baroreceptor dysfunction. Our findings (17,19,54,55). Cardiopulmonary baroreflex control of sym- partly reproduce the known alterations in baroreceptor pathetic nerve activity is also deranged in hypertension and regulation in disease states with endothelial dysfunction. atherosclerosis (12,56,57), adding further evidence to the Indeed, our results are in line with studies investigating concept of endothelium dependency of baroreflex. baroreflex control in hypertensive patients. Grassi et al. (14) Previous studies. Small doses of L-NMMA (50 ␮g/kg/ reported altered regulation of heart rate but not sympathetic min) were reported to have stimulating effects on MSA, nerve activity in hypertension. In heart failure, abnormal whereas high doses decreased MSA (58). Combined infu- baroreceptor responses of heart rate and MSA to falling sion of L-NMMA and nitroprusside increased MSA, central venous pressure, related in part to the severity of whereas mean arterial blood pressure was held constant. JACC Vol. 36, No. 1, 2000 Spieker et al. 217 July 2000:213–8 NO and Baroreceptor Regulation

However, the effects of nitroprusside on central venous Acknowledgment pressure must not be neglected. Additionally, when com- The authors thank Isabella Sudano for helping with the paring sympathetic outflow during L-NMMA and phenyl- experiments and Rosy Hug for organizational assistance. ephrine infusions, it must be taken into account that phenylephrine increases central venous pressure (58,59). Reprint requests and correspondence: Dr. Georg Noll, Cardiol- Elevated central venous pressure activates cardiopulmonary ogy, University Hospital, CH-8091 Zu¨rich, Switzerland. E-mail: baroreceptors and therefore reduces sympathetic outflow. [email protected]. We found no differences in MSA during L-NMMA and phenylephrine when LBNP was used to achieve similar REFERENCES central venous pressure. Further effects of NO synthase inhibition. Our results 1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. 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