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1Department of Cardiology, Erasme Hospital, Brussels, Belgium; and 2Department of Clinical Pharmacology, Faculty of Medicine and University Hospital, Toulouse, France
Submitted 9 July 2007 ; accepted in final form 18 October 2007
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ABSTRACT |
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phosphodiesterase inhibitor; adenosine 3',5'-cyclic monophosphate
Several animal (8, 22, 23, 33–35) and human (4) studies have reported the important role of cAMP in peripheral chemoreceptor sensitivity. Hypoxia, which is the primary natural stimulus of the peripheral chemoreceptors, increased the cAMP content of cat (8) and rabbit (33, 34) carotid bodies in proportion to the stimulus intensity. Moreover, the chemoreflex response to hypoxia was modulated by the cAMP content in the rabbit carotid body (22, 23, 33, 34), probably by inhibition of the O2-sensitive transient chemoreceptor K+ currents (17) and the effects of cAMP on the exocytotic machinery (10). Indeed, increased cAMP levels in rabbit (22, 23, 33, 34) and rat (8) carotid body preparations, after administration of forskolin (adenylate cyclase activator) or methylxanthine [phosphodiesterase (PDE) inhibitor], enhanced the release of catecholamines in a dose-dependent manner, which resulted in a rise in carotid sinus nerve discharge (34). In humans, administration of aminophylline to newborn infants has also been shown to increase peripheral chemoreflex sensitivity, probably by decreasing the breakdown of cAMP in the carotid body cells (4).
Enoximone is a PDE III and IV inhibitor (29) used clinically for the hemodynamic support of patients with severe congestive heart failure (3). Administration of enoximone increases cAMP levels in cardiac cells, which activate protein kinase A. This promotes phosphorylation of several key proteins responsible for the inotropic effect of this molecule (28, 29). Moreover, animal studies (11, 15, 16) revealed a direct chronotropic action of PDE III inhibitors mediated by the increase of the action potential amplitude, the maximal rate of depolarization, and the spontaneous firing frequency of sinus node cells. Accordingly, human studies (18, 24) have also shown a decrease in spontaneous sinus cycle length, sinus node recovery time, and the refractory period of the atrium responsible for the chronotropic effects of enoximone.
On the basis of previous evidence that cAMP is an important modulator of chemoreflex function (8, 22, 23, 33–35), we decided to test the hypothesis that enoximone increases peripheral chemoreflex activity in a single-blind, randomized, crossover, and controlled study of 15 young healthy men. We determined the effects of enoximone and placebo on the ventilatory, hemodynamic, and MSNA responses to 5 min of isocapnic hypoxia. We also studied the MSNA response during voluntary end-expiratory apnea to suppress the inhibitory influences of pulmonary afferents on sympathetic activity (27).
In addition, muscle metaboreflex activation was examined to ensure that the changes were not simply due to a nonspecific increase in ventilatory and sympathetic responses to excitatory stimuli by enoximone. Finally, we studied the effect of enoximone during hyperoxic hypercapnia to exclude any intervention of the central chemoreflex on our results.
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METHODS |
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We recruited 15 young healthy men (24 yr old, range 21–33 yr, median 24, SD 3.7) taking no medication and with normal physical examination. The subjects were told not to drink coffee or tea 48 h before the study. Each subject was asked to empty his bladder before the start of the recordings. The study protocol conformed to the standards set by the Declaration of Helsinki. The Ethical Committee of Erasme Hospital approved the study protocol, and written informed consent was obtained from each subject.
Measurements
We obtained continuous recordings of minute ventilation (pneumotachometer), end-tidal CO2 (Normocap, Datex-Ohmeda, Madison, WI), O2 saturation (Nellcor, Pleasanton, CA), heart rate (HR), and ECG (Siemens, New York, NY). Systolic, diastolic, and mean blood pressure (MBP) were measured every minute during normoxia, isocapnic hypoxia, hyperoxic hypercapnia, and isometric handgrip (Physiocontrol BP-880 sphygmomanometer, Colin, San Antonio, TX). MSNA was recorded continuously using multiunit recordings of postganglionic sympathetic activity measured from a nerve fascicle in the peroneal nerve posterior to the fibular head (21, 26, 31, 32). Cardiac output (CO) was estimated during normoxic periods with the Modelflow method (2) by finger pressure measurement (Finometer, FMS, Amsterdam, The Netherlands).
Interventions
The protocol used to test the chemoreflex responses was identical to that used in previous studies (21, 26, 31, 32). In our laboratory, the coefficient of variation of the ventilatory response to isocapnic hypoxia between two recordings separated by 1 wk in the same subject is 10% (n = 6). Subjects breathed across a low-resistance mouthpiece with a nose clip to ensure exclusive mouth breathing during each sequence.
Isocapnic hypoxia. Measurements were made after 3 min of stable baseline recordings, with subjects breathing room air, followed by 5 min of peripheral chemoreflex activation achieved by exposure to isocapnic hypoxia (10% O2 in 90% N2, with CO2 titrated to maintain isocapnia).
Hyperoxic hypercapnia. Measurements were made after 3 min of stable baseline recordings with subjects breathing room air, followed by 5 min of central chemoreflex activation achieved by exposure to hyperoxic hypercapnia (7% CO2 in 93% O2). Hyperoxia was used to deactivate peripheral chemoreceptors during central chemoreflex sensitivity assessment.
Apnea. Recordings were also made during a voluntary maximal end-expiratory apnea after the baseline period and after hypoxia to correct for the possible inhibitory influence of ventilation on MSNA (27).
Isometric handgrip and local circulatory arrest. We determined the maximal voluntary contraction of the dominant arm for every subject, in triplicate, with a handgrip dynamometer at the beginning of the recording session.
We recorded the above-mentioned variables during 3 min of isometric handgrip exercise of the dominant arm at 30% of maximum voluntary contraction in normoxia. The handgrip test was preceded by a 3-min baseline period with stable ventilation, while subjects were breathing room air. Subjects were requested to minimize any muscle contraction in the resting muscle during handgrip. All interventions were followed by a forearm local circulatory arrest obtained by inflation of a standard blood pressure cuff to 240 mmHg on the exercising arm for 2 min. Subjects were instructed to relax their grip after the cuff was inflated. This procedure traps metabolites released by the muscle contraction and maintains metabolically sensitive muscle afferent activation independently of muscle contraction (mechanoreceptor reflex) and volitional effects (central command) (9). This test was performed to ensure that the eventual changes observed during peripheral chemoreflex activation were not simply due to a nonspecific increase in responses to excitatory stimuli by enoximone.
Measurements were made twice, on 2 separate days 1 wk apart, after administration of enoximone or placebo (0.9% NaCl) in random allocation. Drugs were administered into a peripheral vein in the nondominant arm. The dose of enoximone was 90 µg·kg–1·min–1 over 17 min. Studies were performed at the same time of day for both experimental sessions, 10 min after administration of enoximone or placebo. Peripheral and central chemoreflex sensitivity was assessed in a randomized order, with 15 min separating both interventions. However, the order of peripheral and central chemoreflex activation was left constant within a given subject. Responses to isometric handgrip and local circulatory arrest were always recorded at the end of the study.
Data Analysis
Sympathetic bursts were identified by careful inspection of the mean voltage neurogram (21, 26, 31, 32). This analysis was performed by an investigator fully blinded to the medication taken by the subject. The amplitude of each burst was determined, and sympathetic activity was calculated as bursts per minute and multiplied by mean burst amplitude (arbitrary units). We also calculated sympathetic activity as the number of bursts per 100 heartbeats. We analyzed the baselines, 5 min of isocapnic hypoxia, 5 min of hyperoxic hypercapnia, and isometric handgrip. The sympathetic responses to the apneas were calculated during the entire period of apnea, divided by the duration of the apnea in seconds, and subsequently multiplied by 60 to express the response in changes per minute (21, 26, 31, 32). Changes in MSNA during hypoxia, hypercapnia, and handgrip were expressed as the percent change from baseline. Excellent recordings of MSNA with a signal-to-noise ratio >3 were achieved during enoximone and placebo sessions in 9 of 15 subjects. For technical reasons, we could not obtain such a recording in six subjects. However, age, baseline parameters, and response to interventions in subjects from whom we did not achieve an adequate measurement of MSNA did not differ from those in subjects from whom MSNA was recorded. Inasmuch as the absolute value of the computed CO estimated by the Modelflow method is uncertain unless calibration against another method of measurement can be done (13), changes in CO produced by enoximone and placebo were calculated as percent increase from mean CO recorded during 10 min of rest before drug infusion.
Statistical Analysis
Values are means ± SE. We performed an ANOVA for repeated measures to assess whether cardiovascular, ventilatory, and MSNA responses to isocapnic hypoxia, hyperoxic hypercapnia, isometric handgrip, and local circulatory arrest following exercise differed between enoximone and placebo. Other comparisons were made with a Student's paired t-test (2-tailed). The effects of enoximone on ventilatory and MSNA responses to isocapnic hypoxia and hyperoxic hypercapnia were also tested using multivariate regression analyses. Significance was assumed at P < 0.05.
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RESULTS |
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Enoximone increased CO compared with placebo (Table 1). Enoximone induced a modest decrease in O2 saturation, which was accompanied by a nonsignificant increase in ventilation and a minimal decrease in end-tidal CO2. There was no difference in MBP between the placebo and enoximone sessions. However, HR was higher with enoximone than with placebo. MSNA was larger with enoximone when expressed in number of bursts per minute, but this difference was no longer present when MSNA was expressed in number of bursts per 100 heartbeats.
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Enoximone increased the ventilatory response to acute hypoxia [13.6 ± 1 vs. 11.2 ± 0.7 l/min at 5 min of hypoxia, P = 0.03 vs. placebo (by ANOVA); Fig. 1]. This response was accompanied by a lesser decrease in O2 saturation [83 ± 1 vs. 79 ± 2% at 5 min of hypoxia, P = 0.003 vs. placebo (by ANOVA)]. However, there was no difference in the sympathetic response to hypoxia between enoximone and placebo [P = 0.28 (by ANOVA)]. The increase in HR during hypoxia was similar with enoximone and placebo (P = 0.48), and there was no increase in MBP during both experiments (P = 0.42). End-tidal CO2 was maintained at the same level to ensure isocapnic hypoxia [38 ± 1 and 39 ± 1 mmHg at 5 min of hypoxia for enoximone and placebo, respectively, P = 0.24 (by ANOVA)]. Multivariate regression analyses, however, revealed a significant effect of enoximone on ventilatory and MSNA (both P < 0.001) responses to isocapnic hypoxia (Fig. 2).
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The duration of the apneas and the subsequent reduction in O2 saturation were the same for enoximone and placebo during normoxia (Table 2). Nevertheless, the increase in MSNA was less pronounced after enoximone administration in the same conditions [P = 0.04 (by Student's paired t-test)]. On the other hand, during hypoxia, despite the trend to shorter apnea and less desaturation in the enoximone group (Table 3), there was no difference in the MSNA increase between enoximone and placebo.
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There was no difference in the ventilatory response between enoximone and placebo during hyperoxic hypercapnia [24.5 ± 2 and 25.6 ± 1.9 l/min at 5 min of hypercapnia with enoximone and placebo, respectively, P = 0.8 (by ANOVA); Fig. 3]. End-tidal CO2 [52 ± 1 and 53 ± 1 mmHg at 5 of hypoxia with enoximone and placebo, respectively, P = 0.99 (by ANOVA)] and the O2 saturation [99 ± 1 and 99 ± 1% at 5 min of hypoxia with enoximone and placebo, respectively, P = 0.55 (by ANOVA)] were similar between both groups. MSNA responses to hyperoxic hypercapnia were similar with enoximone and placebo [125 ± 6 and 144 ± 14% at 5 min of hypercapnia, respectively, P = 0.13 (by ANOVA)]. There was no difference in the increase in MBP in the two groups [92 ± 3 and 95 ± 3 mmHg at 5 min of hypercapnia with enoximone and placebo, respectively, P = 0.53 (by ANOVA)]. However, the HR response was larger with enoximone [87 ± 3 vs. 77 ± 2 beats/min at 5 min of hypercapnia, P = 0.02 (by ANOVA)]. Multivariate regression analyses confirmed that enoximone had no effect on ventilatory and MSNA (both P > 0.05) responses to hyperoxic hypercapnia (Fig. 4).
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Enoximone did not affect the ventilatory (Fig. 5), hemodynamic, or sympathetic responses during isometric handgrip (P > 0.22 by ANOVA) or local circulatory arrest (P > 0.15 by ANOVA).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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These findings were particularly evident when the effects of enoximone on chemoreflex function were assessed using a "response vs. stimulus" curve in a multivariate regression model, instead of the classical "response vs. time" curve used in our previous studies (21, 31, 32). This further analysis permits us to report chemoreflex responses without the confounding effect of O2 saturation differences observed between placebo and enoximone during isocapnic hypoxia.
Hyperventilation, acting via pulmonary afferents, inhibits the sympathetic response to hypoxia (27). This inhibition was probably more manifest with enoximone when we consider the higher minute ventilation reported during its administration. Moreover, O2 saturation was also higher with enoximone during hypoxia, resulting in a lower hypoxic stimulus. In regard to previous observations, one would expect a smaller sympathetic activation in response to hypoxia during the enoximone session. However, we found no difference in MSNA between enoximone and placebo in that condition, suggesting a relative sensitization of the MSNA response to the hypoxic stimulus. Moreover, after correction for O2 saturation in a multivariate regression model, enoximone enhanced ventilatory and MSNA responses to isocapnic hypoxia. In addition, similar analyses confirmed the lack of effect of enoximone on ventilatory and MSNA responses during central chemoreflex activation by hyperoxic hypercapnia.
We observed a differential effect of enoximone on the HR response to hypoxia and hypercapnia. Although changes in HR with hypoxia started from a higher baseline, they followed a pattern similar to that observed with placebo, resulting in a further rise. This was not observed during hypercapnia, where HR was also faster during baseline, but did not increase during hypercapnia in the presence of enoximone. This differential response of HR could also result from a selective potentiation of the peripheral chemoreceptors, leading to a more pronounced tachycardia during hypoxia than hypercapnia.
Our results, however, contrast with the clear-cut, dose-dependent increase in peripheral chemoreflex activity under dobutamine also present during end-expiratory maximal voluntary apneas (32). These effects of dobutamine on chemoreflex function were evident at a low dose of 2.5 µg·kg–1·min–1 and despite substantial increases in blood pressure, which are known to restrain the sympathetic response to peripheral chemoreflex activation (27). An increase in CO and pulse pressure, similar to that observed during enoximone administration, could also have an inhibitory effect on sympathetic activity, via baroreflex activation, as reported in healthy subjects (6) and in animal studies (5). This mechanism may have restrained the sympathetic response to the normoxic apnea during enoximone, resulting in a more marked MSNA response to the apnea with placebo. On the other hand, chemoreflex sensitization may have overridden these inhibitory effects during the apneas in hypoxia, resulting in more similar changes in MSNA with enoximone and placebo. Thus, even if the increased ventilatory and sympathetic response to hypoxia with enoximone cannot be explained by a nonspecific facilitation of the ventilatory and sympathetic response to excitatory stimuli, the chemoreflex effects of enoximone are likely to be much less marked than those we observed previously with dobutamine. This hypothesis will, however, require further direct comparison between both inotropic agents at similar levels of CO.
The fact that enoximone did not exert clear-cut facilitatory effects on peripheral chemoreflex function cannot be attributed to our study protocol. Indeed, the dose of enoximone administered to our subjects is similar to that used in clinical practice. This dose produced an increase in cardiac CO and HR in our subjects without side effects. All our interventions were performed within 1.5 h after the infusion of the drug, because the half-life of enoximone varies from 2 to 2.7 h in healthy volunteers (19). We used a single-blind study design for safety reasons. However, all variables were analyzed in a fully blinded manner. Moreover, although differences in the hypoxic ventilatory responses during enoximone vs. placebo were small, they were larger than the day-to-day variability in this response in our laboratory.
During normoxia, enoximone slightly decreased O2 saturation, probably by creating nonspecific vasodilation in the pulmonary vascular bed and ventilation-perfusion mismatch, as described previously (12). This effect was also presumably responsible for the modest increase in minute ventilation and decrease in end-tidal CO2 in our subjects during normoxia after enoximone administration. However, O2 saturation during isocapnic hypoxia was larger with enoximone than with placebo, because enoximone potentiated the ventilatory response to the hypoxic stimulus. There is, therefore, no reason to believe that the modest decrease in O2 saturation with enoximone in normoxia could be responsible for the larger minute ventilation during hypoxia.
Enoximone increased HR during normoxia. Subsequently, MSNA, expressed in bursts per minute, was also increased, inasmuch as changes in HR are accompanied by similar changes in sympathetic discharge frequency (7). A marked participation of baroreflex deactivation in these changes is doubtful, because there were no differences in sympathetic activity between enoximone and placebo after correction for HR, once MSNA was expressed as number of bursts per 100 heartbeats. Moreover, blood pressure was similar between the two groups. We believe, therefore, that a direct chronotropic effect of enoximone on the sinus node reported in several animal and human studies (11, 15, 16, 18, 24, 30) is more likely to explain the increase in baseline HR and MSNA burst frequency.
In conclusion, enoximone increases the chemoreflex response to isocapnic hypoxia. Apnea duration is unaffected by enoximone. Nevertheless, the ventilatory and sympathetic effects of enoximone are specific for peripheral chemoreflex function, inasmuch as central chemoreflex and isometric handgrip responses are not altered by enoximone.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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