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ORIGINAL ARTICLES |
From the Georgia Prevention Institute (F.T., G.K.K., H.D.), Vascular Biology Center (J.S.P., D.M.P.), the Departments of Pediatrics (F.T., G.K.K., H.D.), Pharmacology and Toxicology (J.S.P., D.M.P.), Psychiatry (F.T.), and Surgery (D.M.P.), Medical College of Georgia, Augusta, GA.
Address reprint requests to: Frank A. Treiber, Medical College of Georgia, Georgia Prevention Institute, Building HS1640, Augusta, GA 30912-3710. Email: ftreiber{at}mail.mcg.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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METHODS: Two hundred twenty-two adolescents (mean age = 18.5 ± 2.8 years; 130 [70 males] EAs and 92 [48 males] AAs) completed two stressors (video game, forehead cold). Hemodynamic measures and blood samples were collected at catheter insertion and before and immediately after the two stressors, separated by 20-minute rest periods.
RESULTS: AAs and males exhibited higher levels of SBP and DBP and of TPRI and ET-1 at each sampling point compared with EAs and females, respectively (p values < .001). AAs and males exhibited greater increases in SBP, TPRI, and ET-1 in response to each stressor (p values < .05). Intraindividual correlations between ET-1 and hemodynamic parameters revealed that most individuals exhibited a positive association between ET-1, BP, and TPRI. However, some individuals exhibited a negative association between ET-1 and the above-mentioned hemodynamics, suggesting a compensatory vasodilation mechanism.
CONCLUSION: The findings demonstrate significant sex and ethnicity differences in stress-induced vasoconstrictive peptide release and support the hypothesis that these differences may be important in explaining the ethnicity and sex differences in the prevalence of cardiovascular disease.
Key Words: ET-1, • stress, • cardiovascular reactivity, • ethnicity, • sex.
Abbreviations: ET-1 = endothelin-1;; TPRI = total peripheral resistance indexed by body surface area;; AA = African Americans;; BP = blood pressure;; DBP = diastolic blood pressure;; EA = European Americans;; EH = essential hypertension;; SBP = systolic blood pressure.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cardiovascular reactivity is a candidate risk factor for cardiovascular disease, as several longitudinal studies in adults have found exaggerated blood pressure (BP) reactivity to be predictive of EH and coronary artery disease [see review in (4)]. Studies in youth have found BP reactivity predicts future BP levels and increases left ventricular mass (LVM) 1–6 years later [see review in (5)]. Adult and youth studies have found that AAs and males often exhibit higher TPR-mediated BP reactivity than EAs and females, respectively, to a variety of laboratory stressors (6–12).
Endothelin-1 (ET-1) is a potent vasoactive peptide that is released basolaterally by endothelial cells and elicits smooth muscle cell constriction (13). It may be an underlying biological mediator of increased vascular tone at rest and in response to stress. Studies involving normotensive and hypertensive adults have shown that ET-1 infusion elicits an increase in BP and TPR (14). However, under normal circumstances, ET-1 produced by endothelial cells is released abluminally and therefore the circulating level of ET-1 is thought to be the result of spillover. Although it is expected that the circulating level of ET-1 is proportional to the total ET-1 produced by the endothelial cells, it is possible that this is not the case. Thus, the circulating level of ET-1 may not directly reflect the full physiological impact of ET-1. Among normotensive and hypertensive adults, AAs have been found to exhibit higher plasma levels of ET-1 than EAs (15, 16). Recently, a similar finding was observed among normotensive adolescent males (17). With regard to possible sex differences in ET-1, Polderman et al. (18) found that, among healthy adult EAs, males had higher circulating levels of ET-1 than females. However, among a group of healthy 18–70-year-old EAs and AAs, Evans et al. (16) found plasma ET-1 levels in EA males were significantly lower than in all remaining groups.
Endothelin-1 has been shown to increase in response to acute mental (eg, mental arithmetic) and physical (eg, cold pressor) stress in adults (19–21). Hypertension status as well as family history of EH have been associated with greater release of ET-1 in response to laboratory stressors in adults (21, 22). To our knowledge, only one study, which involved a small sample of adolescent males (N = 41), has evaluated ethnic differences in ET-1 responsivity to laboratory stress (17). AAs exhibited higher plasma basal levels than EAs and greater increases in ET-1 in response to both video game challenge and forehead cold stimulation. To our knowledge, no research has examined possible sex differences, much less the potential interactive effect of ethnicity and sex on ET-1 levels during laboratory stress. Given that AA males are at particular risk for development of EH (1–3) and often exhibit exaggerated vasoconstrictive-mediated BP reactivity to stress (5, 7, 10, 11), examination of the possible interactive effects of ethnicity and sex on ET-1 and BP reactivity to stress is warranted.
Based on previous findings in adults and normotensive adolescents, it was hypothesized that AAs, particularly males, would exhibit higher levels of ET-1 at rest and in response to psychological (eg, video game challenge) and physical (eg, forehead cold stimulation) stress. Further, it was predicted that AAs, particularly males, would show greater TPRI-mediated BP reactivity to the two stressors and that ET-1 release would be associated with BP and TPRI responses. Participants in the study had verified family histories of cardiovascular disease (8, 11, 23) and thus are at increased risk for future development of EH (24). Evaluation of ET-1 levels and hemodynamic function in normotensive at-risk youth may be useful in elucidating the role of the endothelin system in the early pathophysiology of cardiovascular disease.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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For comparison, the sample was divided into two groups: 1) those from whom blood was not obtained at all nine time points (N = 103), primarily due to failure to stabilize the needle in the antecubital vein, and 2) those from whom blood was obtained at all nine time points (N = 248). The groups did not differ significantly in percentage of EAs or males, age, height, weight, body mass index, waist-to-hip ratio; supine resting DBP or TPRI; SBP, DBP, or TPRI reactivity to the video game stressor; SBP reactivity to the ice stressor; or initial ET-1 levels. The group with complete blood data had significantly higher mean values for DBP and TPRI reactivity to the ice stressor and a significantly lower supine resting SBP (all p values < .03). Finally, 26 subjects were excluded because their ET-1 data had been analyzed using an older, less reliable assay.
Procedure
The Institutional Review Committee of the Medical College of Georgia approved all experimental protocols and the process for obtaining informed consent. On arrival at the laboratory, the subject was escorted to a quiet, temperature-controlled room where the subject’s anthropometric measurements (eg, height, weight, waist and hip circumferences, suprailiac, subscapular, and triceps skinfold thicknesses) were recorded using established protocols (25). The subject was then fitted with equipment for recording BP and HR (Dinamap Model 1846 SX, Critikon Inc, Tampa, FL). Cardiac index (CI) was determined using thoracic bioimpedance (NCCOM-3, Bo Med Medical Manufacturing Ltd, Irvine, CA), as previously described (11). TPRI was calculated using concurrently derived BP and CI as follows: [(SBP + 2 x DBP)/3]/CI expressed in Wood units (mm Hg/liter/minute/m2). After attachment of the BP cuff to the right arm, the subject was asked to lie on a hospital bed in a supine position. The left elbow was stabilized with an armboard; a 21-gauge butterfly needle was inserted into the antecubital vein, and a three-way plastic stopcock was attached. Immediately after needle placement, a 5-ml blood sample was drawn, transferred to a 10-ml prechilled EDTA tube vacutainer, and maintained on ice. All subsequent blood collection followed this procedure. One ml of 0.9% saline was infused at 2 to 3 minute intervals to try to help maintain patency. Blood was centrifuged at 4°C and plasma collected and stored at -80°C until analysis.
Hemodynamic Evaluations and Blood Collections
After the initial blood draw, the subject was given standardized instructions to relax as completely as possible for 20 minutes. During minutes 11, 13, 15, 17, and 19 of this period, hemodynamic measurements (ie, SBP, DBP, TPRI) were obtained. At minutes 15 and 20, 5-ml blood samples were drawn. Following the baseline evaluation, the subject engaged in two laboratory stressors (viz, video game and forehead cold) in the supine position using standardized protocols.
The 10-minute video game stressor "Break Out" (Atari Inc, USA) was presented under a monetary incentive challenge (11). The video game controller was secured at a position comfortable for use with the subject’s right hand. The game was presented on a 635-mm diagonal color television located 2 m from the subject at a position comfortable for viewing. Hemodynamic readings were obtained during minutes 1, 3, 5, 7, and 9 of the video game task and a 5-ml blood sample was obtained immediately at its completion. Samples were also taken at minutes 15 and 20 following the game, with hemodynamic measures concomitantly acquired.
The forehead cold-stimulation task was based on a protocol developed in our laboratory (11). A plastic bag containing 6 cups of crushed ice and 1.5 cups of water was placed on the subject’s forehead for 1 minute. Hemodynamic measurements and blood samples were concomitantly obtained immediately on completion of the stressor and at minutes 15 and 20 during recovery.
ET-1 Measurements
Plasma ET-1 levels were determined with ELISA (QuantiGlo, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions except that the standard curve was limited to a maximum of 6 pg/ml. The reported cross-reactivity of the antibody was <0.02% for all big ETs, 7.8% for ET-3, and 27.4% for ET-2. Samples were thawed at room temperature, inverted three times, and centrifuged for 5 minutes at 1500 g at 4°C. All samples and standards were processed in duplicate. Unknown sample data were fitted to a standard curve with commercially available software (Prism 2.0, GraphPad Software, San Diego). The intraassay variability was 4.2%.
Data Reduction and Statistical Analyses
Two (ethnicity) x 2 (sex) x 9 (time) repeated-measures ANOVAs were computed on ET-1, SBP, DBP, and TPRI separately to compare levels of each of these parameters across the experiment. Significant ethnicity and/or sex by time interactions for ET-1 and the hemodynamic variables were examined with a series of follow-up tests on the change scores (computed by subtracting the prior resting measure from the stressor response). Comparable repeated-measures analyses of covariance covarying out the corresponding ET-1 level were computed to determine whether the hemodynamic changes across the experiment could be explained by changes in the ET-1 level alone. Because ET-1 levels were not concordant with assumptions for ANOVA, log transformations were used before analysis.
Pearson correlations were computed for ET-1 levels with SBP, DBP, and TPRI within each individual. These correlations were transformed using Fisher’s r to z transformation to normalize the values and used as dependent variables with sex and ethnicity as independent variables. They were also correlated to anthropometric and hemodynamic variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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DBP Findings
For DBP, ethnicity and time were both significant (both p values < .001), as was the time by sex interaction (p < .001). AAs had higher DBP at each evaluation point than did EAs. Post hoc tests revealed that the interaction of time by sex was due to larger increases in DBP for males than for females to the video game and cold stressors. Otherwise, the two sexes were comparable at each time point. The inclusion of ET-1 level as a covariate did not alter the pattern of significant effects.
TPRI Findings
Significant effects were found for time by ethnicity and time by sex interactions (both p values < .04). As illustrated in Figure 1, although the mean TPRI value for AAs was higher than for EAs at each time, the interaction of time by ethnicity was due to the greater TPRI increase for AAs during the ice stressor than for EAs (p < .05). Similarly, although males had higher TPRI means at each time point than did females, the interaction of time and sex was due to the greater increase in TPRI for males during the cold stressor than for females (p < .05).
Association Between ET-1 and Hemodynamic Responses
Within-subjects correlations were computed correlating ET-1 levels with SBP, DBP, and TPRI across the nine time points. This yielded three correlations (viz, ET-1 with SBP, ET-1 with DBP, ET-1 with TPRI) for each individual. These correlations were used in subsequent data analyses after they were transformed using the normalizing Fisher r to z transformation. Descriptive statistics for the correlations before transformation are given in Table 2 for each ethnicity-sex group. Using the transformed correlations in ethnicity by sex ANOVAs yielded no significant sex or ethnicity differences.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Our results confirm and extend several previous studies that examined ethnic differences in resting levels of plasma ET-1 in older adults. These studies reported that, in normotensives and hypertensives, AAs exhibited higher resting ET-1 levels than EAs (15, 16). We observed lower baseline ET-1 levels in females than males, as did Polderman et al. (18) in a sample of 29 slightly older healthy females. The reason plasma levels of ET-1 are higher in AAs and men is unclear. Differences in the synthesis, degradation rate, and/or release of ET-1 in response to stimulation may relate to the observed findings but will require further exploration.
Endothelin-1 has been shown to increase during exposure to acute behavioral stressors such as mental arithmetic and cold stimulation in adults (19–22). Recently, among adolescent males, AAs were found to exhibit greater ET-1 release compared with EAs in response to a video game challenge and cold pressor stimulation (17). The present findings corroborate and extend that study in a much larger sample of males and females. That is, irrespective of sex, AAs exhibited greater increases in ET-1 than EAs to both stressors. To our knowledge, this is the first study to report a sex difference in ET-1 release in response to stress. Males exhibited higher levels at baseline and maintained these differences in response to both stressors.
With regard to hemodynamic responses to the stressors, previous studies in adults and youth have found that AAs often exhibit greater vasoconstrictive-mediated BP reactivity to acute stress than EAs (6–8, 10, 11). Similarly, adult studies have found that males exhibit greater BP reactivity than females (7, 9, 10, 12). The present findings corroborate these studies in that, among a group of normotensive adolescents and young adults, AAs and males had greater increases in BP and/or vasoconstrictive tone to the two stressors compared with EAs and females, respectively.
The endothelial system might be one underlying biological pathway contributing to ethnic and sex differences in hemodynamic function. This effect could be either through the direct actions of ET-1 on the endothelial system or by potentiation of the effects of other vasoactive systems such as the sympathetic nervous system or the renin-angiotensin-aldosterone system. In the present study, AAs and males had a greater ET-1 release along with higher BP and TPRI at all examination points. However, when controlling for ET-1, the ethnicity and sex differences in BP and TPRI remained, suggesting that hemodynamic changes were not solely dependent on ET-1 release. Furthermore, when intraindividual correlations between ET-1 and hemodynamic responses were computed, we found that most individuals exhibited a vasoconstrictive pattern characterized by a positive association between ET-1 and BP and TPRI responses. Although the means of these correlations for each ethnicity-sex group were all statistically significant, one of the more interesting findings of this study was that some individuals exhibited a pattern characterized by a negative association between ET-1 and the above-mentioned hemodynamics (and others no association). Reasons for the individual differences in these patterns of relationship are unclear. However, they may be due to differences in the synergistic effects between ET-1 and other vasoactive factors that are stimulated by these tasks such as norepinepherine and angiotensin II. Although these intraindividual relationships between ET-1 and the hemodynamic parameters were significantly related to some anthropometric and demographic variables (eg, height, weight, age), the relationships were generally too low and too inconsistent (across groups and across hemodynamic variables) to be considered indicative of causal links. Normally, ET-1-related vasoconstrictive effects are accompanied by release of the endothelium-derived vasodilator nitric oxide in response to flow change, sheer stress, etc (26). Perhaps individual differences in the ratio of nitric oxide to ET-1 release in response to stress are responsible for the observed subject differences in relationships between ET-1 release and hemodynamic changes to stress. Future studies are necessary to evaluate the possible mechanisms underlying the individual differences in response patterns as well as their clinical significance.
Another surprising finding was the fact that neither the average levels of ET-1 nor the hemodynamic variables returned to baseline levels following the first stressor, although the differences for the hemodynamic variables between the baseline and stressor recovery values were generally quite small. The amount of time allocated for each recovery period had been selected based on the expected half-life of ET-1 and our previous experience with regard to hemodynamic recovery to these stressors (8, 11, 17, 23). In our previous studies (not involving needle insertion), the recovery periods used in the present study would have been more than sufficient to allow the large majority of subjects’ hemodynamic values to return to baseline before the initiation of the second stressor. Although it is only speculation, it might be that the placement of an intravenous needle and repeated blood draws influenced the recovery time.
Although the findings are intriguing and supportive of other recent findings, they need to be interpreted cautiously for several reasons. First, all subjects have verified family history of cardiovascular diseases involving EH and/or premature myocardial infarction. Whether the observed patterns of ethnicity and sex differences are moderated by family history of cardiovascular disease is unknown. Second, subjects in whom all nine blood samples were not obtained were excluded from the analyses. However, because demographics, resting hemodynamics, and basal ET-1 levels did not differ from those for included subjects, we do not believe that the results had a selection bias. Finally, pharmacologic studies involving blockade of ET receptors and/or nitric oxide generation are required to ascertain what roles they play in BP reactivity to stress. Nevertheless, given that plasma ET-1 levels have been reported to reflect the extent of atherosclerosis in patients with a family history of EH (27), the observed sex and ethnicity differences in ET-1 release in apparently healthy individuals may be important in explaining the ethnicity and sex differences in the prevalence of cardiovascular disease.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Received for publication March 26, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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