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J Appl Physiol 98: 787-794, 2005; doi:10.1152/japplphysiol.00503.2004
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Gly16 + Glu27 {beta}2-adrenoceptor polymorphisms cause increased forearm blood flow responses to mental stress and handgrip in humans

Ivani C. Trombetta,1 Luciana T. Batalha,2 Maria U. P. B. Rondon,1 Mateus C. Laterza,1 Eliana Frazzatto,2 Maria J. N. N. Alves,1 Amilton C. Santos,1 Patrícia C. Brum,3 Antonio C. P. Barretto,1 Alfredo Halpern,2 Sandra M. F. Villares,2 and Carlos E. Negrão1,3

1Heart Institute (InCor), Medical School, 2Department of Endocrinology, and 3School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil

Submitted 17 May 2004 ; accepted in final form 15 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We hypothesized that the muscle vasodilatation during mental stress and exercise would vary among humans who are polymorphic at alleles 16 and 27 of the {beta}2-adrenoceptors. From 216 preselected volunteers, we studied 64 healthy, middle-aged normotensive women selected to represent three genotypes: homozygous for the alleles Arg16 and Gln27 (Arg16/Gln27, n = 34), Gly16 and Gln27 (Gly16/Gln27, n = 20), and Gly16 and Glu27 (Gly16/Glu27, n = 10). Forearm blood flow (plethysmography) and muscle sympathetic nerve activity (microneurography) were recorded during 3-min Stroop color-word test and 3-min handgrip isometric exercise (30% maximal voluntary contraction). Baseline muscle sympathetic nerve activity, forearm vascular conductance, mean blood pressure, and heart rate were not different among groups. During mental stress, the peak forearm vascular conductance responses were greater in Gly16/Glu27 group than in Gly16/Gln27 and Arg16/Gln27 groups (1.79 ± 0.66 vs. 0.70 ± 0.11 and 0.58 ± 0.12 units, P = 0.03). Similar results were found during exercise (0.80 ± 0.25 vs. 0.28 ± 0.08 and 0.31 ± 0.08 units, P = 0.02). Further analysis in a subset of subjects showed that brachial intra-arterial propranolol infusion abolished the difference in vasodilatory response between Gly16/Glu27 (n = 6) and Arg16/Gln27 (n = 7) groups during mental stress (0.33 ± 0.20 vs. 0.46 ± 0.21 units, P = 0.50) and exercise (0.08 ± 0.06 vs. 0.03 ± 0.03 units, P = 0.21). Plasma epinephrine concentration in Arg16/Gln27 and Gly16/Glu27 groups was similar. In conclusion, women who are homozygous for Gly16/Glu27 of the {beta}2-adrenoceptors have augmented muscle vasodilatory responsiveness to mental stress and exercise.

exercise;; muscle vasodilatation

BECAUSE THE NATURALLY OCCURRING polymorphism of the NH2 terminus of {beta}2-adrenoceptors caused by mutation encoding for amino acid position 16 (Gly for Arg) and position 27 (Glu for Gln) have been described (24), much interest on their functionality and vascular modulation has been addressed. {beta}2-Adrenoceptor polymorphism seems to play an important role in determining vascular reactivity in normotensive subjects (2, 13, 16). In humans with Gly16/Gly16 {beta}2-adrenoceptors, the increase in cardiac output and the decrease in systemic vascular resistance during salbutamol administration are attenuated (13). The increase in calf blood flow during systemic infusion of terbutaline is smaller in Gly16 homozygote than Arg16 homozygote (16). Moreover, forearm vasodilatory response to intra-arterial infusion of isoproterenol is greater in subjects who are homozygous for Glu27 of the {beta}2-adrenoceptor than in subjects who are homozygous for Gln27 of the {beta}2-adrenoceptor (2).

Although the clinical relevance of the vascular responsiveness to exogenous {beta}2-agonist administration in humans who are polymorphic at alleles 16 and 27 of the {beta}2-adrenoceptors are unquestionable, the actual role of these polymorphisms during physiological maneuvers remains poorly understood. Because muscle vasodilatation during some physiological maneuvers depends, in great part, on endogenously mediated {beta}2-adrenoceptor stimulation, it would be legitimate to expect that humans who are polymorphic at alleles 16 and 27 of the {beta}2-adrenoceptors would have altered blood vessel responsiveness during mental stress and exercise.

In the present investigation, we report the muscular vasodilatory response during mental stress and exercise in humans who were homozygous for Arg16 and Gln27 (Arg16/Gln27), Gly16 and Gln27 (Gly16/Gln27), and Gly16 and Glu27 (Gly16/Glu27) of the {beta}2-adrenoceptors. The choice for these genotypes was based on the importance of haplotypes of the {beta}2-adrenoceptor during exogenous stimuli (5) and on the marked linkage disequilibrium between the polymorphism at alleles 16 and 27 of the {beta}2-adrenoceptors in humans (4, 5). Almost all subjects who are homozygous for Glu27 are also homozygous for Gly16, whereas subjects who are homozygous for Gly16 can be homozygous for Gln27 or homozygous for Glu27 (5).

We tested the hypothesis that muscle vasodilatory response during mental stress and exercise would be increased in young adult women who were homozygous for Gly16/Glu27 of the {beta}2-adrenoceptors compared with age-matched women who were homozygous for Arg16/Gln27 and Gly16/Gln27 of the {beta}2-adrenoceptors. To rule out the possibility that the increased forearm vasodilatation in Gly16/Glu27 women was a consequence of lowered sympathetic nerve activation and/or augmented levels of endogenous {beta}2-adrenoceptor agonist, muscle sympathetic nerve activity (MSNA) and plasma catecholamines were evaluated during mental stress and exercise. Finally, to confirm that the muscle vasodilatation was due to the polymorphism in the {beta}2-adrenoceptors, we studied the forearm vasodilatation during mental stress and exercise after regional {beta}-blockade.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects

Two hundred and sixteen preselected healthy female volunteers from the Women Endocrinology Ambulatory of University of São Paulo were genotyped for the Arg16, Gly16, Gln27, and Glu27 {beta}2-adrenoceptor alleles. These alleles were identified by single-strand conformation polymorphism analysis with the use of PCR, as previously described (19). PCR amplification was performed on an automated apparatus (Perkin-Elmer). Because they were heterozygous for alleles 16 or 27, 152 subjects were excluded from the study. Sixty-four subjects who were homozygous for alleles 16 and 27 were involved in the study. Thirty-four were homozygous for Arg16/Gln27, 20 for Gly16/Gln27, and 10 for Gly16/Glu27. Subjects who were homozygous for Arg16 and Glu27 (Arg16/Glu27) were not found. To avoid gender, age, and disease confounding in our study, the eligibility for taking part in the study was determined by gender, healthy status, tobacco consumption, and age. The subjects were screened for cardiovascular, endocrine, and metabolic disorders. They had no apparent cardiovascular disease. Although they were obese, their total cholesterol, LDL cholesterol, HDL cholesterol levels, and glucose levels were close to the normal range. They were women from different ethnic backgrounds, healthy, normotensive, and nonsmokers, whose ages ranged between 20 and 40 yr. There was no significant difference among groups in age (Arg16/Gln27 = 30 ± 1, Gly16/Gln27 = 34 ± 1, and Gly16/Glu27 = 30 ± 1 yr old, P = 0.08) and body mass index (Arg16/Gln27 = 34 ± 0.4, Gly16/Gln27 = 34 ± 0.5, and Gly16/Glu27 = 34 ± 0.6, P = 0.58). Clinical examination, laboratory testing, and cardiopulmonary exercise test determined their health status. All women were studied between the 1st and the 5th day after the onset of menstruation. The Human Subject Protection Committees of the Heart Institute (InCor) and Clinical Hospital, University of São Paulo, Medical School, approved the study protocol. The subjects took no medication 3 mo before the study and abstained from caffeine and alcohol 1 day before the study.

Measurements and Procedures

Forearm blood flow.   Forearm blood flow was measured by venous occlusion plethysmography (21). The nondominant arm was elevated above heart level to ensure adequate venous drainage. A mercury-filled Silastic tube attached to a low-pressure transducer was placed around the forearm and connected to a plethysmograph (Hokanson, Bellevue, WA). Sphygmomanometer cuffs were placed around the wrist and upper arm. At 15-s intervals, the upper cuff was inflated above venous pressure for 7–8 s. Forearm blood flow (ml·min–1·100 ml tissue–1) was determined on the basis of a minimum of four separate readings. Forearm vascular conductance was calculated by dividing forearm blood flow by mean arterial pressure. The reproducibility of forearm blood flow measured at different time intervals in the same individual expressed as milliliters per minute per 100 milliliters tissue in our laboratory is r = 0.93.

MSNA.   Resting MSNA was recorded directly from the peroneal nerve using the technique of microneurography (10). Multiunit postganglionic muscle sympathetic nerve recordings were made using a tungsten microelectrode. Signals were amplified by a factor of 50,000–100,000 and band-passed filtered (700–2,000 Hz). Nerve activity was rectified and integrated (time constant, 0.1 s) to obtain a mean voltage display of sympathetic nerve activity that was recorded on paper. All recordings of MSNA met previously established and described criteria. Muscle sympathetic bursts were identified by visual inspection by a single investigator (C. E. Negrão) blinded to the study protocol and were expressed as burst frequency (bursts per minute). The reproducibility of MSNA measured at different time intervals in the same individual expressed as bursts per minute is r = 0.88 and expressed as bursts/100 heartbeats is r = 0.91 (10). In the present study, we obtained accurate and reliable MSNA recordings in 28 Arg16/Gln27, 18 Gly16/Gln27, and 9 Gly16/Glu27 subjects.

Blood pressure and heart rate.   During mental stress, blood pressure was monitored noninvasively by a finger photoplethysmography device (FinaPress2300; Ohmeda, Englewood, CO) on a beat-to-beat basis (AT/CODAS) at a frequency of 500 Hz (18). During handgrip exercise, blood pressure was monitored noninvasively and intermittently from an automatic and oscillometric cuff (Dixtal, DX 2710; Brazil, Manaus) placed on the ankle with cuff width adjusted to ankle circumference (22, 28). The cuff inflated every 30 s. When a catheter was placed into the brachial artery, blood pressure was monitored directly on a beat-to-beat basis (AT/CODAS) at a frequency of 500 Hz (21). Heart rate was monitored continuously through lead II of ECG.

Insulin resistance.   Plasmatic insulin levels were determined by radioimmunoassay. Insulin resistance was estimated by homeostasis model assessment score and calculated with the following formula: fasting serum insulin (µU/ml) x fasting plasma glucose (mM)/22.5 (18).

Mental stress testing.   Mental stress was elicited by the Stroop color-word test (26). During the Stroop color-word test, subjects were shown a series of names of colors written in a different color ink from the color specified. The subjects were asked to identify the color of the ink, not read the word.

Handgrip exercise.   After the maximal voluntary contraction (MVC; average of three trials) was obtained, handgrip isometric exercise was performed at 30% of MVC with the dominant arm using a handgrip dynamometer. The individuals were instructed to breathe normally during exercise and to avoid inadvertent performance of a Valsalva maneuver.

Catecholamine measurements.   Plasma epinephrine and norepinephrine concentrations were determined by high-pressure liquid chromatography (1, 3).

Propranolol intra-arterial infusion.   Propranolol (0.5 mg/min) was infused into the brachial artery throughout mental stress and handgrip exercise to block the local {beta}2-adrenoceptors (8, 23).

Experimental Protocol

Protocol 1.   The subjects who were involved in this protocol are shown in Table 1. All studies were performed in a quiet temperature-controlled (21°C) room at approximately the same time of day. The arm was positioned for venous plethysmography, and the leg was positioned for microneurography. After an adequate nerve recorded site was obtained, the subject rested quietly for 15 min. Baseline MSNA, blood pressure, forearm blood flow, and heart rate were then recorded for 3 min. Acute mental stress testing was then performed for 3 min (Fig. 1). MSNA, forearm blood flow, blood pressure, and heart rate were recorded continuously during mental stress. The task difficulty was determined on completion of the protocol, using a standard five-point scale: 0, not stressful; 1, somewhat stressful; 2, stressful; 3, very stressful; and 4, very very stressful.


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  		Table 1. Number of subjects enrolled in each experimental protocol

 


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  		Fig. 1. Timeline of experimental protocols (see Experimental Protocol for explanation). MSNA, muscle sympathetic nerve activity; FBF, forearm blood flow; BP, blood pressure; HR, heart rate.

 
Protocol 2.   The subjects who were involved in this protocol are shown in Table 1. After a 15-min rest period, a new baseline MSNA, forearm blood flow, blood pressure, and heart rate were recorded for 3 min. Handgrip isometric exercise was then performed for 3 min (Fig. 1). MSNA and forearm blood flow rate were recorded continuously during handgrip exercise. Blood pressure was monitored noninvasively and intermittently from an automatic and oscillometric cuff placed on the ankle with cuff width adjusted to ankle circumference (22, 28). The cuff inflated every 30 s.

Protocol 3.   The subjects who were involved in this protocol are shown in Table 1. The purpose of this study was to confirm the effect of {beta}2-adrenoceptor polymorphisms on forearm blood flow response. We reasoned that, if any of the studied polymorphisms evoked increased forearm vasodilatation and this response was, in fact, due to polymorphism of the {beta}2-adrenoceptors, brachial intra-arterial infusion of propranolol would abolish the increased forearm vasodilatation. A subset of seven women who were homozygous for the alleles Arg16/Gln27 and a subset of six women who were homozygous for the alleles Gly16/Glu27 were enrolled in this protocol. In this last subset, which was initially composed of 10 women, 3 of them did not want to participate in the study, and we were not able to get the brachial artery of one of them. The subject was positioned, and electrocardiogram leads were placed on the chest. An intra-arterial line was placed into the brachial artery of the nondominant arm. The catheter was attached to a three-way stopcock for ease of intra-arterial drug infusion. Cuffs for forearm blood flow measurements were placed on the nondominant arm. The timeline of the experimental protocols is shown in Fig. 1. After a 15-min rest period, saline was infused (0.5 ml/min) for 5 min, and baseline values for forearm blood flow, blood pressure, and heart rate were recorded for 3 min. Mental stress was then carried out for 3 min. After a 15-min rest period, when all parameters returned to baseline, saline was again infused (0.5 ml/min) for 2.5 min, and baseline values for forearm blood flow, blood pressure, and heart rate were recorded for 3 min. Three minutes of handgrip exercise (30% MVC) were then begun. After a 15-min rest period, when all parameters returned to baseline, intra-arterial infusion of propranolol (0.5 mg/min) was performed for 5 min. Baseline forearm blood flow, arterial pressure, and heart rate were recorded for 3 min. A mental stress test was then carried out for 3 min under propranolol administration. After a 15-min rest period, when all parameters returned to baseline values, an additional intra-arterial infusion of propranolol (0.5 mg/min) was performed for 2.5 min, and a new baseline forearm blood flow, arterial pressure, and heart rate were recorded for 3 min. Handgrip exercise was then performed for 3 min under propranolol administration. Arterial blood withdrawal was performed at baseline and at the peak of mental stress and handgrip exercise for verifying plasma catecholamine concentration in all groups studied.

Statistical Analysis

Data are presented as means ± SE. Data of both demographic characteristics and baseline measurements were subjected to one-way ANOVA. The responses of MSNA, forearm blood flow, forearm vascular conductance, mean blood pressure, and heart rate are presented as the absolute change and were subjected to two-way ANOVA with repeated measures. When significance was found, Scheffé's post hoc comparison was performed. The responses of plasma epinephrine and norepinephrine levels between groups, presented as the absolute change, were subjected to unpaired t-test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline Measurements

Baseline metabolic, hemodynamic, and neurovascular measurements in all three groups studied are shown in Table 2. Total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and glucose levels were similar in Gly16/Glu27, Arg16/Gln27, and Gly16/Gln27 groups. Insulin resistance by homeostasis model assessment was also similar among groups. There were no significant differences in MSNA, mean blood pressure, heart rate, forearm blood flow, and forearm vascular conductance among groups.


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  		Table 2. Baseline measurements

 
Responses to Mental Stress

During mental stress, MSNA, mean blood pressure, and heart rate increased similarly in all three groups studied (Table 3). As expected, forearm blood flow increased during mental stress. However, the response in forearm blood flow was significantly greater in the Gly16/Glu27 group compared with Arg16/Gln27 and Gly16/Gln27 groups (Fig. 2A). There was no significant difference in forearm blood flow between Arg16/Gln27 and Gly16/Gln27 groups. Mental stress significantly increased forearm vascular conductance in all three groups studied, but the magnitude of this increase was significantly greater in the Gly16/Glu27 group than in the Arg16/Gln27 and Gly16/Gln27 groups (Fig. 2B). There was no significant difference in forearm vascular conductance between the Arg16/Gln27 group and Gly16/Gln27 group.


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  		Table 3. Baseline and absolute changes in muscle sympathetic nerve activity, mean blood pressure, and heart rate during mental stress and moderate exercise at 30% of maximal voluntary contraction in women encoding Arg16/Gln27, Gly16/Gln27, and Gly16/Glu27

 


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  		Fig. 2. FBF (A) and forearm vascular conductance (FVC; B) responses (absolute change) during mental stress in women who are homozygous for Arg16/Gln27, Gly16/Gln27, and Gly16/Glu27 of the {beta}2-adrenoceptors. Values are means ± SE. *Significant difference, Gly16/Glu27 vs. Arg16/Gln27 and Gly16/Gln27, P < 0.05. {dagger}Significant difference, Gly16/Glu27 vs. Gly16/Gln27, P < 0.05.

 
Responses to Exercise

During handgrip exercise, MSNA, mean blood pressure, and heart rate increased progressivly and similarly in all three groups studied (Table 3). Forearm blood flow increased throughout the exercise period in all three groups. However, the increase in forearm blood flow was significantly greater in the Gly16/Glu27 group than in the Arg16/Gln27 and Gly16/Gln27 groups (Fig. 3A). No significant difference was found between these last two groups. The increase in forearm vascular conductance was significantly greater in the Gly16/Glu27 group than in the Arg16/Gln27 and Gly16/Gln27 groups (Fig. 3B). Forearm vascular conductance was not different between the Arg16/Gln27 group and Gly16/Gln27 group.



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  		Fig. 3. FBF (A) and FVC (B) responses (absolute change) during 30% of maximal voluntary contraction (MVC) handgrip exercise in women who are homozygous for Arg16/Gln27, Gly16/Gln27, and Gly16/Glu27 of the {beta}2-adrenoceptors. Values are means ± SE. *Significant difference, Gly16/Glu27 vs. Arg16/Gln27 and Gly16/Gln27, P < 0.05.

 
Response to Mental Stress and Exercise Under {beta}2-Adrenoceptors Blockade

During mental stress with intra-arterial saline control infusion, the responses in forearm blood flow (Fig. 4A) and forearm vascular conductance (Fig. 4C) were significantly greater in the Gly16/Glu27 group compared with Arg16/Gln27 group. In contrast, both the responses in forearm blood flow (Fig. 4B) and forearm vascular conductance (Fig. 4D) with intra-arterial infusion of propranolol decreased substantially in the Gly16/Glu27 group. Moreover, the difference between the Gly16/Glu27 group and Arg16/Gln27 group was no longer observed. The same results were obtained during handgrip exercise. The responses in forearm blood flow (Fig. 5A) and forearm vascular conductance (Fig. 5C) with intra-arterial infusion of saline were significantly greater in the Gly16/Glu27 group compared with the Arg16/Gln27 group. However, the responses in forearm blood flow (Fig. 5B) and forearm vascular conductance (Fig. 5D) with intra-arterial infusion of propranolol decreased dramatically in the Gly16/Glu27 group, and the differences between groups were no longer observed.



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  		Fig. 4. FBF (A and B) and FVC responses (absolute change) (C and D) during mental stress with brachial intra-arterial infusion of saline control (A and C) and propranolol (B and D) in women who are homozygous for Arg16/Gln27 and Gly16/Glu27 of the {beta}2-adrenoceptors. Values are means ± SE. *Significant difference, Gly16/Glu27 vs. Arg16/Gln27, P ≤ 0.05.

 


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  		Fig. 5. FBF (A and B) and FVC responses (absolute change) (C and D) during handgrip exercise at 30% of MVC with brachial intra-arterial infusion of saline control (A and C) and propranolol (B and D) in women who are homozygous for Arg16/Gln27 and Gly16/Glu27 of the {beta}2-adrenoceptors. Values are means ± SE. *Significant difference, Gly16/Glu27 vs. Arg16/Gln27 and Gly16/Gln27, P ≤ 0.05.

 
Plasma Catecholamine Levels During Mental Stress and Exercise

The magnitude of the increase in plasma epinephrine concentrations at the peak of mental stress was similar in the Arg16/Gln27 and Gly16/Glu27 groups (8.33 ± 0.29 vs. 7.00 ± 0.26 pg/ml, 18.3 vs. 15.5%, respectively, P = 0.29). Similarly, no significant difference was observed in the magnitude of the increase in plasma epinephrine concentrations at the peak of handgrip exercise in the Arg16/Gln27 and Gly16/Glu27 groups (4.17 ± 0.83 and 4.17 ± 0.17 pg/ml, 8.7 vs. 10.2%, respectively, P = 1.00). The magnitude of the increase in plasma norepinephrine concentrations at the peak of mental stress (9.00 ± 9.94 vs. 17.83 ± 7.08 pg/ml, 19.7 vs. 35.3%, respectively, P = 0.50) and handgrip exercise (23.00 ± 5.32 vs. 29.43 ± 8.66 pg/ml, 36 vs. 35.1%, respectively, P = 0.56) was not significantly different in the Arg16/Gln27 and Gly16/Gln27 groups.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The novelty of the present study is the augmented reflex muscle vasodilatory response during mental stress and handgrip exercise in women who are homozygous for Gly16/Glu27 of the {beta}2-adrenoceptors.

The modulation of {beta}2-adrenoceptor polymorphisms on systemic and regional vascular response to exogenous {beta}2-adrenergic agonists has been previously reported (2, 13). The present study extends our knowledge of the relevance of {beta}2-adrenoceptor polymorphism during physiological maneuvers in humans. In addition, our results suggest that it is the allele Glu27 that augments forearm vasodilatation during mental stress and exercise. Forearm blood flow is lower in women who are homozygous for Gln27 than in women who are homozygous for Glu27, regardless of the mutation encoding for amino acids at position 16.

It would be legitimate to argue that the increased vasodilatory response in Gly16/Glu27 women was due to an attenuation of vasoconstriction {alpha}1-noradrenergic stimulation rather than a mutation in the {beta}2-adrenoceptor. This seems to be unlikely because both the responses of MSNA and the plasma norepinephrine concentrations during mental stress and handgrip exercise were similar in the Gly16/Glu27 and Arg16/Gln27 groups, although these responses do not rule out the chance of a difference in {alpha}-receptor responsiveness among groups. The most striking evidence in favor of {beta}2-adrenoceptor mutation was the fact that the brachial intra-arterial infusion with propranolol during mental stress and handgrip exercise dramatically reduced forearm vasodilatory response in Gly16/Glu27 women. Moreover, propranolol abolished the difference in forearm vasodilatory response between Gly16/Glu27 women and Arg16/Gln27 women.

The plasma epinephrine concentrations at the peak of mental stress and handgrip exercise were virtually the same in women who were homozygous for Gly16/Glu27 and Arg16/Gln27 (wild type). These findings rule out the possibility that the increased forearm vasodilatation in Gly16/Glu27 women was a consequence of the augmented levels of endogenous plasma {beta}2-adrenoceptor agonist.

We are confident that the increase in muscle blood flow in Gly16/Glu27 women is locally blood vessel mediated rather than determined by other possible hemodynamic adjustments. The heart rate response, which is the main component of the increase in cardiac output during mental stress and isometric exercise in humans (11), was similar among all three groups studied. In addition, the multivariate analysis showed no significant association between heart rate response and forearm blood flow response in Gly16/Glu27 women during mental stress and exercise (r = 0.28, P = 0.58; and r = 0.44, P = 0.64, respectively).

The substitutions of Gly16 for Arg16 provoked no change in vasodilatory response. This could be viewed as an unexpected result, because previous studies have demonstrated that, in human airway smooth muscle cells (14), Gly16 is associated with an increase in agonist-induced downregulation of the {beta}2-adrenoceptor. Also, more recently, substitution of Gly16 for Arg16 increases forearm vasodilatation during brachial artery infusion of the {beta}-agonist isoproterenol, which appears to be dependent on differences in endothelial generation of nitric oxide (2, 12). The increased forearm vasodilatation in Gly16/Gly16 was no longer observed after intra-arterial blockade with NG-monomethyl-L-arginine (12). These apparently paradoxical results may be attributed to the nature of {beta}-agonist stimulation. Inasmuch as they involve two different experimental conditions, a direct comparison between a vasodilatation elicited by exogenous {beta}-agonist with a vasodilatation provoked by physiological maneuvers may be difficult to achieve.

Despite the fact that previous studies have linked the substitutions of Gly for Arg of {beta}2-adrenoceptor at the allele 16 to an increase (17, 27) or a decrease (20) in blood pressure levels in humans, our findings show no difference in resting blood pressure levels between women homozygous for Gly16 and women homozygous for Arg16, as also found in a recent study (7). Moreover, sympathetic nerve activity, which is related to blood pressure levels in young patients, was not different between Gly16 and Arg16 polymorphisms presently studied.

Our results show no apparent association of Gly16 and Arg16 polymorphisms with blood pressure and heart rate responses to mental stress and isometric handgrip exercise. Eisenach and collaborators (7) found similar results in blood pressure during mental stress and cold pressor test, although the heart rate response was greater in Gly16 homozygote during exercise. One possible explanation for the difference in heart rate response between these two studies is the exercise protocol. In the Eisenach study, the subjects were exercised up to exhaustion, whereas, in the present study, the exercise protocol was limited to 3 min. The cardiovascular responses are matched to exercise duration and/or intensity (25). Thus the increased heart rate during prolonged exercise in Gly16 homozygote, in contrast to short-term exercise, may serve to maintain the pressor response in the face of intense vasodilatation.

Twelve distinct haplotypes of the {beta}2-adrenoceptor have been recently described in humans represented by the four major ethnic groups (6), with divergent agonist efficacies and magnitude of responses in vivo (6, 9). In our study, we investigated three haplotypes. Thus it is possible that other interactions of multiple single-nucleotide polymorphisms within a haplotype also affect the vasodilatory response during physiological maneuvers in humans.

The augmented forearm blood flow in women who are homozygous for Gly16/Glu27 could be attributed to a greater level of perceived stress during the color-word test. This seems to be unlikely, because the perception of stress during the color-word test was similar in Gly16/Glu27, Arg16/Gln27, and Gly16/Gln27 groups. In addition, the response in MSNA, which is also closely related to task difficulty, was similar among groups.

Because our sampling included only women, gender was not an intervening factor. However, we recognize that this strategy may limit the generalization of our findings.

Perspectives

The present findings bring about some relevant questions. Are the homozygous subjects for Gly16/Glu27 of the {beta}2-adrenoceptors less susceptible for future cardiovascular disease? Because these individuals have greater vasodilatory response during physiological maneuvers, it is reasonable to think that they will be more protected against cardiovascular disorders. Heckbert et al. (15) have recently reported that the Glu27 allele of the {beta}2-adrenoceptor was associated with lower risk of incident coronary events in elderly subjects. If confirmed in other populations, these findings demonstrate that the genetic variation in the {beta}2-adrenoceptor may affect the risk of cardiovascular events during the life span. Another obvious and important question would be whether Gly16/Glu27 polymorphism of the {beta}2-adrenoceptor predicts increased functional capacity in humans. It is well described that muscle energy generation depends, in part, on muscle blood flow. Because subjects who are homozygous for Glu27 have greater vasodilatory response during physiological maneuver, someone could expect that they would have augmented aerobic capacity, which facilitates exercise training adaptations.

In conclusion, the augmented muscle vasodilatory responsiveness to mental stress and exercise in women who are homozygous for Gly16/Glu27 provides evidence for the understanding of the genetic regulation of vascular responses mediated by {beta}2-adrenoceptors during physiological maneuvers in humans.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (no. 1998/15983–8) for funding the present investigation. We also express our gratitude to Fundação Zerbini for support in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. E. Negrão, Instituto do Coração (InCor), Unidade de Reabilitação Cardiovascular e Fisiologia do Exercício, Av. Dr. Enéas de Carvalho Aguiar, 44, Cerqueira César, São Paulo, SP, CEP 05403–000, Brazil (E-mail: cndnegrao{at}incor.usp.br)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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