Aging has a profound effect on autonomic cardiovascular control. Cardiac parasympathetic tone is reduced1,2 while sympathetic activity is increased as demonstrated by increases in resting plasma norepinephrine,3-5 whole body norepinephrine spillover, cardiac norepinephrine spillover,3,6,7 and peripheral muscle sympathetic nerve activity.6,8,9 This increase in sympathetic activity with aging may lead to a decrease in beta-receptor number (i.e., downregulation)10,11 and thereby to decreased responses to beta-adrenergic stimulation, a mechanism well described in congestive heart failure12-14 and hypertension.15 Abundant evidence has shown a reduction in the response to beta-adrenergic stimulation with aging, as manifested by a reduced heart rate, blood pressure, ejection fraction, and cardiac output response to both isoproterenol and exercise in older subjects.16 An age-associated reduction in the response to alpha-stimulation analogous to that seen with beta-stimulation also been suggested based on human in vivo studies of forearm blood flow17 and human in vitro studies.2,18
An agent that reduces sympathetic tone would be predicted to increase alpha and beta-receptor responsiveness by the upregulation of both alpha and beta receptor numbers. Clonidine acts in the central nervous system on postsynaptic alpha-2 receptors to inhibit alpha and beta adrenergic outflow from the brainstem.
In the current study, we used the reduction in sympathetic tone induced by clonidine to examine the effect of normal aging on the upregulation of the alpha (αAR) and beta-receptor (βAR) responses. The hypothesis of this study was that reduction of sympathetic tone by clonidine administration would lead simultaneously to increased alpha-adrenergic responsiveness to phenylephrine (a selective alpha-1 receptor agonist) and to increased beta-adrenergic responsiveness to isoproterenol (a beta-1 and beta-2 receptor agonist). Due to the greater sympathetic activity in older subjects at baseline, we hypothesized that reduced sympathetic activity with clonidine would result in a greater improvement in both alpha and beta-adrenergic responsiveness in older adults than in young subjects.
The subjects studied consisted of 12 young healthy adult (aged 22 to 33 years; 12 males) and 18 older healthy adult (aged 63 to 80 years; 11 males) subjects. Subjects were excluded if they had any history of angina, myocardial infarction, stroke, hypertension, chronic pulmonary disease, diabetes, current medication use (prescription or over the counter), or current smoking. Entry requirements included a normal blood pressure, a normal physical exam, normal resting electrocardiogram, normal M-mode and two-dimensional echocardiograms showing no more than mild valvular regurgitation, a normal Bruce protocol treadmill maximal exercise stress test, and normal hematocrit, fasting blood glucose, total cholesterol, and creatinine. Two female subjects were excluded on the basis of this screening and one older female subject did not receive any infusion secondary to problems with line placement, leaving a total of 12 young and 15 older subjects participating in the study.
This study was conducted at the Seattle Veterans Affairs Medical Center and was approved by the Human Subjects Committee. All subjects gave informed consent.
Subjects were studied before and after using a clonidine patch (TTS-2) for 2 weeks; no adverse effects were experienced. All subjects were in the fasted state during each experimental session (performed supine between 10 AM and 12 PM). Intravenous catheters were inserted into a right hand vein and a right antecubital vein of each subject. Subjects rested supine for 30 minutes before collection of baseline data. All repeat studies were performed with the clonidine patch still in place. After the collection of baseline data, an intravenous infusion of isoproterenol was then followed by a phenylephrine infusion using a Medfusion 2010 infusion pump. For safety reasons, isoproterenol was initially started at 7 ng/kg/min before being increased to the goal infusion dose of 35 ng/kg/min (each infusion dose lasted 14 minutes). There was a 30-minute washout period after which phenylephrine infusion was started. Phenylephrine was initially started at 0.5 μg/kg/min (again for safety reasons) before being increased to the goal infusion dose of 1.0 μg/kg/min (each infusion dose lasted 10 minutes). Steady-state levels of isoproterenol and phenylephrine have been demonstrated at these durations of infusion.19 Phenylephrine is a selective alpha-1 receptor agonist, which causes peripheral vasoconstriction resulting in an increase in blood pressure and systemic vascular resistance. Isoproterenol, a beta-receptor agonist, acts primarily to increase heart rate, cardiac contractility, and vasodilation. Infusions were halted if the subject's blood pressure exceeded 200/100 mm Hg (which occurred in only one older subject who did not receive the 1.0 μg/kg/min phenylephrine dose). Each individual subject received the same doses of phenylephrine and isoproterenol before and after the 2 weeks of clonidine. No complications occurred, and all younger subjects received all doses. We chose doses of isoproterenol19 that were not expected to elevate heart rate beyond 80% of maximum, for safety reasons.
Data Collection and Processing
Cardiac blood pool images were acquired at rest for 15 million counts and for the last 3 minutes of the goal infusion dose (35 ng/kg/min of isoproterenol or 1.0 μg/kg/min phenylephrine). Mean heart rate (three-lead electrocardiogram) and blood pressure (Ohmeda 2300 Finapres) were recorded for 2 minutes both at rest and during the last 2 minutes of the goal infusion dose. Blood was obtained at the time of intravenous catheter placement and labeled with 20-30 mCi of 99mTc as previously described20 for radionuclide angiography. A high-sensitivity parallel hole collimator and a General Electric 300 small-field-of-view camera interfaced to a Microdelta imaging terminal were used to obtain images in the left anterior oblique projection, which offered the best septal definition. Radionuclide images were acquired in 20-msec frames by forward and backward reconstruction with ±20% arrhythmia rejection; a single beat was dropped after each rejected beat.20 Previously described methods20 were used to calculate ejection fraction (EF), end-diastolic volume index (EDVI), and end-systolic volume index (ESVI). During the acquisition, cardiac index (CI) was obtained by multiplying the stroke volume index (SVI) times the mean heart rate (HR). The interpretation of all nuclear studies was done in a blinded fashion.
Plasma concentrations of norepinephrine (NE) were measured in a blinded fashion using high-performance liquid chromatography as described previously.16 The drop in norepinephrine with two weeks of clonidine patch (DeltaNE) was calculated by subtracting the preclonidine NE level from the postclonidine NE level.
Derived Measurements: Beta-Adrenergic Responses
The βAR for heart rate, ejection fraction, cardiac index, and stroke volume index was assessed by the change in each variable from baseline with isoproterenol infusion. Upregulation in the beta-adrenergic response (UpBeta) was the βAR postclonidine minus the βAR preclonidine.
Derived Measurements: Alpha-Adrenergic Responses
The alpha-adrenergic responses (αAR) were assessed by the response to phenylephrine (PE) infusion of systolic blood pressure (SBP), mean arterial pressure, diastolic blood pressure (DBP), and systemic vascular resistance (SVR) from baseline. Upregulation in the alpha-adrenergic response (UpAlpha) was the αAR postclonidine minus the αAR preclonidine. Systemic vascular resistance (SVR) was defined as (mean arterial pressure × 80)/cardiac output.21
All data analysis was done in a blinded fashion. Results are expressed as the mean ± standard error. Paired t-tests (SPSS 11) were used to determine the effect of clonidine on baseline measures as well as changes in cardiovascular measures from baseline with isoproterenol and phenylephrine.22,23 Due to the small sample sizes, all comparisons between young and old groups with respect to adrenergic responses were done by nonparametric tests (SPSS 11). The Mann-Whitney U Test was used to determine the effects of age (young versus old) on the αAR (upregulation in HR, EF, CI, and SVI responses), the βAR (upregulation in the SBP, DBP, and SVR responses) and the drop in norepinephrine levels.24 A value of P ≤ 0.05 was considered significant.22-24
Study Group Characteristics and Baseline Resting Measurements
Older subjects were significantly shorter than young subjects (P < 0.05) but there was no significant difference in weight, body surface area, or body mass index (Table 1).
Effect of Clonidine on Baseline Resting Measurements
Two weeks of clonidine resulted in a significant drop in heart rate (young, −12 ± 2%, P = 0.001; older, −7 ± 3%, P = 0.02) and SBP (young, −6 ± 2%, P = 0.002; older, −6 ± 2%, P = 0.01) (Table 2). Clonidine also significantly lowered DBP (−8 ± 4%, P = 0.05) and cardiac index (−14 ± 4%, P = 0.01) in young subjects and there was a trend towards lower DBP in the older group (−7 ± 3%, P = 0.06). Clonidine resulted in a drop in plasma norepinephrine levels from 158 ± 17 to 119 ± 10 pg/mL in young subjects (P = 0.02) and from 248 ± 22 to 132 ± 13 in older subjects (P < 0.0001). The reduction in norepinephrine level with clonidine patch was significantly greater in the older group as compared to the younger group (−47 ± 3 versus −26 ± 6%, P = 0.001).
Upregulation of the Isoproterenol Response
Isoproterenol elevated heart rate, SBP, cardiac index, stroke volume index, and ejection fraction (Table 2) and decreased DBP, end-diastolic volume index, and end-systolic volume index in all subjects both before and after clonidine (Fig. 1; Table 2). After using the clonidine patch for 2 weeks, UpBeta was significantly higher in young subjects for heart rate (+10.7 ± 1.5 versus +4.6 ± 1.5 bpm; U = 21.00; P = 0.01). For every 10-pg/mL drop in norepinephrine level with clonidine, upregulation of the βAR for HR increased by 4.1 bpm in the younger subjects as compared to only 0.51 bpm in the older subjects. There was no significant difference in upregulation of the βAR (Young versus Old) for EF (+1.33 versus -0.46%; U = 47.00; P = 0.47), CI (+0.96 versus +0.08 L/min/m2; U = 41.00; P = 0.26), or SVI (+3.67 versus -0.18 mL/m; U = 48.00; P = 0.51).
Upregulation of the Phenylephrine Response
SBP, DBP, end-diastolic volume index, end-systolic volume index, stroke volume index, and SVR increased while heart rate, cardiac index, and ejection fraction decreased with phenylephrine before and after clonidine in both age groups (Fig. 2, Table 3). There was a significant upregulation of the αAR in both age groups for SBP, DBP, and SVR. There was no statistical difference in upregulation (Young versus Old) in the αAR for the SBP (+7.8 versus +14.4 mm Hg; U = 31.50; P = 0.31), DBP (+5.6 versus +3.4 mm Hg; U = 33.00; P = 0.40) or SVR (+431 dynes/sec.cm5; U = 35.00; P = 0.49) between the two groups (Fig. 2).
Sex differences in the phenylephrine and isoproterenol responses cannot be commented upon because there were too few female subjects. All results were unchanged when the analysis excluded female subjects.
With the reduced sympathetic activity induced by clonidine, this study found evidence of a lesser upregulation in the chronotropic beta-adrenergic response to isoproterenol in older subjects but preserved upregulation of the alpha-adrenergic response to phenylephrine. These results are consistent with impaired upregulation of beta-1 receptors but preserved upregulation of alpha-1 receptors in older adults.
Beta-adrenergic Resensitization: Effects of Normal Aging
Reduced sympathetic activity for 2 weeks resulted in an upregulation of the response of heart rate and cardiac index to intravenous isoproterenol in both young and older subjects. This is concordant with previous work done on young subjects.19,25 Although both the young and older groups had evidence of upregulation of responses, the degree of upregulation was significantly less in the older group. To our knowledge, this is the first demonstration that upregulation of the βAR may be impaired with normal aging.
All measures of sympathetic neuronal outflow have been shown to increase with aging, including resting plasma norepinephrine,3-5 whole body norepinephrine spillover, cardiac norepinephrine spillover,3,6,7 and peripheral muscle sympathetic nerve activity.6,8,9 This increase in sympathetic nervous system activity is felt to result in a peripheral densensitization of beta-adrenoceptor response, as demonstrated by a reduced cardiac reactivity to orthostasis, exercise, and mental challenge.16,26-29 Beta receptor densensitization involves several mechanisms, including agonist-induced dissociation of the receptor from the cell membrane, inactivation of intracellular messengers, decreased synthesis of receptor protein, and phosphorylation of the receptor.30-34 A combination of a reduction in reduced beta-1 receptor numbers10,11 and intracellular uncoupling of the receptor32 leads to a reduction in the beta-adrenergic response in the face of chronic sympathetic nervous system activity in the older subject.
When beta-agonist activity is removed or reduced, this results in a resensitization (upregulation) of the beta-adrenergic response, as shown by the increase in heart rate and cardiac index responses to beta-adrenergic stimulation with isoproterenol in the present study. Although less well studied than the desensitization (downregulation) process, current evidence indicates that beta-receptor resensitization is an active process involving receptor sequestration from the cell membrane.35,36 Once the receptor is internalized, uncoupled receptors are dephosphorylated and recycled to the cell surface,37,38 allowing resensitization. This is in comparison with the downregulation process during prolonged agonist exposure, where internalized beta-receptor traffic is sent to the degradation pathway.39 Once receptors are internalized, different residues in the tail of the receptors seem to be responsible for trafficking receptors back to the cell surface (resensitization) or to lysosomes for degradation (desensitization).35,40,41
The results of our study suggest that the ability to resensitize the beta-adrenergic response decreases with age, suggesting that normal aging results in a derangement of one of the processes that redirect beta-receptor traffic back to the cell surface. Resensitization of the heart rate response in older subjects was only 43% of the resensitization seen in younger subjects (10.7 versus 4.6 beats per minute, P = 0.011). This indicates that merely reducing sympathetic overactivity in older adults is not sufficient to correct the age-associated drop in the beta-adrenergic response with aging.
Alpha-adrenergic Resensitization: Effects of Normal Aging
Contrary to the beta-adrenergic results, age had no impact on resensitization of the alpha-response by central sympathetic downregulation. Previous work has shown an increased SBP response after clonidine in young perioperative subjects25,42 and increased forearm vasoconstriction with norepinephrine after an acute ganglionic blockade with guanadrel.17,43 These results are in agreement with the present study, although none of these studies examined the SVR response to phenylephrine or the effect of normal aging on the resensitization of the alpha-adrenergic response.
Little work has been done on the mechanisms underlying both desensitization and resensitzation of the alpha-adrenoceptors.35 Both resensitization and desensitization of alpha-1 receptors are felt to operate in a way analogous to that of beta-adrenoceptors, involving changes in receptor number at the cell surface10,11 and modifications to intracellular coupling of the alpha-1 receptor to the G protein due to receptor phosphorylation/dephosphorylation.35,40,41 The results of the present study demonstrate that the mechanisms underlying resensitization of the αAR remain intact in the older adult population.
Normal aging is known to diminish both the alpha-adrenergic2,17,18 and beta-adrenergic responses,16 presumably due to the increase in sympathetic activation with age.3-9 This manifests clinically as a reduced chronotropic and pressor response to mental stress, orthostasis, and exercise.16,26-29 The results of this study demonstrate that merely decreasing sympathetic overactivation in the older adult population will not be effective in restoring the beta-adrenergic response because the aging process itself also seems to impair the process of resensitizing cardiac beta-1 receptors. The results of the present study do not allow us to draw conclusions about the exact mechanism behind the age-associated impairment in the beta-1 resensitization process.
The number of subjects in this study was small, and it is possible there was an age-associated attenuation in αAR upregulation that was not detected due to low statistical power. However, this does not change the primary finding of an age-associated impairment in the beta-resensitization process. This study used radionuclide angiographic methods that are relatively insensitive measures of contractility when compared to more invasive measures. Such invasive measures are not ethically justifiable in a sample of normal healthy persons. The number of female subjects in this study was not large enough to determine sex differences in the effects of clonidine. The current study did not measure alpha and beta-receptor numbers, which would have been of great interest but beyond the scope of this trial.
Clonidine administration has shown a biphasic response in terms of blood pressure, demonstrating hypotension at low doses but a peripheral postsynaptic pressor response at higher doses during the initial stages of an intravenous or intrathecal clonidine bolus. This biphasic response has not been observed during low-dose 24-hour patch dosing.44 Despite the fact that all subjects received the same dose of clonidine (TTS-2) regardless of weight, we do not believe that this was a confounding factor in the current study because the maximum possible clonidine dose administered to both groups was not high enough to provoke a pressor response.44 This is supported by the fact that none of the subjects in the present study demonstrated a hypertensive response to clonidine (Table 2).
A reduction in sympathetic activity induced by clonidine led to an increase in both alpha- and beta-receptor responsiveness, compatible with an upregulation of receptor numbers in both age groups. The novel finding of the present study is that normal aging results in an impairment in the resensitization of the beta-adrenergic response, whereas alpha-adrenergic resensitization was preserved with age. This suggests that simple sympathetic downregulation is not sufficient to fully reverse the age-associated changes in the adrenergic response.
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