2-Adrenoceptor Genotype and Function Affect Hemodynamic Profile Heterogeneity in Postural Tachycardia Syndrome
the J. Recanati Autonomic Dysfunction Center (J.G.), Department of Internal Medicine A, Rambam Medical Center & Technion-IIT, Haifa, Israel
Autonomic Dysfunction Center (E.M.G., F.C., C.M.S., H.-G.X., R.M.R., I.B., D.R.), Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn.
Abstract
Previous studies suggest that the 2-adrenoceptor functions abnormally in patients with postural tachycardia syndrome (POTS) and may contribute to their altered hemodynamic profile. To test the hypothesis that the 2-adrenoceptor response is decreased in POTS, we studied: (1) the arterial vasodilation response to the agonist, isoproterenol, and (2) the distribution of common polymorphisms (codons 16 and 27) of the gene coding the receptor (2-AR) in a large population with POTS. We measured plasma catecholamines and monitored hemodynamics and changes in forearm and leg blood flow to incremental doses of intraarterial isoproterenol in 9 patients with POTS compared with 8 healthy subjects. For polymorphism assessment we collected DNA from 57 patients with POTS and compared with 67 age-sex matched healthy subjects. Circulating catecholamines were significantly higher in POTS subjects compared with controls. Intrabrachial and intrafemoral isoproterenol infusion elicited a dose-dependent increase in blood flow. In healthy subjects, blood flow increased (mean±SEM) 400±70% in the forearm and 170±40% in the leg, but only 280±60% in forearms and 120±20% in legs of patients with POTS (ANOVA for both P<0.001). The genotype and allele distributions for codons 16 and 27 2-AR variants were not different in the 2 groups. However, the blood pressure and plasma norepinephrine levels diverged in patients according to their genotype. Patients with Gly16Gly and patients with Glu27Glu had lower plasma catecholamines and higher supine and upright blood pressure, compared with other genotypes. Therefore, both decreased 2-adrenoceptor-related vasodilation and 2-AR polymorphisms may contribute to the hemodynamic diversity of patients with POTS.
Key Words: receptors adrenergic receptor agonists norepinephrine tachycardia polymorphism
Introduction
Postural tachycardia syndrome (POTS), is one of the most frequent forms of chronic orthostatic intolerance in the general population.1 It affects &500 000 Americans with a female to male ratio of 5:1, and most cases occur between the ages of 15 and 50 years.2 Clinically it is characterized by frequent orthostatic symptoms, such as dizziness, blurred vision, syncope and fatigue associated with blood pressure (BP) fluctuations, orthostatic tachycardia, and often high circulating plasma catecholamines. The remarkable increase in heart rate on standing without a significant drop in blood pressure is the hallmark of this syndrome.2 The etiology of POTS remains elusive. Different investigators have described various aspects of the pathophysiology that underlies the cardiovascular irregularities in these patients. For instance, baroreflex dysfunction,3 partial autonomic denervation (neuropathic POTS),4 hyporeninemic hypovolemia,5 antiganglionic nicotinic receptor antibodies,6 and norepinephrine transporter deficiency7 have been reported.
Conflicting data exist on cardiovascular adrenoceptor (-AR) sensitivity in patients with POTS. Although a normal or increased chronotropic response to 1-AR stimulation occurs in patients with POTS (despite the high circulating plasma catecholamines),8,9 no data are available on vascular 2-AR dilatory function. The coexistence of exaggerated blood pressure oscillations and absence of a significant drop in BP on standing in these patients could suggest that the vascular 2-AR is not functioning normally. The continued exposure of receptors to adrenergic agonists leads to receptor desensitization; therefore the presence of high circulating catecholamines in patients with POTS might result in less responsive vascular 2-AR.
There has been great interest in the potential role of 2-AR polymorphisms in diseases in which the 2-AR is thought to be involved, such as hypertension, asthma, and obesity. The common single nucleotide polymorphisms (SNPs) in 2-AR at amino acid positions 16 (Arg16Gly) and 27 (Gln27Glu) affect receptor function. During continuous infusion of isoproterenol, subjects homozygous for the Arg16 allele demonstrate marked agonist-mediated desensitization, whereas subjects homozygous for Gly16 do not show desensitization.10 The Arg16 and Glu27 variants have been linked with a greater vasodilator response to adrenergic agonists. However, the genetic variation of 2-AR has not previously been explored in POTS. We therefore investigated the role of the Arg16Gly and Gln27Glu 2-AR polymorphisms in POTS in a case-control association study.
We initially tested the hypothesis that the 2-AR vasodilatory response is decreased in patients with POTS as compared with age and sex-matched healthy subjects. Second, we proposed that functionally relevant 2-AR polymorphisms may contribute to susceptibility to POTS and to the magnitude of the variations in hemodynamic and catecholamine responses in patients.
Methods
Subjects
Patients with POTS were recruited from consecutive referrals to the Autonomic Dysfunction Center at Vanderbilt University Medical Center between 1996 and 2000.
The first study examined vascular dilatory function (2-AR sensitivity: isoproterenol study) and included 9 patients. Patients were enrolled if they met the following stringent criteria: (1) a history of daily typical symptoms (eg, dizziness, blurred vision, presyncope or frank syncope, fatigue, palpitations, chest discomfort, irritability, clamminess) for at least 6 months, (2) an increase in heart rate of at least 30 bpm within 5 minutes of standing, without a concomitant decrease in blood pressure greater than 20/10 mm Hg and (3) a plasma norepinephrine level of at least 600 pg/mL (3.18 nmol/L) with standing. This group was compared with 8 age- and sex-matched completely healthy subjects.
The second study determined the 2-AR SNP distribution (genetic study) in 57 consecutive patients with POTS (91% female and 98% white) who met the above criteria, unrelated to their plasma norepinephrine levels. Genotypes of this group were compared with those of 68 healthy volunteers (80% female and 87% white), recruited from the Nashville, Tenn, community, whose DNA was stored at our institution’s DNA bank. Female to male ratio and white to nonwhite ratio were not statistically different between the groups (P=0.14 and 0.24, respectively).
All subjects underwent a physical examination; ECG; hematologic, biochemical, and autonomic laboratory tests; and an interview using an autonomic questionnaire to determine the type and extent of clinical symptoms. Patients with systemic illnesses capable of affecting the autonomic nervous system function were excluded. The Vanderbilt Investigational Review Board approved all study procedures, and subjects gave written informed consent.
Experimental Design
All subjects undergoing 2-AR sensitivity testing and the patients who participated in the genotyping portion of the study were admitted and studied after consuming a restricted diet (150 mEq Na+ and 70 mEq K+ per day, free of caffeine and low in monoamines) for at least 3 days. Medications were discontinued at least 2 weeks before admission, and smoking was not permitted during the study. All of these subjects underwent measurements of blood pressure (cuff sphygmomanometer), heart rate, and venous plasma norepinephrine and epinephrine during supine and upright posture. Blood for catecholamine assays was collected in a chilled tube and assayed as previously described.11 Participants’ blood was also sampled for the extraction of DNA.
Local 2-AR Sensitivity
Nine patients and 8 healthy controls were studied after resting supine and fasting overnight. A cardiologist catheterized both brachial and femoral arteries, and a large antecubital vein was used for blood sampling. Ipsilateral forearm and leg blood flow (to the arterial lines) were determined by venous occlusion air plethysmography as previously described.12 ECG and brachial arterial blood pressure (through a 3-way valve connected to a transducer [DT-4812, Omheda]) were monitored on signal conditioners (Gould) and displayed on a thermal array recorder (model TA-6000, Gould). Subjects rested quietly for 60 minutes following placement of the catheters, and then local 2-AR sensitivity in both arms and legs was determined in each subject. Isoproterenol was infused intraarterially in increasing doses (from 10 to 300 ng/min). Each dose was infused for 5 minutes, and blood flow in the forearms and legs was recorded for at least 5 cycles during the last minute of the infused dose. The infusion was stopped after reaching the maximal dilatory effect or an undesired systemic side effect. Dose response curves were assessed, and the maximal effect (Emax) and the dose of agonist producing half-maximal effect (ED50) were extrapolated from the individual sigmoidal (nonlinear regression) curves. Noteworthy to mention, this study was performed and analyzed before genotype determination.
2-AR Genotyping
Genotyping was performed by allele specific oligonucleotide (ASO) hybridization as described elsewhere.13,14 Briefly, genomic DNA was extracted from 10 mL of whole blood by standard methods. A 234-bp fragment spanning the polymorphisms of interest of the 2-AR was generated by polymerase chain reaction (PCR). The PCR reaction contained 1 e of genomic DNA (normalized to 0.3 e蘥/e), 5 e of 2 mmol/L dNTP (200 eol/L final concentration of each dNTP), 5 e of PCR 10x buffer, 3 e of 25 mmol/L MgCl (1.5 mmol/L final concentration), 2 eol/L of each primer, and water to a total of 50 e. One unit of Taq polymerase was added per reaction. The primer sequences used were upstream CCC AGC CAG TGC GCT TAC CT and downstream CCG TCT GCA GAC GCT CGA AC. The reaction consisted of 36 cycles (melting temperature 94°C, 90 s; annealing temperature 60°C, 90 s; extension at 72°C after last cycle). PCR product was then applied to Hybond N+ filters by use of a dot blot apparatus, and genotype was finally determined by allele-specific oligonucleotide hybridization. The 32P-labeled probes used for hybridization were Gln27 (CAC GCA GCA AAG GGA CGA G), Glu27 (CAC GCA GGA AAG GGA CGA G), Arg16 (GCA CCC AAT AGA AGC CAT G), and Gly16 (GCA CCC AAT GGA AGC CAT G). Ten-fold excess of cold probe was used in the initial part of the hybridization for the Arg16Gly polymorphism, and 30-fold excess of cold probe was used for the Gln27Glu27 polymorphism. Probe filters were exposed to x-ray film for 2 to 3 hours, and genotype was determined. Genotyping was repeated on different occasions for several subjects and results were confirmed.
Statistical Analysis and Calculations
Vascular resistance was calculated from mean individual arterial BP [0.33(SBP-DBP)+DBP]/local blood flow (unit=mm Hg/mL/min/dL tissue). Results are expressed as mean±SEM. Paired and unpaired t tests were used for comparisons between groups and within groups before and after stimuli as appropriate. ManneCWhitney U test and Wilcoxon signed-rank test were used for data that were not normally distributed. Repeated measures ANOVA was used to assess the isoproterenol dose response and 2-way ANOVA for repeated measurement was used to compare hemodynamic and catecholamine data between the genotypes. A nonlinear regression analysis (adapted sigmoid curve) was performed for each subject to establish the maximal response, Emax, and the dose required to increase the flow to 50% of the Emax, the ED50 (GraphPad, Prism version 4.03, San Diego, CA). For analysis of binary data, including genotype and allele frequencies, we used the 2 test. Because few cases or controls were male or nonwhite, no adjustments were made for sex or race. All P values are 2-sided; P<0.05 was considered to be statistically significant.
Results
Local 2-AR Sensitivity
Demographic characteristics and the female/male ratio were similar between the groups. General characteristics, hemodynamic data and plasma catecholamine concentrations are shown in Table 1. The most frequent symptoms among patients were dizziness and lightheadedness, blurred and tunnel vision, syncope, fatigue, irritability and clamminess on standing, headache relieved with supine rest, abdominal discomfort and nausea unrelated to meals, and prominent exercise intolerance. Patients with POTS had a marked increase in heart rate on standing, as expected.
Mean brachial arterial BP was 71±2.5 and 69±1.5 mm Hg in patients and controls, respectively. Forearm and leg resistance were higher in patients compared with controls (Table 1). Supine plasma norepinephrine tended to be higher in patients (Table 1) on standing, and both norepinephrine and epinephrine concentrations increased more substantially in patients than controls, as shown in Figure 1 a and 1b.
Intraarterial infusion of isoproterenol caused a dose-dependent increase in forearm and leg blood flow (Figure 2 a and 2b). Isoproterenol increased blood flow 400±70% in the forearm and 170±40% in legs of control subjects but only 280±60% in the forearm and 120±20% in the leg in patients with POTS (ANOVA, P=0.001 for both). The isoproterenol ED50 was higher for patients than for healthy controls, for both forearm and leg blood flow. The heart rate increased by 5 bpm in both groups at the higher doses, but there were no detectable effects on systolic and diastolic BP.
2-AR Genotype in Patients With POTS
Genotype frequencies were in Hardy-Weinberg equilibrium in cases and controls for both the Arg16Gly and Gln27Glu 2-AR polymorphisms (P<0.05). The genotype and allele distributions were not different in the 2 population groups (Table 2).
Hemodynamic and plasma catecholamine data were available at supine rest and during orthostatic stress in patients, but not in controls. The hemodynamic responses of patients with POTS and their circulating plasma norepinephrine and epinephrine, stratified according to genotype, are detailed in Table 3. Mean ages did not differ between genotypes.
Gly16Gly genotype was associated with significantly higher supine and upright systolic BP and lower supine norepinephrine and upright epinephrine compared with 16Arg groups. Glu27Glu genotype had the highest supine and upright systolic BP with lower upright plasma norepinephrine and epinephrine concentrations compared with 27Gln groups. However, none of the tested genotypes affected the supine or the upright heart rate.
Discussion
The most novel finding in this study is the markedly impaired vasodilation to a -adrenergic receptor agonist in patients with POTS. Moreover, whereas neither of the 2 most common 2-AR SNPs was predominant in POTS, patients homozygous for Gly16 or Glu27 had different hemodynamic and circulating plasma catecholamine profiles compared with the other genotypes.
2-AR Desensitization in POTS
Orthostatic tolerance, blood pressure, and heart rate regulation are affected by a number of homeostatic mechanisms.15 An abnormality in one of these mechanisms could cause orthostatic intolerance evident clinically as POTS.16,17 Thus, disturbances in blood volume regulation, autonomic control of the cardiovascular system, or both may lead to the pathophysiology of POTS. None of the recently described pathophysiologies is able to explain all the diverse clinical features, emphasizing the heterogeneity of the syndrome.
Both idiopathic18 and hyporeninemic hypovolemia5 are frequently found in patients with POTS. The predominant regulator of the cardiovascular system on standing (ie, the sympathetic nervous system) has been evaluated extensively in patients with POTS by many investigators in the last decade, and many putative mechanisms described. Partial denervation of the autonomic nervous system underlies neuropathic POTS.4 Impaired baroreflex responses,3,8 norepinephrine transporter (NET) deficiency,7 centrally mediated increase in sympathetic activity, and abnormal NO release19 may underlie some of the pathophysiologies of the syndrome. To shed light on the high circulating catecholamines in patients with POTS, we previously studied norepinephrine kinetics in these patients. Our results revealed that an augmented increase in norepinephrine spillover and a significant decrease in its clearance underlie the high plasma norepinephrine levels.8
This hyperadrenergic state (ie, continuous exposure to agonist) could cause desensitization of 2-ARs in the vasculature of patients with POTS. Both plasma epinephrine and norepinephrine are high in patients with POTS, but some patients present higher plasma epinephrine than others.20 Through action on vascular and 2-adrenoceptors, this heterogeneity could give rise to different hemodynamic profiles in these patients. Endogenous activation of 2-ARs is primarily through epinephrine, but a high circulating norepinephrine (albeit with lower affinity) also contributes.21 This is supported by the fact that the 2-AR is a humoral receptor (located extrasynaptically and activated by circulating agonists) rather than an innervated receptor.
Patients with POTS had higher vascular resistance (lower conductance) compared with controls. This baseline vasomotor balance could affect the amount of the increase in vascular conductance elicited by isoproterenol.22 This balance is the result of multiple physiological factors, principally -AR vasoconstrictor tone, 2-AR vasodilatory effect, and endothelial-relaxation capability (eg, NO), which ultimately may be determined by genotype.19,23,24 However, intraarterial infusion of phenylephrine (selective 1-AR agonist) causes similar vasoconstriction in our patients with POTS as compared with healthy controls (G. Jacob, I. Biaggioni, D. Robertson, unpublished data, 2005). An hypothesis recently presented by Medow et al proposes that endothelial function is altered in the vasculature of some patients with POTS, perhaps via perturbations in NO synthesis or breakdown.19 However, the response to systemic infusion of sodium nitroprusside was normal in our previous studies in this group of patients with POTS.8,25
Systemic infusion of isoproterenol solicits a similar drop in BP in patients with POTS compared with controls, before and after ganglionic blockade.8,25 Although this may suggest that the 2-AR functions normally in these patients, isoproterenol activates both 1 and 2-AR, which could mask eventual vascular 2-AR hyposensitivity.
The 2-AR is expressed ubiquitously and mediates physiological responses, including arterial and venous dilatation and bladder and lung smooth-muscle relaxation, as well as metabolic effects such as glycolysis and lipolysis in liver and skeletal muscle.26 It also intensifies norepinephrine release, through the activation of the presynaptic 2-AR by circulating epinephrine during stress or by baroreflex activation caused by systemic vasodilatation.21 Because altered 2-AR function could explain some of the findings in POTS, we hypothesized that one of the common 2-AR polymorphisms might be a causative factor for POTS or might be a marker in linkage disequilibrium with a causative polymorphism.
Association of 2-AR Polymorphisms With POTS
The attenuated blood flow response to isoproterenol in our patients would be consistent with a relative decrease in Arg16 or Glu27 alleles, which are associated with an enhanced vasodilation response.10,27eC29 Normotensive whites homozygous for Gln27 have a lower baseline forearm blood flow as well as an attenuated response to isoproterenol compared with Glu27 homozygotes.27 Furthermore, individuals homozygous for Arg16 demonstrate marked desensitization following continuous infusion of isoproterenol.10 These results might predict that POTS patients, in whom isoproterenol-stimulated blood flow is blunted, are enriched with the Gln27 and/or Arg16 alleles. However, the genotype distributions for the 2-AR polymorphisms at codons 16 and 27 did not differ in our patient compared with controls.
POTS is heterogeneous and has a diverse presentation, and as a complex phenotype, any genetic contribution might be too small to detect except in a much larger study. Furthermore, the 2 polymorphisms that were studied are very common in the general population. The relatively low prevalence of POTS and the probability that different pathophysiologies underlie this syndrome might also make it difficult to detect an association with a particular genotype. It should also be noted that the codon 16 and codon 27 polymorphisms are in partial linkage disequilibrium, so those individuals homozygous for Glu27 are almost always also homozygous for Gly16.13,30 The linkage disequilibrium between these 2 polymorphisms makes it difficult to detect the contribution of each genotype to a particular phenotype. Haplotype analysis might provide additional information on the genetic component of POTS.
In other patient populations, such as asthma, hypertension, and congestive heart failure, although these variants are not major contributors to the occurrence of the disease, they can affect the severity, course, and response to treatment. For instance, the Gly16 variant is associated with more severe asthma31 and decreased response to an 2-AR agonist.32 Conflicting data exist on the effect of 2-AR polymorphisms on blood pressure in the general population.28,30,33 Nevertheless, healthy whites with Gly16 and Glu27 variants present higher blood pressure.30
The Arg16Gly and Gln27Glu polymorphisms influence both agonist-mediated responsiveness and agonist-mediated desensitization, and it is unclear how the 2 might interact to produce the phenotype in POTS of decreased blood flow and reduced responsiveness to isoproterenol. Ideally, we would like to have studied the relationship between 2-AR genotypes and vascular 2-AR dilatory function in POTS, but again the same patients did not undergo 2-AR sensitivity testing and genotyping in this study.
Although our study does not support a significant association between POTS and either the Arg16Gly or Gln27Glu genotype, these polymorphisms did contribute to the diverse hemodynamic and catecholamine profile of POTS patients. In agreement with some of the previous studies on blood pressure, in healthy subjects, POTS patients who were homozygous for Gly16 had significantly higher blood pressure than did patients with an Arg16 allele. Since our 9 Glu27 homozygous patients were also homozygous for Gly16, the Glu27Glu genotype was also associated with elevated blood pressure. The majority of patients with POTS are normotensive. However, during orthostatic stress, anxiety, and other stimuli that increase sympathetic activity, we frequently observe an exaggerated fluctuation in systolic and particularly in diastolic BP in addition to the increment in heart rate.34 The mechanism underlying these phenomena could be explained, at least partially, by the findings of the present study (ie, blunted vasodilator responses to 2-AR agonists). Thus, sympathetic activation causes vasoconstriction through -adrenoceptor stimulation without the possibility to counterbalance the increase in BP, because of the 2-AR desensitization. In support of our explanation, Stein et al have tested a similar hypothesis in patients with borderline hypertension and found that vascular 2-AR was downregulated in the setting of increased systemic norepinephrine spillover. Unopposed -AR activation, because of the former association, was claimed to contribute to the high BP in their subjects.35 Some subgroups of patients with POTS have a blunted baroreflex buffering ability, which may also contribute to this hypertensive fluctuation.3 Moreover, a subgroup of patients with POTS has increased systemic 1-AR responsiveness that cannot be explained in the setting of high circulating catecholamines except by partial baroreflex failure.8 Our findings with the two common 2-AR SNPs and blood pressure in POTS suggest that there might also be a genetic component to the exaggerated blood pressure response in this syndrome.
In conclusion, patients with POTS and high circulating catecholamines have a blunted vasodilatory response to the infusion of 2-AR agonist. Although the 2-AR genotype distribution in POTS was similar to that of healthy subjects, it affects the hemodynamic phenotype, as it does in healthy individuals, and is also related to plasma catecholamine levels. Therefore, decreased 2-AReCrelated vascular dilatation and common genetic polymorphisms of the 2-AR could contribute to the altered and the heterogeneous hemodynamic profile of patients with POTS.
Perspectives and Limitation
As noted above, recruitment of our patients with POTS for assessment of vascular 2-AR sensitivity did not involve genetic selection. Certainly, such an approach could have provided better support for the study conclusions. This approach requires higher numbers of invasive studies. This limitation is common to many studies that simultaneously study genotype and phenotype in patients. Although patients with POTS defined in terms of their 2-AR genotype could have different hemodynamic and plasma catecholamine profiles, the syndrome itself did not appear to be caused by the haplotypes studied. Future studies that recruit patients with POTS according to stringent criteria may well require such prospective genetic assessment.
Isoproterenol activates 2-ARs in muscle, cutaneous, and other vascular beds. The increment in flow, as assessed by plethysmography, is the sum of all. Therefore, we are unable to differentiate between the effects on skin and muscle. Noteworthy to mention, most of the skin flow is excluded through the wrist occlusion. Cardiac 1-AR activation by the highest doses of isoproterenol could contribute to the hemodynamic responses.
POTS is highly heterogeneous and our findings in hyperadrenergic patients might not apply to others POTS populations. Some authors have classified patients with POTS according to low, normal, or high blood flow in their legs.19 This heterogeneity of leg flow could be influenced by the 2-AR genotype, an hypothesis that requires testing.
Management of patients with POTS requires pharmacological and nonpharmacological intervention. There is no definitive treatment modality. Usually patients are encouraged to build their lower body muscles in association with a high salt diet. If needed, low doses of -blockers, fludrocortisone, vasoconstrictors (eg, midodrine), and clonidine (partial 2-AR agonist) are administered.36 The findings of the present study encourage efforts toward reduction of plasma catecholamine levels, especially in patients with the hyperadrenergic form of POTS.
Acknowledgments
This study was supported by grants from Yael Foundation (Biosence, Israel) and National Institutes of Health MO1 RR00095 and 5P01 HL56693.
References
Schondorf R, Low PA. Idiopathic postural tachycardia syndrome. Neurology. 1993; 43: 132eC137.
Jacob G, Biaggioni I. Idiopathic orthostatic intolerance and postural tachycardia syndromes. Am J Med Sci. 1999; 317: 88eC101.
Farquhar WB, Taylor JA, Darling SE, Chase KP, Freeman R. Abnormal baroreflex responses in patients with idiopathic orthostatic intolerance. Circulation. 2000; 102: 3086eC3091.
Jacob G, Costa F, Shannon JR, Robertson RM, Wathen M, Stein M, Biaggioni I, Ertl A, Black B, Robertson D. The neuropathic postural tachycardia syndrome. N Engl J Med. 2000; 343: 1008eC1014.
Jacob G, Robertson D, Mosqueda-Garcia R, Ertl CA, Robertson RM, Biaggioni I. Hypovolemia in syncope and orthostatic intolerance: Role of renin-angitensin system. Am J Med. 1997; 103: 128eC133.
Vernino S, Low PA, Fealey RD, Stewart JD, Farrugia G, Lennon VA. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies. N Engl J Med. 2000; 343: 847eC855.
Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, Blakely RD, Robertson D. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N Engl J Med. 2000; 342: 541eC549.
Jacob G, Shannon JR, Costa F, Furlan R, Biaggioni I, Mosqueda-Garcia R, Robertson RM, Robertson D. Abnormal norepinephrine clearance and adrenergic receptor sensitivity in idiopathic orthostatic intolerance. Circulation. 1999; 99: 1706eC1712.
Singer W, Shen WK, Opfer-Gehrking TL, McPhee BR, Hilz MJ, Low PA. Evidence of an intrinsic sinus node abnormality in patients with postural tachycardia syndrome. Mayo Clin Proc. 2002; 77: 246eC252.
Dishy V, Sofowora GG, Xie HG, Kim RB, Byrne DW, Stein CM, Wood AJ. The effect of common polymorphisms of the 2-adrenergic receptor on agonist-mediated vascular desensitization. N Engl J Med. 2001; 345: 1030eC1035.
Goldstein DS, Eisenhofer G, Stuff R. Plasma dihydroxyphenylglycerol and the intraneuronal disposition of norepinephrine in humans. J Clin Invest. 1988; 81: 231eC240.
Raine NM, Sneddon JC. A simple water-filled plethysmograph for measurement of limb blood flow in humans. Adv Physiol Educ. 2002; 26: 120eC128.
Dewar JC, Wheatley AP, Venn A, Morrison JF, Britton J, Hall IP. 2-adrenoceptor polymorphisms are in linkage disequilibrium, but are not associated with asthma in an adult population. Clinical & Experimental Allergy. 1998; 28: 442eC448.
Xie HG, Stein CM, Kim RB, Xiao ZS, He N, Zhou HH, Gainer JV, Brown NJ, Haines JL, Wood AJ. Frequency of functionally important -2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals. Pharmacogenetics. 1999; 9: 511eC516.
Rowell LB. Human Cardiovascular Control. New York: Oxford; 1993.
Stewart JM, Montgomery LD. Regional blood volume and peripheral blood flow in postural tachycardia syndrome. Am J Physiol Heart Circ Physiol. 2004; 287: H1319eCH1327.
Stewart JM, Medow MS, Montgomery LD. Local vascular responses affecting blood flow in postural tachycardia syndrome. Am J Physiol Heart Circ Physiol. 2003; 285: H2749eCH2756.
Fouad FM, Thadena-Thome L, Bravo E, Tarazi RC. Idiopathic hypovolemia. Ann Int Med. 1986; 298eC303.
Medow MS, Minson CT, Stewart JM. Decreased microvascular nitric oxide-dependent vasodilation in postural tachycardia syndrome. Circulation. 2005; 112: 2611eC2618.
Goldstein DS, Eldadah B, Holmes C, Pechnik S, Moak J, Sharabi Y. Neurocirculatory abnormalities in chronic orthostatic intolerance. Circulation. 2005; 111: 839eC845.
Hoffman BB. Catecholamines, symapthomimetic drugs, and adrenergic receptor antagonists. In: Hrdaman JG, Limberd LL, eds. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. New York: McGraw-Hill; 2001.
Barbato E, Piscione F, Bartunek J, Galasso G, Cirillo P, De Luca G, Iaccarino G, De Bruyne B, Chiariello M, Wijns W. Role of 2 adrenergic receptors in human atherosclerotic coronary arteries. Circulation. 2005; 111: 288eC294.
Rascado RR, Bendhack LM. Activation of (2)-adrenoceptors is necessary to induce nitric oxide release in isoprenaline-induced relaxation. Vascul Pharmacol. 2005; 42: 63eC68.
Whalen EJ, Johnson AK, Lewis SJ. -adrenoceptor dysfunction after inhibition of NO synthesis. Hypertension. 2000; 36: 376eC382.
Jordan J, Shannon JR, Diedrich A, Black BK, Robertson D. Increased sympathetic activation in idiopathic orthostatic intolerance: role of systemic adrenoreceptor sensitivity. Hypertension. 2002; 39: 173eC178.
Insel PA. Seminars in medicine of the Beth Israel Hospital, Boston. Adrenergic receptorseCevolving concepts and clinical implications. N Engl J Med. 1996; 334: 580eC585.
Cockcroft JR, Gazis AG, Cross DJ, Wheatley A, Dewar J, Hall IP, Noon JP. (2)-adrenoceptor polymorphism determines vascular reactivity in humans. Hypertension. 2000; 36: 371eC375.
Gratze G, Fortin J, Labugger R, Binder A, Kotanko P, Timmermann B, Luft FC, Hoehe MR, Skrabal F. -2 Adrenergic receptor variants affect resting blood pressure and agonist-induced vasodilation in young adult Caucasians. Hypertension. 1999; 33: 1425eC1430.
Hoit BD, Suresh DP, Craft L, Walsh RA, Liggett SB. 2-adrenergic receptor polymorphisms at amino acid 16 differentially influence agonist-stimulated blood pressure and peripheral blood flow in normal individuals. Am Heart J. 2000; 139: 537eC542.
Bray MS, Krushkal J, Li L, Ferrell R, Kardia S, Sing CF, Turner ST, Boerwinkle E. Positional genomic analysis identifies the (2)-adrenergic receptor gene as a susceptibility locus for human hypertension. Circulation. 2000; 101: 2877eC2882.
Reihsaus E, Innis M, MacIntyre N, Liggett SB. Mutations in the gene encoding for the 2-adrenergic receptor in normal and asthmatic subjects. Am Journal of Respiratory Cell & Molecular Biology. 1993; 8: 334eC339.
Lima JJ, Thomason DB, Mohamed MH, Eberle LV, Self TH, Johnson JA. Impact of genetic polymorphisms of the 2-adrenergic receptor on albuterol bronchodilator pharmacodynamics. Clinical Pharmacology & Therapeutics. 1999; 65: 519eC525.
Pereira AC, Floriano MS, Mota GF, Cunha RS, Herkenhoff FL, Mill JG, Krieger JE. 2 adrenoceptor functional gene variants, obesity, and blood pressure level interactions in the general population. Hypertension. 2003; 42: 685eC692.
Schondorf R. Evaluation of the patient with orthostatic intolerance. In: Robertson D, ed. Primer on the Autonomic Nervous System. Amsterdam, The Netherlands: Elsevier; 2004.
Stein CM, Nelson R, Deegan R, He H, Wood M, Wood AJ. Forearm adrenergic receptor-mediated vasodilation is impaired, without alteration of forearm norepinephrine spillover, in borderline hypertension. J Clin Invest. 1995; 96: 579eC585.
Jacob G, Shannon JR, Black B, Biaggioni I, Mosqueda-Garcia R, Robertson RM, Robertson D. Effects of volume loading and pressor agents in idiopathic orthostatic tachycardia. Circulation. 1997; 96: 575eC580., http://www.100md.com(Giris Jacob; Emily M. Gar)
Autonomic Dysfunction Center (E.M.G., F.C., C.M.S., H.-G.X., R.M.R., I.B., D.R.), Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn.
Abstract
Previous studies suggest that the 2-adrenoceptor functions abnormally in patients with postural tachycardia syndrome (POTS) and may contribute to their altered hemodynamic profile. To test the hypothesis that the 2-adrenoceptor response is decreased in POTS, we studied: (1) the arterial vasodilation response to the agonist, isoproterenol, and (2) the distribution of common polymorphisms (codons 16 and 27) of the gene coding the receptor (2-AR) in a large population with POTS. We measured plasma catecholamines and monitored hemodynamics and changes in forearm and leg blood flow to incremental doses of intraarterial isoproterenol in 9 patients with POTS compared with 8 healthy subjects. For polymorphism assessment we collected DNA from 57 patients with POTS and compared with 67 age-sex matched healthy subjects. Circulating catecholamines were significantly higher in POTS subjects compared with controls. Intrabrachial and intrafemoral isoproterenol infusion elicited a dose-dependent increase in blood flow. In healthy subjects, blood flow increased (mean±SEM) 400±70% in the forearm and 170±40% in the leg, but only 280±60% in forearms and 120±20% in legs of patients with POTS (ANOVA for both P<0.001). The genotype and allele distributions for codons 16 and 27 2-AR variants were not different in the 2 groups. However, the blood pressure and plasma norepinephrine levels diverged in patients according to their genotype. Patients with Gly16Gly and patients with Glu27Glu had lower plasma catecholamines and higher supine and upright blood pressure, compared with other genotypes. Therefore, both decreased 2-adrenoceptor-related vasodilation and 2-AR polymorphisms may contribute to the hemodynamic diversity of patients with POTS.
Key Words: receptors adrenergic receptor agonists norepinephrine tachycardia polymorphism
Introduction
Postural tachycardia syndrome (POTS), is one of the most frequent forms of chronic orthostatic intolerance in the general population.1 It affects &500 000 Americans with a female to male ratio of 5:1, and most cases occur between the ages of 15 and 50 years.2 Clinically it is characterized by frequent orthostatic symptoms, such as dizziness, blurred vision, syncope and fatigue associated with blood pressure (BP) fluctuations, orthostatic tachycardia, and often high circulating plasma catecholamines. The remarkable increase in heart rate on standing without a significant drop in blood pressure is the hallmark of this syndrome.2 The etiology of POTS remains elusive. Different investigators have described various aspects of the pathophysiology that underlies the cardiovascular irregularities in these patients. For instance, baroreflex dysfunction,3 partial autonomic denervation (neuropathic POTS),4 hyporeninemic hypovolemia,5 antiganglionic nicotinic receptor antibodies,6 and norepinephrine transporter deficiency7 have been reported.
Conflicting data exist on cardiovascular adrenoceptor (-AR) sensitivity in patients with POTS. Although a normal or increased chronotropic response to 1-AR stimulation occurs in patients with POTS (despite the high circulating plasma catecholamines),8,9 no data are available on vascular 2-AR dilatory function. The coexistence of exaggerated blood pressure oscillations and absence of a significant drop in BP on standing in these patients could suggest that the vascular 2-AR is not functioning normally. The continued exposure of receptors to adrenergic agonists leads to receptor desensitization; therefore the presence of high circulating catecholamines in patients with POTS might result in less responsive vascular 2-AR.
There has been great interest in the potential role of 2-AR polymorphisms in diseases in which the 2-AR is thought to be involved, such as hypertension, asthma, and obesity. The common single nucleotide polymorphisms (SNPs) in 2-AR at amino acid positions 16 (Arg16Gly) and 27 (Gln27Glu) affect receptor function. During continuous infusion of isoproterenol, subjects homozygous for the Arg16 allele demonstrate marked agonist-mediated desensitization, whereas subjects homozygous for Gly16 do not show desensitization.10 The Arg16 and Glu27 variants have been linked with a greater vasodilator response to adrenergic agonists. However, the genetic variation of 2-AR has not previously been explored in POTS. We therefore investigated the role of the Arg16Gly and Gln27Glu 2-AR polymorphisms in POTS in a case-control association study.
We initially tested the hypothesis that the 2-AR vasodilatory response is decreased in patients with POTS as compared with age and sex-matched healthy subjects. Second, we proposed that functionally relevant 2-AR polymorphisms may contribute to susceptibility to POTS and to the magnitude of the variations in hemodynamic and catecholamine responses in patients.
Methods
Subjects
Patients with POTS were recruited from consecutive referrals to the Autonomic Dysfunction Center at Vanderbilt University Medical Center between 1996 and 2000.
The first study examined vascular dilatory function (2-AR sensitivity: isoproterenol study) and included 9 patients. Patients were enrolled if they met the following stringent criteria: (1) a history of daily typical symptoms (eg, dizziness, blurred vision, presyncope or frank syncope, fatigue, palpitations, chest discomfort, irritability, clamminess) for at least 6 months, (2) an increase in heart rate of at least 30 bpm within 5 minutes of standing, without a concomitant decrease in blood pressure greater than 20/10 mm Hg and (3) a plasma norepinephrine level of at least 600 pg/mL (3.18 nmol/L) with standing. This group was compared with 8 age- and sex-matched completely healthy subjects.
The second study determined the 2-AR SNP distribution (genetic study) in 57 consecutive patients with POTS (91% female and 98% white) who met the above criteria, unrelated to their plasma norepinephrine levels. Genotypes of this group were compared with those of 68 healthy volunteers (80% female and 87% white), recruited from the Nashville, Tenn, community, whose DNA was stored at our institution’s DNA bank. Female to male ratio and white to nonwhite ratio were not statistically different between the groups (P=0.14 and 0.24, respectively).
All subjects underwent a physical examination; ECG; hematologic, biochemical, and autonomic laboratory tests; and an interview using an autonomic questionnaire to determine the type and extent of clinical symptoms. Patients with systemic illnesses capable of affecting the autonomic nervous system function were excluded. The Vanderbilt Investigational Review Board approved all study procedures, and subjects gave written informed consent.
Experimental Design
All subjects undergoing 2-AR sensitivity testing and the patients who participated in the genotyping portion of the study were admitted and studied after consuming a restricted diet (150 mEq Na+ and 70 mEq K+ per day, free of caffeine and low in monoamines) for at least 3 days. Medications were discontinued at least 2 weeks before admission, and smoking was not permitted during the study. All of these subjects underwent measurements of blood pressure (cuff sphygmomanometer), heart rate, and venous plasma norepinephrine and epinephrine during supine and upright posture. Blood for catecholamine assays was collected in a chilled tube and assayed as previously described.11 Participants’ blood was also sampled for the extraction of DNA.
Local 2-AR Sensitivity
Nine patients and 8 healthy controls were studied after resting supine and fasting overnight. A cardiologist catheterized both brachial and femoral arteries, and a large antecubital vein was used for blood sampling. Ipsilateral forearm and leg blood flow (to the arterial lines) were determined by venous occlusion air plethysmography as previously described.12 ECG and brachial arterial blood pressure (through a 3-way valve connected to a transducer [DT-4812, Omheda]) were monitored on signal conditioners (Gould) and displayed on a thermal array recorder (model TA-6000, Gould). Subjects rested quietly for 60 minutes following placement of the catheters, and then local 2-AR sensitivity in both arms and legs was determined in each subject. Isoproterenol was infused intraarterially in increasing doses (from 10 to 300 ng/min). Each dose was infused for 5 minutes, and blood flow in the forearms and legs was recorded for at least 5 cycles during the last minute of the infused dose. The infusion was stopped after reaching the maximal dilatory effect or an undesired systemic side effect. Dose response curves were assessed, and the maximal effect (Emax) and the dose of agonist producing half-maximal effect (ED50) were extrapolated from the individual sigmoidal (nonlinear regression) curves. Noteworthy to mention, this study was performed and analyzed before genotype determination.
2-AR Genotyping
Genotyping was performed by allele specific oligonucleotide (ASO) hybridization as described elsewhere.13,14 Briefly, genomic DNA was extracted from 10 mL of whole blood by standard methods. A 234-bp fragment spanning the polymorphisms of interest of the 2-AR was generated by polymerase chain reaction (PCR). The PCR reaction contained 1 e of genomic DNA (normalized to 0.3 e蘥/e), 5 e of 2 mmol/L dNTP (200 eol/L final concentration of each dNTP), 5 e of PCR 10x buffer, 3 e of 25 mmol/L MgCl (1.5 mmol/L final concentration), 2 eol/L of each primer, and water to a total of 50 e. One unit of Taq polymerase was added per reaction. The primer sequences used were upstream CCC AGC CAG TGC GCT TAC CT and downstream CCG TCT GCA GAC GCT CGA AC. The reaction consisted of 36 cycles (melting temperature 94°C, 90 s; annealing temperature 60°C, 90 s; extension at 72°C after last cycle). PCR product was then applied to Hybond N+ filters by use of a dot blot apparatus, and genotype was finally determined by allele-specific oligonucleotide hybridization. The 32P-labeled probes used for hybridization were Gln27 (CAC GCA GCA AAG GGA CGA G), Glu27 (CAC GCA GGA AAG GGA CGA G), Arg16 (GCA CCC AAT AGA AGC CAT G), and Gly16 (GCA CCC AAT GGA AGC CAT G). Ten-fold excess of cold probe was used in the initial part of the hybridization for the Arg16Gly polymorphism, and 30-fold excess of cold probe was used for the Gln27Glu27 polymorphism. Probe filters were exposed to x-ray film for 2 to 3 hours, and genotype was determined. Genotyping was repeated on different occasions for several subjects and results were confirmed.
Statistical Analysis and Calculations
Vascular resistance was calculated from mean individual arterial BP [0.33(SBP-DBP)+DBP]/local blood flow (unit=mm Hg/mL/min/dL tissue). Results are expressed as mean±SEM. Paired and unpaired t tests were used for comparisons between groups and within groups before and after stimuli as appropriate. ManneCWhitney U test and Wilcoxon signed-rank test were used for data that were not normally distributed. Repeated measures ANOVA was used to assess the isoproterenol dose response and 2-way ANOVA for repeated measurement was used to compare hemodynamic and catecholamine data between the genotypes. A nonlinear regression analysis (adapted sigmoid curve) was performed for each subject to establish the maximal response, Emax, and the dose required to increase the flow to 50% of the Emax, the ED50 (GraphPad, Prism version 4.03, San Diego, CA). For analysis of binary data, including genotype and allele frequencies, we used the 2 test. Because few cases or controls were male or nonwhite, no adjustments were made for sex or race. All P values are 2-sided; P<0.05 was considered to be statistically significant.
Results
Local 2-AR Sensitivity
Demographic characteristics and the female/male ratio were similar between the groups. General characteristics, hemodynamic data and plasma catecholamine concentrations are shown in Table 1. The most frequent symptoms among patients were dizziness and lightheadedness, blurred and tunnel vision, syncope, fatigue, irritability and clamminess on standing, headache relieved with supine rest, abdominal discomfort and nausea unrelated to meals, and prominent exercise intolerance. Patients with POTS had a marked increase in heart rate on standing, as expected.
Mean brachial arterial BP was 71±2.5 and 69±1.5 mm Hg in patients and controls, respectively. Forearm and leg resistance were higher in patients compared with controls (Table 1). Supine plasma norepinephrine tended to be higher in patients (Table 1) on standing, and both norepinephrine and epinephrine concentrations increased more substantially in patients than controls, as shown in Figure 1 a and 1b.
Intraarterial infusion of isoproterenol caused a dose-dependent increase in forearm and leg blood flow (Figure 2 a and 2b). Isoproterenol increased blood flow 400±70% in the forearm and 170±40% in legs of control subjects but only 280±60% in the forearm and 120±20% in the leg in patients with POTS (ANOVA, P=0.001 for both). The isoproterenol ED50 was higher for patients than for healthy controls, for both forearm and leg blood flow. The heart rate increased by 5 bpm in both groups at the higher doses, but there were no detectable effects on systolic and diastolic BP.
2-AR Genotype in Patients With POTS
Genotype frequencies were in Hardy-Weinberg equilibrium in cases and controls for both the Arg16Gly and Gln27Glu 2-AR polymorphisms (P<0.05). The genotype and allele distributions were not different in the 2 population groups (Table 2).
Hemodynamic and plasma catecholamine data were available at supine rest and during orthostatic stress in patients, but not in controls. The hemodynamic responses of patients with POTS and their circulating plasma norepinephrine and epinephrine, stratified according to genotype, are detailed in Table 3. Mean ages did not differ between genotypes.
Gly16Gly genotype was associated with significantly higher supine and upright systolic BP and lower supine norepinephrine and upright epinephrine compared with 16Arg groups. Glu27Glu genotype had the highest supine and upright systolic BP with lower upright plasma norepinephrine and epinephrine concentrations compared with 27Gln groups. However, none of the tested genotypes affected the supine or the upright heart rate.
Discussion
The most novel finding in this study is the markedly impaired vasodilation to a -adrenergic receptor agonist in patients with POTS. Moreover, whereas neither of the 2 most common 2-AR SNPs was predominant in POTS, patients homozygous for Gly16 or Glu27 had different hemodynamic and circulating plasma catecholamine profiles compared with the other genotypes.
2-AR Desensitization in POTS
Orthostatic tolerance, blood pressure, and heart rate regulation are affected by a number of homeostatic mechanisms.15 An abnormality in one of these mechanisms could cause orthostatic intolerance evident clinically as POTS.16,17 Thus, disturbances in blood volume regulation, autonomic control of the cardiovascular system, or both may lead to the pathophysiology of POTS. None of the recently described pathophysiologies is able to explain all the diverse clinical features, emphasizing the heterogeneity of the syndrome.
Both idiopathic18 and hyporeninemic hypovolemia5 are frequently found in patients with POTS. The predominant regulator of the cardiovascular system on standing (ie, the sympathetic nervous system) has been evaluated extensively in patients with POTS by many investigators in the last decade, and many putative mechanisms described. Partial denervation of the autonomic nervous system underlies neuropathic POTS.4 Impaired baroreflex responses,3,8 norepinephrine transporter (NET) deficiency,7 centrally mediated increase in sympathetic activity, and abnormal NO release19 may underlie some of the pathophysiologies of the syndrome. To shed light on the high circulating catecholamines in patients with POTS, we previously studied norepinephrine kinetics in these patients. Our results revealed that an augmented increase in norepinephrine spillover and a significant decrease in its clearance underlie the high plasma norepinephrine levels.8
This hyperadrenergic state (ie, continuous exposure to agonist) could cause desensitization of 2-ARs in the vasculature of patients with POTS. Both plasma epinephrine and norepinephrine are high in patients with POTS, but some patients present higher plasma epinephrine than others.20 Through action on vascular and 2-adrenoceptors, this heterogeneity could give rise to different hemodynamic profiles in these patients. Endogenous activation of 2-ARs is primarily through epinephrine, but a high circulating norepinephrine (albeit with lower affinity) also contributes.21 This is supported by the fact that the 2-AR is a humoral receptor (located extrasynaptically and activated by circulating agonists) rather than an innervated receptor.
Patients with POTS had higher vascular resistance (lower conductance) compared with controls. This baseline vasomotor balance could affect the amount of the increase in vascular conductance elicited by isoproterenol.22 This balance is the result of multiple physiological factors, principally -AR vasoconstrictor tone, 2-AR vasodilatory effect, and endothelial-relaxation capability (eg, NO), which ultimately may be determined by genotype.19,23,24 However, intraarterial infusion of phenylephrine (selective 1-AR agonist) causes similar vasoconstriction in our patients with POTS as compared with healthy controls (G. Jacob, I. Biaggioni, D. Robertson, unpublished data, 2005). An hypothesis recently presented by Medow et al proposes that endothelial function is altered in the vasculature of some patients with POTS, perhaps via perturbations in NO synthesis or breakdown.19 However, the response to systemic infusion of sodium nitroprusside was normal in our previous studies in this group of patients with POTS.8,25
Systemic infusion of isoproterenol solicits a similar drop in BP in patients with POTS compared with controls, before and after ganglionic blockade.8,25 Although this may suggest that the 2-AR functions normally in these patients, isoproterenol activates both 1 and 2-AR, which could mask eventual vascular 2-AR hyposensitivity.
The 2-AR is expressed ubiquitously and mediates physiological responses, including arterial and venous dilatation and bladder and lung smooth-muscle relaxation, as well as metabolic effects such as glycolysis and lipolysis in liver and skeletal muscle.26 It also intensifies norepinephrine release, through the activation of the presynaptic 2-AR by circulating epinephrine during stress or by baroreflex activation caused by systemic vasodilatation.21 Because altered 2-AR function could explain some of the findings in POTS, we hypothesized that one of the common 2-AR polymorphisms might be a causative factor for POTS or might be a marker in linkage disequilibrium with a causative polymorphism.
Association of 2-AR Polymorphisms With POTS
The attenuated blood flow response to isoproterenol in our patients would be consistent with a relative decrease in Arg16 or Glu27 alleles, which are associated with an enhanced vasodilation response.10,27eC29 Normotensive whites homozygous for Gln27 have a lower baseline forearm blood flow as well as an attenuated response to isoproterenol compared with Glu27 homozygotes.27 Furthermore, individuals homozygous for Arg16 demonstrate marked desensitization following continuous infusion of isoproterenol.10 These results might predict that POTS patients, in whom isoproterenol-stimulated blood flow is blunted, are enriched with the Gln27 and/or Arg16 alleles. However, the genotype distributions for the 2-AR polymorphisms at codons 16 and 27 did not differ in our patient compared with controls.
POTS is heterogeneous and has a diverse presentation, and as a complex phenotype, any genetic contribution might be too small to detect except in a much larger study. Furthermore, the 2 polymorphisms that were studied are very common in the general population. The relatively low prevalence of POTS and the probability that different pathophysiologies underlie this syndrome might also make it difficult to detect an association with a particular genotype. It should also be noted that the codon 16 and codon 27 polymorphisms are in partial linkage disequilibrium, so those individuals homozygous for Glu27 are almost always also homozygous for Gly16.13,30 The linkage disequilibrium between these 2 polymorphisms makes it difficult to detect the contribution of each genotype to a particular phenotype. Haplotype analysis might provide additional information on the genetic component of POTS.
In other patient populations, such as asthma, hypertension, and congestive heart failure, although these variants are not major contributors to the occurrence of the disease, they can affect the severity, course, and response to treatment. For instance, the Gly16 variant is associated with more severe asthma31 and decreased response to an 2-AR agonist.32 Conflicting data exist on the effect of 2-AR polymorphisms on blood pressure in the general population.28,30,33 Nevertheless, healthy whites with Gly16 and Glu27 variants present higher blood pressure.30
The Arg16Gly and Gln27Glu polymorphisms influence both agonist-mediated responsiveness and agonist-mediated desensitization, and it is unclear how the 2 might interact to produce the phenotype in POTS of decreased blood flow and reduced responsiveness to isoproterenol. Ideally, we would like to have studied the relationship between 2-AR genotypes and vascular 2-AR dilatory function in POTS, but again the same patients did not undergo 2-AR sensitivity testing and genotyping in this study.
Although our study does not support a significant association between POTS and either the Arg16Gly or Gln27Glu genotype, these polymorphisms did contribute to the diverse hemodynamic and catecholamine profile of POTS patients. In agreement with some of the previous studies on blood pressure, in healthy subjects, POTS patients who were homozygous for Gly16 had significantly higher blood pressure than did patients with an Arg16 allele. Since our 9 Glu27 homozygous patients were also homozygous for Gly16, the Glu27Glu genotype was also associated with elevated blood pressure. The majority of patients with POTS are normotensive. However, during orthostatic stress, anxiety, and other stimuli that increase sympathetic activity, we frequently observe an exaggerated fluctuation in systolic and particularly in diastolic BP in addition to the increment in heart rate.34 The mechanism underlying these phenomena could be explained, at least partially, by the findings of the present study (ie, blunted vasodilator responses to 2-AR agonists). Thus, sympathetic activation causes vasoconstriction through -adrenoceptor stimulation without the possibility to counterbalance the increase in BP, because of the 2-AR desensitization. In support of our explanation, Stein et al have tested a similar hypothesis in patients with borderline hypertension and found that vascular 2-AR was downregulated in the setting of increased systemic norepinephrine spillover. Unopposed -AR activation, because of the former association, was claimed to contribute to the high BP in their subjects.35 Some subgroups of patients with POTS have a blunted baroreflex buffering ability, which may also contribute to this hypertensive fluctuation.3 Moreover, a subgroup of patients with POTS has increased systemic 1-AR responsiveness that cannot be explained in the setting of high circulating catecholamines except by partial baroreflex failure.8 Our findings with the two common 2-AR SNPs and blood pressure in POTS suggest that there might also be a genetic component to the exaggerated blood pressure response in this syndrome.
In conclusion, patients with POTS and high circulating catecholamines have a blunted vasodilatory response to the infusion of 2-AR agonist. Although the 2-AR genotype distribution in POTS was similar to that of healthy subjects, it affects the hemodynamic phenotype, as it does in healthy individuals, and is also related to plasma catecholamine levels. Therefore, decreased 2-AReCrelated vascular dilatation and common genetic polymorphisms of the 2-AR could contribute to the altered and the heterogeneous hemodynamic profile of patients with POTS.
Perspectives and Limitation
As noted above, recruitment of our patients with POTS for assessment of vascular 2-AR sensitivity did not involve genetic selection. Certainly, such an approach could have provided better support for the study conclusions. This approach requires higher numbers of invasive studies. This limitation is common to many studies that simultaneously study genotype and phenotype in patients. Although patients with POTS defined in terms of their 2-AR genotype could have different hemodynamic and plasma catecholamine profiles, the syndrome itself did not appear to be caused by the haplotypes studied. Future studies that recruit patients with POTS according to stringent criteria may well require such prospective genetic assessment.
Isoproterenol activates 2-ARs in muscle, cutaneous, and other vascular beds. The increment in flow, as assessed by plethysmography, is the sum of all. Therefore, we are unable to differentiate between the effects on skin and muscle. Noteworthy to mention, most of the skin flow is excluded through the wrist occlusion. Cardiac 1-AR activation by the highest doses of isoproterenol could contribute to the hemodynamic responses.
POTS is highly heterogeneous and our findings in hyperadrenergic patients might not apply to others POTS populations. Some authors have classified patients with POTS according to low, normal, or high blood flow in their legs.19 This heterogeneity of leg flow could be influenced by the 2-AR genotype, an hypothesis that requires testing.
Management of patients with POTS requires pharmacological and nonpharmacological intervention. There is no definitive treatment modality. Usually patients are encouraged to build their lower body muscles in association with a high salt diet. If needed, low doses of -blockers, fludrocortisone, vasoconstrictors (eg, midodrine), and clonidine (partial 2-AR agonist) are administered.36 The findings of the present study encourage efforts toward reduction of plasma catecholamine levels, especially in patients with the hyperadrenergic form of POTS.
Acknowledgments
This study was supported by grants from Yael Foundation (Biosence, Israel) and National Institutes of Health MO1 RR00095 and 5P01 HL56693.
References
Schondorf R, Low PA. Idiopathic postural tachycardia syndrome. Neurology. 1993; 43: 132eC137.
Jacob G, Biaggioni I. Idiopathic orthostatic intolerance and postural tachycardia syndromes. Am J Med Sci. 1999; 317: 88eC101.
Farquhar WB, Taylor JA, Darling SE, Chase KP, Freeman R. Abnormal baroreflex responses in patients with idiopathic orthostatic intolerance. Circulation. 2000; 102: 3086eC3091.
Jacob G, Costa F, Shannon JR, Robertson RM, Wathen M, Stein M, Biaggioni I, Ertl A, Black B, Robertson D. The neuropathic postural tachycardia syndrome. N Engl J Med. 2000; 343: 1008eC1014.
Jacob G, Robertson D, Mosqueda-Garcia R, Ertl CA, Robertson RM, Biaggioni I. Hypovolemia in syncope and orthostatic intolerance: Role of renin-angitensin system. Am J Med. 1997; 103: 128eC133.
Vernino S, Low PA, Fealey RD, Stewart JD, Farrugia G, Lennon VA. Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies. N Engl J Med. 2000; 343: 847eC855.
Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, Blakely RD, Robertson D. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N Engl J Med. 2000; 342: 541eC549.
Jacob G, Shannon JR, Costa F, Furlan R, Biaggioni I, Mosqueda-Garcia R, Robertson RM, Robertson D. Abnormal norepinephrine clearance and adrenergic receptor sensitivity in idiopathic orthostatic intolerance. Circulation. 1999; 99: 1706eC1712.
Singer W, Shen WK, Opfer-Gehrking TL, McPhee BR, Hilz MJ, Low PA. Evidence of an intrinsic sinus node abnormality in patients with postural tachycardia syndrome. Mayo Clin Proc. 2002; 77: 246eC252.
Dishy V, Sofowora GG, Xie HG, Kim RB, Byrne DW, Stein CM, Wood AJ. The effect of common polymorphisms of the 2-adrenergic receptor on agonist-mediated vascular desensitization. N Engl J Med. 2001; 345: 1030eC1035.
Goldstein DS, Eisenhofer G, Stuff R. Plasma dihydroxyphenylglycerol and the intraneuronal disposition of norepinephrine in humans. J Clin Invest. 1988; 81: 231eC240.
Raine NM, Sneddon JC. A simple water-filled plethysmograph for measurement of limb blood flow in humans. Adv Physiol Educ. 2002; 26: 120eC128.
Dewar JC, Wheatley AP, Venn A, Morrison JF, Britton J, Hall IP. 2-adrenoceptor polymorphisms are in linkage disequilibrium, but are not associated with asthma in an adult population. Clinical & Experimental Allergy. 1998; 28: 442eC448.
Xie HG, Stein CM, Kim RB, Xiao ZS, He N, Zhou HH, Gainer JV, Brown NJ, Haines JL, Wood AJ. Frequency of functionally important -2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals. Pharmacogenetics. 1999; 9: 511eC516.
Rowell LB. Human Cardiovascular Control. New York: Oxford; 1993.
Stewart JM, Montgomery LD. Regional blood volume and peripheral blood flow in postural tachycardia syndrome. Am J Physiol Heart Circ Physiol. 2004; 287: H1319eCH1327.
Stewart JM, Medow MS, Montgomery LD. Local vascular responses affecting blood flow in postural tachycardia syndrome. Am J Physiol Heart Circ Physiol. 2003; 285: H2749eCH2756.
Fouad FM, Thadena-Thome L, Bravo E, Tarazi RC. Idiopathic hypovolemia. Ann Int Med. 1986; 298eC303.
Medow MS, Minson CT, Stewart JM. Decreased microvascular nitric oxide-dependent vasodilation in postural tachycardia syndrome. Circulation. 2005; 112: 2611eC2618.
Goldstein DS, Eldadah B, Holmes C, Pechnik S, Moak J, Sharabi Y. Neurocirculatory abnormalities in chronic orthostatic intolerance. Circulation. 2005; 111: 839eC845.
Hoffman BB. Catecholamines, symapthomimetic drugs, and adrenergic receptor antagonists. In: Hrdaman JG, Limberd LL, eds. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. New York: McGraw-Hill; 2001.
Barbato E, Piscione F, Bartunek J, Galasso G, Cirillo P, De Luca G, Iaccarino G, De Bruyne B, Chiariello M, Wijns W. Role of 2 adrenergic receptors in human atherosclerotic coronary arteries. Circulation. 2005; 111: 288eC294.
Rascado RR, Bendhack LM. Activation of (2)-adrenoceptors is necessary to induce nitric oxide release in isoprenaline-induced relaxation. Vascul Pharmacol. 2005; 42: 63eC68.
Whalen EJ, Johnson AK, Lewis SJ. -adrenoceptor dysfunction after inhibition of NO synthesis. Hypertension. 2000; 36: 376eC382.
Jordan J, Shannon JR, Diedrich A, Black BK, Robertson D. Increased sympathetic activation in idiopathic orthostatic intolerance: role of systemic adrenoreceptor sensitivity. Hypertension. 2002; 39: 173eC178.
Insel PA. Seminars in medicine of the Beth Israel Hospital, Boston. Adrenergic receptorseCevolving concepts and clinical implications. N Engl J Med. 1996; 334: 580eC585.
Cockcroft JR, Gazis AG, Cross DJ, Wheatley A, Dewar J, Hall IP, Noon JP. (2)-adrenoceptor polymorphism determines vascular reactivity in humans. Hypertension. 2000; 36: 371eC375.
Gratze G, Fortin J, Labugger R, Binder A, Kotanko P, Timmermann B, Luft FC, Hoehe MR, Skrabal F. -2 Adrenergic receptor variants affect resting blood pressure and agonist-induced vasodilation in young adult Caucasians. Hypertension. 1999; 33: 1425eC1430.
Hoit BD, Suresh DP, Craft L, Walsh RA, Liggett SB. 2-adrenergic receptor polymorphisms at amino acid 16 differentially influence agonist-stimulated blood pressure and peripheral blood flow in normal individuals. Am Heart J. 2000; 139: 537eC542.
Bray MS, Krushkal J, Li L, Ferrell R, Kardia S, Sing CF, Turner ST, Boerwinkle E. Positional genomic analysis identifies the (2)-adrenergic receptor gene as a susceptibility locus for human hypertension. Circulation. 2000; 101: 2877eC2882.
Reihsaus E, Innis M, MacIntyre N, Liggett SB. Mutations in the gene encoding for the 2-adrenergic receptor in normal and asthmatic subjects. Am Journal of Respiratory Cell & Molecular Biology. 1993; 8: 334eC339.
Lima JJ, Thomason DB, Mohamed MH, Eberle LV, Self TH, Johnson JA. Impact of genetic polymorphisms of the 2-adrenergic receptor on albuterol bronchodilator pharmacodynamics. Clinical Pharmacology & Therapeutics. 1999; 65: 519eC525.
Pereira AC, Floriano MS, Mota GF, Cunha RS, Herkenhoff FL, Mill JG, Krieger JE. 2 adrenoceptor functional gene variants, obesity, and blood pressure level interactions in the general population. Hypertension. 2003; 42: 685eC692.
Schondorf R. Evaluation of the patient with orthostatic intolerance. In: Robertson D, ed. Primer on the Autonomic Nervous System. Amsterdam, The Netherlands: Elsevier; 2004.
Stein CM, Nelson R, Deegan R, He H, Wood M, Wood AJ. Forearm adrenergic receptor-mediated vasodilation is impaired, without alteration of forearm norepinephrine spillover, in borderline hypertension. J Clin Invest. 1995; 96: 579eC585.
Jacob G, Shannon JR, Black B, Biaggioni I, Mosqueda-Garcia R, Robertson RM, Robertson D. Effects of volume loading and pressor agents in idiopathic orthostatic tachycardia. Circulation. 1997; 96: 575eC580., http://www.100md.com(Giris Jacob; Emily M. Gar)