Functional correlates of {alpha}2A-adrenoceptor gene polymorphism in the HANE study

Martin C. Michel, Christopher Plogmann, Thomas Philipp and Otto-Erich Brodde1

Department of Medicine, University of Essen, Essen, Germany

Correspondence and offprint requests to: Dr Martin C. Michel, Nephrology Laboratory IG1, Klinikum, Hufelandstr. 55, D-45122 Essen, Germany.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The aim of this study was to test an association between alleles of the {alpha}2A-adrenoceptor gene with hereditary hypertension and with alterations of lipid and carbohydrate metabolism.

Methods. Genomic DNA was isolated from 147 hypertensive patients and digested with DraI. Genotypes at the {alpha}2A-adrenoceptor were identified by restriction fragment length polymorphism. Genotype at each locus was related to blood pressure, family history of hypertension and various clinical chemistry parameters.

Results. The {alpha}2A-adrenoceptor polymorphism was not significantly associated with blood pressure or a family history of hypertension. Patients with the d allele of the {alpha}2A-adrenoceptor had significantly lower HbA1 (5.6 vs 6.2%, P=0.0344) and HbA1c (3.4 vs 3.9%, P=0.0237) and total cholesterol (212 vs 229 mg/dl, P=0.0333) than those without. Similar trends, which failed to reach statistical significance, were seen for glucose, triglycerides and LDL cholesterol.

Conclusions. We propose that alleles at the {alpha}2A-adrenoceptor locus might contribute to interindividual differences in the regulation of human lipid and glucose metabolism.

Keywords: {alpha}2A-adrenoceptor; cholesterol; gene polymorphism; HbA1



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
{alpha}2A-Adrenoceptors mediate many of the physiological effects of the sympatho-adrenal transmitters, noradrenaline and adrenaline, including presynaptic regulation of transmitter release, central blood pressure and baroreflex modulation, vasoconstriction, modulation of renin release and inhibition of lipolysis and insulin release [1]. The responsiveness to {alpha}2-adrenergic stimulation differs markedly between individuals. Some of this variation may be due to receptor regulation processes, e.g. agonist-induced down-regulation in phaeochromocytoma [2] and end-stage renal disease [3] or following drug treatment [4]. On the other hand, twin studies, e.g. on human platelet {alpha}2-adrenoceptor expression [5] and on sensitivity of vasoconstriction of dorsal hand veins in vivo to noradrenaline [6], demonstrate that interindividual variance of {alpha}2-adrenoceptor responsiveness involves a considerable hereditary component.

During recent years, we have been interested in hereditary effects on {alpha}2-adrenoceptor expression which may relate to the pathophysiology of hypertension and the frequently associated metabolic syndrome [7]. While studies comparing platelet {alpha}2-adrenoceptor density between normotensive and hypertensive subjects have yielded conflicting results [8], three studies consistently have reported that subjects with a family history of hypertension have higher platelet {alpha}2-adrenoceptor expression than those without; this was found in both normotensive and hypertensive subjects and could already be detected in children [911]. In this respect, a `gene dose–response curve' may exist, as children with two essential hypertensive parents have more platelet {alpha}2-adrenoceptors than those with one hypertensive parent who in turn have more than those with two normotensive parents [11]. Moreover, the variance of platelet {alpha}2-adrenoceptor density in children with one hypertensive parent is greater than in children with two normotensive parents [10], as would be expected since only half of these children should have inherited the respective genes. A genetic component of {alpha}2-adrenoceptor regulation has also been suggested in rat studies on renal {alpha}2-adrenoceptor expression (for review, see [8]). Thus, the expression of {alpha}2-adrenoceptors clearly involves a hereditary component and this may relate to the pathogenesis of essential hypertension.

Three subtypes of {alpha}2-adrenoceptors exist in man which are encoded by distinct genes located on human chromosomes 10, 2 and 4, and are designated {alpha}2A, {alpha}2B and {alpha}2C, respectively [12]. The above studies on a hereditary component in the regulation of human {alpha}2-adrenoceptors at the protein level have been performed using platelets, which express a homogeneous population of {alpha}2A-adrenoceptors [1214]. A cDNA encoding the human platelet {alpha}2A-adrenoceptor was cloned originally by Kobilka et al. [15]. The corresponding gene is located in the region q24–q26 of human chromosome 10 [12] and does not contain introns [16]. Using a 5.5 kb fragment of the {alpha}2A-adrenoceptor gene as the probe, two alleles of the human {alpha}2A-adrenoceptor gene can be distinguished by restriction fragment length polymorphism (RFLP) upon digestion of genomic DNA by the endonuclease DraI, while a range of 47 other restriction enzymes did not detect polymorphic restriction fragments [17]. The frequencies of the two alleles appear to be consistent across various Caucasian populations from the US [1719] and Australia [20], while African Americans appear to have a different allele distribution [18,19].

Therefore, the present study was designed to answer two questions: firstly, are alleles at the {alpha}2A-adrenoceptor locus as detected by DraI RFLP associated with a family history of hypertension? Second, do such alleles correlate with parameters which are regulated physiologically by {alpha}2A-adrenoceptors, e.g. those of lipid and glucose metabolism? Thus, we have determined the genotype at the {alpha}2A-adrenoceptor locus in 147 subjects and tested for a possible association of the alleles at this locus with a family history of hypertension and with several metabolic parameters. These subjects were a subgroup of the participants of the HANE study, a multicentre intervention trial on the treatment of essential hypertension [21]. Thus, our study is based solely on hypertensive subjects. This is justified because several previous studies have demonstrated that family history is a better predictor of the hereditary component of {alpha}2A-adrenoceptor expression than the absence or presence of hypertension [911].



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The HANE study was a multicentre intervention trial in essential hypertensive patients, who participated after having given informed written consent [21]. The protocol of the HANE study had been approved by the ethics committee of the German Society of Hypertension. In the HANE trial, blood pressure was determined in all subjects with an automated sphygmomanometric device (Tonoprint, Speidel and Keller, Jungingen, Germany) after they had been free of any blood pressure-related medication and had taken placebo for 2 weeks. On three separate days, five measurements each were taken at 2 min intervals. Patients were included if their mean diastolic pressure ranged between 95 and 120 mmHg on all 3 days. From these patients, Caucasian subjects of either sex living in Austria, Germany or Switzerland were included in the present study if they fulfilled the following criteria: (i) absence of secondary hypertension; (ii) age 20–50 years; and (iii) absence of concomitant congestive heart failure, asthma, diabetes or hypercholesterolaemia requiring drug treatment, Broca-index >130%, hyperuricaemia, severe renal insufficieny or malignomas. Family history of hypertension was determined by the recruiting physicians using a standardized questionnaire. A positive family history was assumed if at least one parent or sibling had developed essential hypertension requiring drug treatment prior to the age of 50 years. Positive family histories were confirmed by telephone conversation with the parent's or sibling's physician. A negative family history was assumed in all other cases. The size of the population sample was chosen to exceed those of previous studies which have detected effects of family history on {alpha}2A-adrenoceptor density [911] or an association between genotype at this locus and physiological parameters [18,22].

{alpha}2A-Adrenoceptor genotyping
Genomic DNA was isolated from 4 ml of EDTA-anticoagulated blood, which had been stored at -70°C, using an extraction procedure with phenol and chloroform/isoamylalcohol (24:1). Samples were stored at 4°C until enzymatic digestion. Approximately 1.5 µg of DNA were digested with the restriction enzyme DraI according to the enzyme manufacturer's instructions. The digests were separated on 0.6% agarose gels, and transferred onto nylon membranes according to Southern [23].

The {alpha}2A-adrenoceptor gene locus probe was prepared using random primer labelling [24] from the 5.5 kb genomic DNA fragment isolated by Kobilka et al. [15] which contains the whole 1350 bp coding region and 5'- and 3'-untranslated regions.

Non-specific DNA-binding sites on the nylon membrane were blocked by pre-hybridization for 2 h at 65°C in pre-hybridization buffer [4x SSPE (0.72 M NaCl, 40 mM NaH2PO4, 4 mM Na2EDTA), 6% polyethylenglycol 20 000, 0.5% SDS, 100 µg/ml salmon sperm DNA, 2x Denhardt solution (0.2 mg/ml Ficoll 400, 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml bovine serum albumin fraction V)]. Following short boiling, aliquots of the labelled probe (~106 c.p.m./ml of pre-hybridization buffer) were added to the nylon membranes. Hybridization was performed under constant agitation at 65°C overnight. Thereafter, the blots were washed using solutions of decreasing ionic strength starting with four times for 5 min with 2x SSC buffer at room temperature and a final stringency of 10 min with 1x SSC buffer at 65°C with 0.1% SDS being added to the SSC buffer. Thereafter, the blots were wrapped in foil and used for exposure of autoradiographic films for 3–5 days at -80°C.

Clinical chemistry
From each patient, a blood sample was obtained after an overnight fast. Plasma renin activity was determined in our laboratory using a commerically available radioimmunoassay (Sorin, Saluggia, Italy). The following serum parameters were determined in the laboratory of Drs W. Eicke and L. Röcker (Berlin, Germany): Na+, K+, Ca2+, Mg2+, inorganic phosphate, creatinine, uric acid, glucose, HbA1, HbA1c, total cholesterol, HDL cholesterol, triglycerides, GPT and alkaline phosphatase. Serum LDL cholesterol was calculated using the equation LDL cholesterol=total cholesterol–HDL cholesterol–(triglycerides/5).

For technical reasons, the measurement of serum parameters was done in two batches of similar size ~1 year apart. The mean values were similar between both batches for most parameters, but a statistically significant difference existed for HbA1 (7.2±0.2 vs 5.0±0.1%, P <0.0001), HbA1c (4.4±0.1 vs 3.2±0.1%, P <0.0001) and HDL (49.7±2.3 vs 44.0±1.7 mg/dl, P=0.0450). The frequency of genotypes at the {alpha}2A-adrenoceptor locus was similar in both batches (data not shown).

Chemicals
Proteinase K, Klenow enzyme and DraI were obtained from Boehringer Mannheim (Mannheim, Germany), Biodyne A nylon membranes from Pall Corp. (Portsmouth, UK), agarose from Bethesda Research Laboratories (Bethdesda, MD), hexanucleotides from Pharmacia Biotec (Freiburg, Germany) and [32P]dCTP from New England Nuclear (Dreieich, Germany).

Data analysis
A small number of patients were excluded from the analysis since their data indicated protocol violations, e.g. total cholesterol of >500 mg/dl or fasting glucose of >180 mg/dl. The statistical significance of differences between patients of differing genotypes was tested using Fisher's exact test for family history and unpaired two-tailed t-tests for the other parameters. All statistical calculations were performed using the InStat program (GraphPAD Software, San Diego, CA), and P <0.05 was considered significant. Corrections for multiple comparisons were not performed, and the resulting P-values are to be interpreted in a descriptive manner. All data are the mean±SEM of n subjects.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Following digestion of genomic DNA by DraI and hybridization with the 5.5 kb {alpha}2A-adrenoceptor probe, bands of 6.3 and/or 6.7 kb were detected (Figure 1Go), and the corresponding alleles were designated `d' and `D', respectively. Out of 147 patients, only four were homozygous for the d allele (dd) of the {alpha}2A-adrenoceptor, 110 were homozygous for the D allele (DD) and 33 were heterozygous (dD). Thus, the genotypes DD, dD and dd were present in ratios of 75:22:3, and the allele frequencies were 86% for D and 14% for d. Thus, genotype distribution did not deviate from Hardy–Weinberg equilibrium. Since the dd genotype was rare, data from dd and dD patients were pooled for all further statistical analysis, in accordance with other previous studies on this polymorphism [18,22].



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1. Autoradiograph of a representative Southern blot in which DraI-digested DNA had been electrophoretically separated and probed with a 5.5 kb genomic fragment from the human {alpha}2A-adrenoceptor gene. Each lane shows digested and probed DNA from one subject.

 
Genotype at the {alpha}2A-adrenoceptor was not significantly related to age, height, body weight or body mass index (Table 1Go) or to plasma renin activity and serum uric acid, creatinine, sodium, potassium, calcium, magnesium, phosphate, GPT or alkaline phosphatase (Table 2Go). Similarly, systolic, diastolic or mean arterial pressure and heart rate did not differ significantly between genotypes within this hypertensive population (Table 1Go). A family history regarding essential hypertension could be obtained in 97 patients, but the prevalence of a positive family history did not differ significantly among genotypes (Table 1Go).


View this table:
[in this window]
[in a new window]
 
Table 1. Physical patient characterization according to genotype
 

View this table:
[in this window]
[in a new window]
 
Table 2. Clinical chemistry values of patients according to genotype
 
Among the indicators of glucose metabolism, HbA1 and HbA1c were significantly lower in those with the d allele of the {alpha}2A-adrenoceptor than in those without (5.6±0.3 vs 6.2±0.2%, P=0.0344 and 3.4±0.2 vs 3.9±0.1%, P=0.0237; Table 2Go). A similar trend was seen for serum glucose (92.6±2.5 vs 95.6±1.6 mg/dl; Table 2Go) but did not reach statistical significance (P=0.3339) with the given number of subjects. Since for unknown reasons HbA1 and HbA1c were higher in the first batch of samples than in the second batch independent of genotypes (see Subjects and methods), we additionally have performed a separate analysis for each batch. Patients with a d allele of the {alpha}2A-adrenoceptor consistently had significantly lower HbA1 (batch 1: 6.4±0.4 vs 7.5±0.2%, P=0.0033; batch 2: 3.9±0.3 vs 4.6±0.1%, P=0.0204) and HbA1c values than those without (batch 1: 4.6±0.2 vs 5.1±0.1%, P=0.0289; batch 2: 2.9±0.2 vs 3.3±0.1%, P=0.0059).

Among the parameters of lipid metabolism patients with a d allele of the {alpha}2A-adrenoceptor had significantly less total cholesterol than those without (212±8 vs 229±4 mg/dl, P=0.0333; Table 2Go). Similarly, triglycerides and LDL were an average of 10 mg/dl and 13 mg/dl, respectively, lower in patients with a d allele than in those without, but these differences did not reach statistical significance (Table 2Go), while HDL was similar in both genotypes (44.6±2.3 vs 47.5±1.8 mg/dl, P=0.3763). A batch-specific analysis of HDL levels did not reveal consistent differences between genotypes at any of the receptor loci (data not shown).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The responsiveness to neurohumoral stimulation differs markedly between individuals. This can be explained partly by pathophysiological or drug treatment-induced intraindividual regulation of receptor expression, a process which has been studied in most detail for ß-adrenoceptors [25]. Since interindividual variation of receptor responsiveness may also involve hereditary factors, the present study has investigated the functional consequences of a polymorphism in the {alpha}2A-adrenoceptor gene. Polymorphisms in the {alpha}2A-adrenoceptor gene primarily have been studied based on RFLP following digestion of genomic DNA with the endonuclease DraI [1720,22]. Based on the size of the probe being used (5.5 kb), the size of the intron-free coding region of the {alpha}2A-adrenoceptor (1.3 kb [15]) and the sizes of the only detectable bands (6.3 and 6.7 kb), the alternative restriction site is unlikely to be located within the coding region of the gene. Indeed work by other investigators has now demonstrated that the second DraI site is found in the 3'-non-coding portion of the {alpha}2A-adrenoceptor gene (Lockette, personal communication). Therefore, these alleles are unlikely to result in structurally different proteins or directly in an altered promoter function. On the other hand, the 3'-non-coding region of a gene can be involved in regulating message stability. In this context, it is interesting that the {alpha}2A-adrenoceptor gene polymorphism involves loss of a DraI recognition site (TTTAAA), and the sequence ATTTA encodes a rapid degradation signal in complementary mRNA. Therefore, it is possible that an alteration in the 3' region of the {alpha}2A-adrenoceptor gene results in an altered expression level due to an enhanced stability of its mRNA. Finally, it is possible that the base exchange recognized by the DraI polymorphism is in linkage disequilibrium with another, as yet undefined base exchange in the {alpha}2A-adrenoceptor gene, which in turns affects the quantity or quality of the resulting protein. This hypothesis, however, remains to be tested in future studies.

The observed frequencies of the two alleles at the {alpha}2A-adrenoceptor gene locus in the present study were very similar to those in three independent Caucasian populations from the US [1719] and one from Australia [20], indicating that a possible founder effect did not markedly affect allele distribution in our study. Since {alpha}-adrenoceptors may be involved in the hereditary component of essential hypertension [7], several groups of investigators have determined whether alleles of the human {alpha}2A-adrenoceptor gene as detected upon DraI digestion of genomic DNA might be associated with essential hypertension. While a significant association between genotype at the DraI site and hypertension in Caucasians from the US was reported in one study [19], two studies with Caucasian populations from Australia [20] and the US [18] have not detected significant differences of allele frequencies at this locus between normotensive and hypertensive subjects. Similarly, the present study has not detected an association of genotype at this locus and blood pressure within a hypertensive population based on Caucasian subjects from Austria, Germany and Switzerland. An altered frequency of the d allele was reported in hypertensive relative to normotensive African Americans in one [18] but not in another study [19]. Therefore, the results regarding an association between genotype at the {alpha}2A-adrenoceptor locus and hypertension are not conclusive at the moment, but the majority of studies in Caucasians including the present one have not detected such an association.

The above conflicting data are not very surprising since studies on altered platelet {alpha}2A-adrenoceptor protein expression in hypertensive patients have also yielded controversial results [8]. On the other hand, an association between family history of hypertension and increased platelet {alpha}2A-adrenoceptors has been reported consistently in normotensive and hypertensive populations [911]. Therefore, we have studied whether the genotypes at the {alpha}2A-adrenoceptor locus are associated with an altered prevalence of a family history of hypertension. However, we have not detected such an association, which is in line with two studies in Caucasian populations from Australia [20] and the US [18], which have investigated the combined effect of hypertension and family history thereof. Therefore, it appears that a family history of hypertension may be associated with increased platelet {alpha}2A-adrenoceptor expression but this is not reflected by the genotype defined by the DraI polymorphism.

Irrespective of a possible association of the {alpha}2A-adrenoceptor gene polymorphism with hypertension or a family history thereof, it may be clinically relevant. Thus, subjects with the d allele were reported to have an enhanced platelet aggregation in response to adrenaline, an increased heart rate response to lower body negative pressure, and a decreased sodium excretion in response to immersion in thermally neutral water [22], findings which are consistent with an enhanced {alpha}2A-adrenoceptor function. These data clearly indicate cardiovascular and renal consequences of the {alpha}2A-adrenoceptor gene polymorphism.

Our data indicate that HbA1 and HbA1c as indicators of glucose metabolism and total cholesterol as an indicator of lipid metabolism are significantly lower in patients with the d allele. Other parameters of glucose and lipid metabolism (glucose, LDL cholesterol, triglycerides) were also lower in such patients but these differences failed to reach statistical significance. It may be argued that the detection of a statistically significant association of genotype at the {alpha}2A-adrenoceptor locus with three parameters may not be surprising in light of the number of parameters which were investigated. While this possibility cannot be excluded, we nevertheless consider the observed associations to be specific for several reasons. Firstly, the same significant association was seen consistently within both batches of patient samples. Second, a trend in the same direction was observed for other parameters of glucose and lipid metabolism, but did not reach statistical significance with the given number of subjects. Third, significant genotype–phenotype associations were observed only for parameters which are regulated by {alpha}2A-adrenoceptors, while parameters which are considered to be unrelated to {alpha}2-adrenoceptors (e.g. uric acid, electrolytes and alkaline phosphatase) were not consistently different between the two groups. Thus, functional studies have shown that inhibition of lipolysis in human adipocytes [26] and inhibition of insulin release in mice [27] and rats [28] occur via an {alpha}2A-adrenoceptor. The {alpha}2A-adrenoceptor also is the dominant {alpha}2-adrenoceptor subtype in the human pancreas at the mRNA level [29]. Fourth, the observed assocations were not found in our population with polymorphisms of the AT1 angiotensin receptor (unpublished observations) or the Y1 neuropeptide Y receptor [30], although the latter is involved in the regulation of lipid and glucose metabolism in similar ways as the {alpha}2A-adrenoceptor [31]. Thus, we propose that alleles at the human {alpha}2A-adrenoceptor gene locus are associated specifically with alterations in parameters of lipid and glucose metabolism and might possibly play a modulatory role in the syndrome of insulin resistance. It might be argued that the observed differences between parameters in patients with and without the d allele are too small to be relevant. However, we feel that the observed size of the differences makes them more plausible as all of the above parameters are fine-tuned by a multitude of regulatory pathways. A very large alteration in any one of these parameters would indicate that {alpha}2A-adrenoceptors play a dominant role in its regulation, which is unlikely for all of the parameters investigated here.

The association of the polymorphism with enhanced platelet responsiveness to adrenaline [22] indicates that it may lead to enhanced {alpha}2A-adrenoceptor function. Since {alpha}2-adrenoceptor stimulation inhibits pancreatic insulin release [27,28], this should result in worsened carbohydrate metabolism, while the opposite was observed in the present study. Moreover, the d allele of the {alpha}2A-adrenoceptor is associated with enhanced platelet aggregation [22] and decreased cholesterol (present study), while previous studies have suggested that high cholesterol is associated with enhanced platelet aggregation in response to {alpha}2-adrenoceptor stimulation [32]. Elucidation of these apparent discrepancies will require further studies.

The associations between genotype at the {alpha}2A- adrenoceptor locus and the various cardiovascular, renal and metabolic parameters could have two causes: firstly, it is possible that the {alpha}2A-adrenoceptor gene locus is in linkage disequilibrium with a nearby locus involved in lipid and glucose metabolism. Secondly, it is possible that the alleles of the {alpha}2A-adrenoceptor gene somehow are related to the function of this receptor and thus indirectly might affect lipid and glucose metabolism. Although we have no definitive proof of this, we consider the first possibility unlikely for several reasons. Numerous gene alterations resulting in a disturbed lipid and/or glucose metabolism have been identified [33] but none of these loci is close to the {alpha}2A-adrenoceptor locus on chromosome 10 q24–q26. Moreover, these alterations are usually associated with large changes in plasma lipids, and our study design has systematically excluded patients with clearly pathological plasma lipids or glucose levels. However, mutations in such genes with smaller effects on lipid or glucose metabolism might have contributed to the background `noise' in our population.

In conclusion, we have confirmed that two major genotypes of the human {alpha}2A-adrenoceptor gene can be detected using RFLP based on the DraI endonuclease. Our data and the majority of the published data indicate that these genotypes are not associated with alterations of blood pressure or family history of hypertension in Caucasian subjects. On the other hand, they are associated with alterations of cardiovascular and renal function [22] and alterations of some parameters of lipid and glucose metabolism which are known to be regulated by {alpha}2A-adrenoceptors. While the molecular basis of the associations remains to be studied, receptor polymorphisms might be important not only pathophysiologically; they might also contribute to interindividual differences in the response to therapeutically administered {alpha}2-adrenoceptor agonists and antagonists.



   Acknowledgments
 
The help of Mrs Annette Kötting, the laboratory of Drs Eicke and Röcker, and the many physicians of the HANE study, who were involved in patient recruitment, are greatly appreciated. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft.



   Notes
 
1 Present address: Department of Pharmalogy, Martin-Luther-University, Halle, Germany. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Ruffolo RRJ, Hieble JP. {alpha}-Adrenoceptors. Pharmacol Ther 1994; 61: 1–64[ISI][Medline]
  2. Brodde O-E, Bock KD. Changes in platelet alpha2-adrenoceptors in human phaeochromocytoma. Eur J Clin Pharmacol 1984; 26: 265–267[ISI][Medline]
  3. Daul AE, Wang XL, Michel MC, Brodde O-E. Arterial hypotension in chronic hemodialyzed patients. Kidney Int 1987; 32: 728–735[ISI][Medline]
  4. Michel MC, Mindermann G, Daul A, Brodde O-E. Effects of antihypertensive therapy on human {alpha}- and ß-adrenoceptors. J Hypertension 1991; 9: 601–606[ISI][Medline]
  5. Propping P, Friedl W. Genetic control of adrenergic receptors on human platelets. Hum Genet 1983; 64: 105–109[ISI][Medline]
  6. Luthra A, Borkowski KR, Carruthers SG. Genetic aspects of variability in superficial vein responsiveness to norepinephrine. Clin Pharmacol Ther 1991; 49: 356–361
  7. Michel MC, Insel PA, Brodde O-E. Renal {alpha}-adrenergic receptor alterations: a cause of essential hypertension? FASEB J 1989; 3: 139–144[Abstract/Free Full Text]
  8. Michel MC, Brodde O-E, Insel PA. Peripheral adrenergic receptors in hypertension. Hypertension 1990; 16: 107–120[Abstract]
  9. Fritschka E, Kribben A, Haller H et al. Familial aggregation of altered adrenoceptor density and free intracellular calcium in patients with essential hypertension. J Cardiovasc Pharmacol 1987; 10 [Suppl 4]: S122–S125[ISI][Medline]
  10. Michel MC, Galal O, Stoermer J, Bock KD, Brodde O-E. {alpha}- and ß-adrenoceptors in hypertension. II. Platelet {alpha}2- and lymphocyte ß2-adrenoceptors in children of parents with essential hypertension. A model for the pathogenesis of the genetically determined hypertension. J Cardiovasc Pharmacol 1989; 13: 432–439[ISI][Medline]
  11. van Hooft IMS, Grobbee DE, Schiffers P, Boomsma F, Hofman A. Raised {alpha}2-receptor with normal ß2-receptor density in youngsters at risk for hypertension. The Dutch hypertension and offspring study. Ric Sci Educ Perm 1989; 76 [Suppl]: 363
  12. Bylund DB. Subtypes of {alpha}1- and {alpha}2-adrenergic receptors. FASEB J 1992; 6: 832–839[Abstract/Free Full Text]
  13. Motomura S, Schnepel B, Seher U, Michel MC, Brodde O-E. Properties of {alpha}2-adrenoceptors in human myometrium and kidney: similarities with human platelets, but differences to rat kidney. J Hypertension 1989; 7 [Suppl 6]: S50–S51[ISI]
  14. Erdbrügger W, Raulf M, Otto T, Michel MC. Does [3H]2-methoxy-idazoxan (RX 821002) detect more alpha-2-adrenoceptor agonist high-affinity sites than [3H]rauwolscine? A comparison of nine tissues and cell lines. J Pharmacol Exp Ther 1995; 273: 1287–1294[Abstract]
  15. Kobilka BK, Matsui H, Kobilka TS et al. Cloning, sequencing, and expression of the gene coding for the human platelet {alpha}2-adrenergic receptor. Science 1987; 238: 650–656[ISI][Medline]
  16. Handy DE, Gavras H. Promoter region of the human {alpha}2A adrenergic receptor gene. J Biol Chem 1992; 267: 24017–24022[Abstract/Free Full Text]
  17. Hoehe MR, Berrettine WH, Lentes K-U. DraI identifies a two allele DNA polymorphism in the human alpha2-adrenergic receptor gene (ADRAR), using a 5.5 kb probe (p ADRAR). Nucleic Acids Res 1988; 16: 9070[ISI][Medline]
  18. Lockette W, Chosh S, Farrow S et al. {alpha}2-Adrenergic receptor gene polymorphism and hypertension in blacks. Am J Hypertension 1995; 8: 390–394[ISI][Medline]
  19. Svetkey LP, Timmons PZ, Emovon O, Anderson NB, Preis L, Chen Y-T. Association of hypertension with ß2- and {alpha}2c10-adrenergic receptor genotype. Hypertension 1996; 27: 1210–1215[Abstract/Free Full Text]
  20. Zee RYL, Morris BJ, Griffiths LR. Association analysis of RFLPs for the {alpha}2- and ß1-adrenoceptor genes in essential hypertension. Hypertension Res 1992; 15: 57–60
  21. Philipp T, Anlauf M, Distler A, Holzgreve H, Michaelis J, Wellek S. Randomised, double blind, multicentre comparison of hydrochlorothiazide, atenolol, nitrendipine, and enalapril in antihypertensive treatment: results of the HANE study. Br Med J 1997; 315: 154–159[Abstract/Free Full Text]
  22. Freeman K, Farrow S, Schmaier A, Freedman R, Schork T, Lockette W. Genetic polymorphism of the {alpha}2-adrenergic receptor is associated with increased platelet aggregation, baroreceptor sensitivity, and salt excretion in normotensive humans. Am J Hypertension 1995; 8: 863–869[ISI][Medline]
  23. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol 1979; 68: 152–176[Medline]
  24. Feinberg AP, Vogelstein A. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984; 137: 266–267[ISI][Medline]
  25. Insel PA. Adrenergic receptors—evolving concepts and clinical implications. N Engl J Med 1996; 334: 580–585[Free Full Text]
  26. Tarkovacs G, Blandizzi C, Vizi ES. Functional evidence that {alpha}2A-adrenoceptors are responsible for antilipolysis in human abdominal fat cells. Naunyn-Schmiedeberg's Arch Pharmacol 1994; 349: 34–41[ISI][Medline]
  27. Angel I, Niddam R, Langer SZ. Involvement of alpha-2 adrenergic receptor subtypes in hyperglycemia. J Pharmacol Exp Ther 1990; 254: 877–882[Abstract]
  28. Niddam R, Angel I, Bidet S, Langer SZ. Pharmacological characterization of alpha-2 adrenergic receptor subtype involved in the release of insulin from isolated rat panreatic islets. J Pharmacol Exp Ther 1990; 254: 883–887[Abstract]
  29. Berkowitz DE, Price DT, Bello EA, Page SO, Schwinn DA. Localization of messenger RNA for three distinct {alpha}2-adrenergic receptor subtypes in human tissues. Anesthesiology 1994; 81: 1235–1244[ISI][Medline]
  30. Michel MC, Hänze J, Bischoff A, Rascher W. Are alleles of the human Y1 neuropeptide Y receptor gene associated with alterations of cardiovascular or metabolic parameters? Br J Clin Pharmacol 1996; 41: 476P
  31. Wahlestedt C Reis DJ. Neuropeptide Y-related peptides and their receptors—are the receptors potential therapeutic drug targets? Annu Rev Pharmacol Toxicol 1993; 32: 309–352
  32. Baldassarre D, Mores N, Colli S, Pazzucconi F, Sirtori CR, Tremoli E. Platelet {alpha}2-adrenergic receptors in hypercholesterolemia: relationship between binding studies and epinephrine-induced platelet aggregation. Clin Pharmacol Ther 1997; 61: 684–691[ISI][Medline]
  33. Rosseneu M, Labeur C. Physiological significance of apolipoprotein mutants. FASEB J 1995; 9: 768–776[Abstract/Free Full Text]
Received for publication: 29. 9.98
Accepted in revised form: 25. 6.99