Introduction

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Adrenergic receptors belong to the superfamily of seven transmembrane domain receptors that produce their effects through coupling with guanine nucleotide-binding proteins (G proteins). In response to activation by the physiologic neurohormones epinephrine and norepinephrine or synthetic adrenergic agonists, these receptors regulate a variety of cellular processes, including cellular metabolism, hormone production, neuronal firing, cardiac function, and blood pressure homeostasis (Raymond et al., 1990; Insel, 1996). -Adrenergic receptors are broadly divided into ₁- and ₂-adrenergic receptors, based on their ability to be activated or blocked by various compounds, i.e., their pharmacological specificity. Each class is further divided into several subtypes (_1A, _1B, _1D; _2A, _2B, _2C), as determined from results of studies with pharmacologic and molecular cloning approaches. The human _1B-adrenergic receptor gene consists of two exons and a single large intron of at least 20 kilobases (Ramarao et al., 1992) (Fig. 1) that interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence consists of 517 amino acids. In previous studies (Liggett et al., 1993; Coteccia et al., 1990) domains of the adrenergic receptors have been identified that are critical for agonist and antagonist binding, G protein coupling, and turning off the receptor signal (termed "desensitization"). From these studies it is clear that small changes in the amino acid sequence can result in substantial changes in the function of the receptors.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Schematic relation of primers to the domain structure of the human 1B-adrenergic receptor gene. Two sets of primers, each, have been used to generate two fragments for exon 1 and exon 2 of the 1B-adrenergic receptor. The binding positions for primers were based on the published sequence 834 to 852 nucleotides (P1, 5'-CGGGGGAAGCAAAGTTTCA-3'), 1581 to 1600 nucleotides (P2, 5'-CGGCAGTACATGACTAGAAT-3'), 1465 to 1483 nucleotides (P3, 5'-CTCTCCTTGGGTGGAA GGA-3'), 1919 to 1938 nucleotides (P4, 5'-AGCTCATCAGTAAACCCAAG-3'), 906 to 929 nucleotides (P5, 5'-GAG AGCTTGACTACTCACAAATTG-3'), 1334 to 1356 nucleotides (P6, 5'-TTCCACTCGGGGAAGGCGCA CAG-3'), 1222 to 1241 nucleotides (P7, 5'-CAAGGACTCGCTGGACGAC-3'), 1534 to 1555 nucleotides (P8, 5'-CAG GGGCATGTTGCTTTTGAAG-3'). Fragments overlapped with at least 100 base pairs. TM, transmembrane-spanning domain.

It has been suggested that there may be an association between different diseases and dysfunctions of adrenergic receptors (Insel, 1996). For example, altered ₁-adrenergic receptors may be involved in prostatic hypertrophy (Shibata et al., 1996), arrhythmias in myocardial ischemia (Billman, 1994), and pathogenesis of essential hypertension (Michel et al., 1989; Cavalli et al., 1997). Studies by Cavalli et al. (1997), who generated mice that lack the _1B-adrenergic receptor, provide strong evidence that the _1B-adrenergic receptor is a mediator of pressor and aortic contractile responses induced by ₁ agonists. Compared with wild-type animals, receptor knockout-mice showed a 45% reduction of the mean arterial pressure (MAP) in response to the ₁-adrenergic agonist phenylephrine.

Interindividual differences in response to phenylephrine in humans has been demonstrated in both normotensive (NT) and hypertensive (HT) subjects (Freedman et al., 1987; Eichler et al., 1989, 1990; Minatoguchi et al., 1995; Tham et al., 1996). With graded infusion of phenylephrine, both the pressor responses and the changes in total peripheral resistance appear to be greater in patients with borderline hypertension than in NT subjects (Minatoguchi et al., 1995). Other data in humans implicate a role for _1B-adrenergic receptors in mediating contraction of peripheral arteries (Hatano et al., 1994) and have suggested a possible role for genetic alterations in _1B-adrenergic receptors in HTs. Kailasam et al. (1998) have reported results from an analysis of first-degree relatives of HT family members of a linkage of -adrenergic pressor response responsiveness with chromosome 5q31-34, a region that contains the _1B-adrenergic receptor. A similar conclusion was reached in a study by Krushkal et al. (1989) who reported on the association between markers in the same chromosome region and intersubjection variation in systolic blood pressure.

Based on such data, we hypothesized that genetic variations in _1B-adrenergic receptors might account for differences in response to phenylephrine in humans. Because no previous data are available regarding such variations, we developed a polymerase chain reaction (PCR)-based method, with sequence analysis of a limited number of PCR fragments for the identification of polymorphisms in the coding sequence of the human _1B-adrenergic receptor. We report herein the results regarding _1B-adrenergic receptor polymorphisms in individuals stratified on the basis of differences in responses to phenylephrine infusion.

	Materials and Methods

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Characteristics of Patients. The subjects of this study were 24 male patients with uncomplicated essential hypertension (12 Caucasians, 12 African Americans) and 21 male NT, first-degree relatives of patients with hypertension (12 Caucasians, 9 African Americans) who were not related to the HT subjects. The diagnosis of hypertension was based on at least three outpatient determinations of diastolic blood pressure 95 mm Hg. Secondary causes of hypertension were excluded by history, physical examination, and laboratory evaluation. Six healthy volunteers with no history of hypertension were used as control. Blood pressure was measured while subjects were supine with an automated Dinamap sphygmomanometer (Critikon Inc., Tampa, FL) on the right arm. ₁-Adrenergic pressor sensitivity was assessed by recording the change in MAP in response to i.v. phenylephrine, as described previously (Wu et al., 1994; Kailasam et al., 1995). "High" response to phenylephrine was defined as rise in MAP 20 mm Hg, whereas "low" response was defined as change in MAP <20 mm Hg. For the 45 subjects, we found a natural cut point at a phenylephrine-promoted MAP of 20 mm Hg by KMEANS cluster anaysis (F = 70.36, p < .001). Each individual underwent a physical examination and gave a written informed consent according to a protocol approved by the Insitutional Review Board at the University of California-San Diego.

Blood Sample Preparation. To obtain human genomic DNA, 3 ml of blood was drawn from six healthy volunteers and the 45 individuals described above, and DNA was isolated with a commercial DNA isolation kit (Puregene; Gentra Systems Inc., Research Triangle Park, NC). Aliquots of 100 ng of purified DNA were stored at 4°C and used for PCR. DNA containing exon 1 and exon 2 of the _1B-adrenergic receptor were amplified by PCR (Perkin-Elmer thermocyler), in which we generated overlapping fragments, fragments A and B of exon 1 and fragments C and D of exon 2 of the coding sequence of the _1B-adrenergic receptor gene (Fig. 1). PCR fragments were generated with a pair of purified forward and reverse oligonucleotide primers, which were designed with "Macvector" software (Oxford Molecular, Oxford, UK) on a Macintosh computer. The sequences of these primers are shown in the legend to Fig. 1. PCR conditions were as follows: 100 ng of human genomic DNA was added to a solution of 1 µM, each, forward and reverse primer, 2.5 mM MgCl₂ (Perkin-Elmer), 1× PCR buffer (50 mM KCl, 10 mM Tris·HCl, pH 8.3; Perkin-Elmer), 0.2 mM, each, deoxynucleotide triphosphate (Pharmacia, Uppsala, Sweden), 5 U of Amplitaq Gold Polymerase (Perkin-Elmer), and distilled H₂0 in a final volume of 100 µl. Temperature cycling proceeded as follows: once at 95°C for 10 min to activate the enzyme, then 95°C for 30 s (naturation), 60°C for 90 s (annealing), and 72°C for 90 s (extension) in 35 cycles, and finally one cycle of extension at 72°C for 10 min. For the G-C-rich region of exon 2 of the _1B-adrenergic receptor, the PCR conditions were slightly modified: 5% dimethyl sulfoxide was added to the PCR and the annealing temperature was increased to 64°C. After completion of the PCR, 1 volume of the PCR products was purified with a Centricon 100 concentrator (Amicon, Bedford, MA). The purified gene fragments were sequenced automatically (ABI automated DNA sequencer, model 377). Oligonucleotides P1, P2, and P3 for exon 1 of the human _1B-adrenergic receptor and P5 and P7 for exon 2 of the _1B-adrenergic receptor were used for sequencing. The PCR primers and conditions reported herein are unique in their ability to generate 300- to 800-bp pieces of the human _1B-adrenergic receptor gene. They were chosen after substantial trial and error in selection of potential primers and experimental conditions for PCR and sequencing.

Data Analysis. Results of the phenylephrine study are expressed as mean ± S.E. A value of p < .05 was considered to be statistically significant. Statistical analyses were performed with the SYSTAT program (Systat Inc., Evanston, IL). Proportions and 95% confidence intervals were computed with the program INSTAT (GraphPAD Software for Sci., San Diego, CA).

	Results

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Results for Phenylephrine Infusion. Characteristics of the group of low and high responders, in terms of response to a 200-µg bolus infusion of phenylephrine are shown in Table 1. The low-response group consisted of 23 individuals and the high-response group consisted of 22 individuals. The two groups were well matched for age, racial composition, body mass index, resting heart rate, baseline systolic blood pressure, diastolic blood pressure, MAP, but differed markedly in phenylephrine responsiveness (p < .001). Subjects were analyzed in further subgroups based on blood pressure status (NT versus HT) and response to phenylephrine [low response (LR) versus high response (HR)]. Blood pressure values for the 21 NT subjects and 24 HT subjects were all significantly different (p < .001): systolic blood pressure (NT, 125 ± 3; HT, 151 ± 3 mm Hg), diastolic blood pressure (NT, 71 ± 2; HT, 92 ± 2 mm Hg), and MAP (NT, 89 ± 2; HT, 112 ± 2 mm Hg). Values for  MAP in response to phenylephrine were NT, LR = 12.4 ± 0.9 mm Hg; NT, HR = 22.7 ± 1.2; HT, LR = 13.8 ± 1.1, and HT, HR = 26.6 ± 1.8. Statistical analysis by two-factor ANOVA yielded a p value of 0.06 for response to phenylephrine between the HT and NT groups.

View this table: [in this window] [in a new window]   TABLE 1 Baseline subject characteristics For continuous variables, values are means ± 1 S.E. Phenylephrine-stimulated effects are expressed as  values of the absolute changes of MAP in mm Hg and refer to a bolus of 200 µg of phenylephrine

Sequencing Results. With the primers and the PCR conditions described above, we generated single, strong PCR products, overlapping the entire coding sequence of the human _1B-adrenergic receptor (Fig. 2). With an automated sequencer, we sequenced these products on both strands for exon 1 from 51 individuals and found only infrequent polymorphisms in exon 1. None of the polymorphisms led to an amino acid substitution. In four individuals we found two different "silent" polymorphisms. The most common polymorphism was located on amino acid position 183 (Gly) in which a guanine (GGG) of the wild type _1B-adrenergic receptor was replaced by adenine (GGA) in a heterozygous or homozygous manner. One of the hypertensive patients was homozygous for this change, whereas one NT and one HT individual were heterozygous. Another "silent" heterozygous polymorphism was identified in one NT individual: guanine (AAG) in nucleic acid position 294 (Lys) was replaced by adenine (AAA). Thus, the four polymorphisms were found in each of two NT and two HT subjects. Repeated sequencing of PCR products of the same individuals was performed on both sense and antisense strands and generally revealed a perfect reliability of our PCR method without requirement for repeat isolation of PCR fragments. As shown in a previous study (Büscher et al., 1998), the method used is capable of discriminating between homozygous and heterozygous polymorphisms. None of the _1B-adrenergic receptors of six healthy volunteers without a family history of hypertension showed a polymorphic site. Thus, exon 1 was sequenced on a total of 102 chromosomes (51 individuals), with a total of five polymorphisms, for a polymorphism rate of 4.9% (95% confidence interval, 0-11%).

View larger version (59K): [in this window] [in a new window]   Fig. 2.   PCR products of the human 1B-adrenergic receptor gene from human genomic DNA templets were subjected to electrophoresis on 1% Seakem GTG-agarose gels. PCR was performed with different primer combinations as described in Fig. 1. Lane A, P1 and P2 (fragment A); lane B, P3 and P4 (fragment B); lane C, P5 and P6 (fragment C); lane D, P7 and P8 (fragment D). Lane A + B indicate exon 1 and lane C + D indicate exon 2 of the coding sequence of the human 1B-adrenergic receptor. Molecular weight standards indicate the positions of all four PCR products.

View larger version (60K): [in this window] [in a new window]   Fig. 3.   Sequencing correction of exon 2 of the human 1B-adrenergic receptor and comparison with previously published sequences. The upper line of each panel refers to data presented in the current manuscript compared with two previously published reports of this region (lower lines). The  over amino acid positions 367 to 369 indicates the position of sequencing differences. The highlighted amino acid 368 is an additional arginine, which we have identified by sequencing exon 2 on both strands. This position is marked with an arrowhead in the single-letter base sequence.

View larger version (72K): [in this window] [in a new window]   Fig. 4.   Untranslated and translated amino acid sequence (present report) for the DNA region encoding the human 1B-adrenergic receptor. Differences with previously published sequences are highlighted in boxes. Arrow, position of the exon-intron junction of the receptor. Corrected sequence was deposited in GenBank.

	Discussion

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Studies on adrenergic receptors other than the _1B-adrenergic receptor, e.g., the ₂-adrenergic receptor (Liggett, 1997) or the ₃-adrenergic receptor (Strosberg, 1997) have demonstrated that small changes in the amino acid sequence can result in substantial changes in the function of the receptors. Moreover, it has been suggested that there may be an association between different diseases and polymorphisms of - and -adrenergic receptors (Walston et al., 1995; Shibata et al., 1996; Svetkey et al., 1996; Kotanko et al., 1997; Liggett, 1997; Büscher et al., 1999). Because no previous data have been available for _1B-adrenergic receptors, this study investigated the hypothesis that polymorphisms are detectable in the coding sequence of the human _1B-adrenergic receptor and that such polymorphisms contribute to altered responses to the _1B-adrenergic agonist phenylephrine and to the hypertensive phenotype. This hypothesis is supported by a study of Krushkal et al. (1998) who showed in an association and linkage study that the region on chromosome 5q31-34 between markers D5S2093 and D5S462 is significantly linked to one or more polymorphic genes that might influence interindividual variation in systolic blood pressure. Comparable findings have been reported by Kailasam et al. (1998). Because the _1B-adrenergic receptor gene is located in this area and this receptor participates in the control of blood pressure, variations in this receptor could contribute to these blood pressure variations. Given the equal and limited expression in NT and HT subjects and among subjects with different responses to phenylephrine, we conclude that frequent polymorphisms of the coding sequence of the _1B-adrenergic receptor appear not to be associated with either variable response to phenylephrine or with essential hypertension.

The rationale for this analysis was based on at least three different observations. First, interindividual differences in response to exogenous phenylephrine, a relatively selective ₁-adrenergic receptor agonist, exist in humans (Freedman et al., 1987; Eichler et al., 1989, 1990; Minatoguchi et al., 1995; Tham et al., 1996). Augmented vascular responsiveness to ₁-adrenergic agonists has been associated with essential hypertension, although this is a controversial finding in that not all groups observe this effect (Freedman et al., 1987; Eichler et al., 1989, 1990; Minatoguchi et al., 1995; Tham et al., 1996). Our own data suggest substantial variability in ₁-adrenergic responsiveness in this cohort consisting of patients with essential hypertension and currently NT subjects at genetic risk of hypertension. Stepniakowski et al. (1996) demonstrated that fatty acids enhance neurovascular reflex responses to ₁-adrenergic receptor activation or phenylephrine infusion and that abnormalities in plasma nonesterified fatty acids may contribute to increased vascular -adrenergic tone in HT patients, thus providing an explanation other than receptor polymorphism for intersubject variability in response to phenylephrine. Second, studies by Cavalli et al. (1997) with a mouse model lacking the _1B-adrenergic receptor provided strong evidence that the _1B-adrenergic receptor contributes to vascular tone and contractile responses induced by ₁ agonists. The current data suggest that although expression of _1B-adrenergic receptor may be important for such effects, hypertension and variations in humans in response to phenylephrine do not frequently result from coding sequence polymorphisms in the receptor. And third, studies have shown that the third intracellular loop of the _1B-adrenergic receptor is responsible for coupling to the G protein and therefore is critical in eliciting the intracellular response to the agonist (Coteccia et al., 1990; Kjelsberg et al., 1992). Mutations in this region of the _1B-adrenergic receptor constitutively activate the receptor, resulting in G protein coupling in the absence of agonist (Kjelsberg et al., 1992). The fact that all 19 possible amino acid substitutions at a single site (amino acid 293, alanine) demonstrate a higher affinity for agonists and increased activation of second messenger pathways suggests that this region may function to constrain G protein coupling of the receptor, a constraint that is relieved by agonist occupancy. We were curious whether we might identify alterations in Ala293, but we did not find changes at this location or in other amino acids in the _1B-adrenergic receptor coding sequence. Thus, although the rationale for the analysis of the _1B-adrenergic receptor as a candidate in human diseases is attractive, the current study indicates that coding sequence polymorphisms are not common, even considering chromosomes derived from African American and Caucasian populations. We had more difficulty in sequencing exon 2 and only obtained unequivocal results from 16 individuals. This relatively small number suggests that we cannot be definitive regarding the observation of polymorphisms in this region but we suggest that the expression of such polymorphisms would not be very common (11% allele frequency). Our findings are perhaps somewhat surprising because the _1B-adrenergic receptor is located close to the ₂-adrenergic receptor and the dopamine D1 receptor on chromosome 5q 31-32 and both of these G protein-coupled receptors are polymorphic (Cichon et al., 1996; Kotanko et al., 1997; Liggett, 1997).

Polymorphisms in the noncoding sequence of a gene occur approximately every 1000 bases and are thought to occur somewhat less frequently for coding sequences (Kruglyak, 1997), values much higher than those for the polymorphisms that we have identified (<1 polymorphism/1554 bases × 102 chromosomes from 51 subjects) in the _1B-adrenergic receptor coding sequence. We conclude that the human _1B-adrenergic receptor, unlike the human ₂-adrenergic and ₃-adrenergic receptor (Liggett, 1997; Strosberg, 1997) is not highly polymorphic. Studies of the human angiotensinogen gene suggest that a nucleotide substitution in the promoter region of the gene is associated with essential hypertension and affects basal transcription in vitro (Inoue et al., 1997). We cannot exclude the possibility that the promoter region of the human _1B-adrenergic receptor is polymorphic, but we have sequenced 20 bases upstream of the 5' initiation codon without identifying a polymorphic site in ~40 patients (data not shown). More detailed studies to characterize this region are in progress.

Based on data obtained thus far we conclude that although phenylephrine response varies in humans, individual variations within the coding region of the human _1B-adrenergic receptor appear not to account for this variable response or for essential hypertension.