Angiotensin I–Converting Enzyme Genotypes and Angiotensin II Receptors
Response to Therapy
In the present study, we studied angiotensin II type 1 (AT1) and type 2 (AT2) receptor messengers by quantitative reverse transcriptase–polymerase chain reaction. We examined peripheral blood mononuclear cells from 30 healthy subjects and 50 subjects with primary hypertension, in whom angiotensin I–converting enzyme genotype was determined, before and after 15 days of treatment with different antihypertensive drugs. The medication included a calcium channel antagonist, an angiotensin I–converting enzyme inhibitor, and a β1-blocker. We also studied the relationship between AT1 receptor gene expression and biochemical parameters of the renin-angiotensin system. AT1 receptor messenger levels were positively correlated with plasma renin activity in both normotensive and untreated hypertensive subjects. Increases of this messenger and plasma angiotensin II levels were correlated with the D allele in the same individuals. AT1 receptor messenger levels decreased significantly with angiotensin I–converting enzyme inhibitor treatment in subjects with the DD genotype, and a significant decrease was observed in subjects with the II and ID genotypes treated with a calcium antagonist. No changes were observed in mRNA with the β1-blocker. We conclude that the AT2 receptor is not expressed in peripheral leukocytes and that AT1 receptor messenger levels vary in relation to angiotensin I–converting enzyme genotype and pharmacological treatment. These results suggest that angiotensin I–converting enzyme genotype may be an important factor when deciding on antihypertensive therapy in individuals with primary hypertension.
- receptors, angiotensin II
- angiotensin-converting enzyme
- antihypertensive therapy
- polymerase chain reaction
The renin-angiotensin system is implicated in a wide variety of physiological responses.1 Human AT1 receptor, which belongs to the G protein–coupled receptor superfamily, has been recently cloned2 3 4 5 6 7 8 9 10 and characterized. AT1 receptor is considered to be the mediator for the cardiovascular effects of Ang II. These effects include vascular smooth muscle constriction, aldosterone and catecholamine secretion, renal response,11 pressor and tachycardic responses, and pathogenic vascular and cardiac hypertrophy.12 In contrast, the role of the AT2 receptor, which is highly expressed in growing tissues, remains unclear.13 14 15 Ang II receptors show a great variability and can be detected in a wide variety of tissues; therefore, it is difficult to relate them to primary hypertension. One of these difficulties has been to select a target tissue in humans. Some studies have used platelet Ang II receptors, and only a few of them have examined these receptors in mononuclear leukocytes.16
It has been shown that ACE (kininase II, EC 18.104.22.168) presents a polymorphism consisting of the presence or absence of a 250-bp DNA fragment. This polymorphism has been correlated to serum immunoreactive ACE concentrations, which are higher in the presence of the D allele.17 The relationship between the D allele and the high risk of a cardiovascular event18 and familial hypertension19 has been studied recently. However, the importance of this polymorphism in the onset and treatment of hypertension still needs to be clarified.
Quantification of human Ang II receptors has been done by Scatchard analysis in platelets,20 Northern blotting, and RT-PCR. Gene quantification by PCR is based on a linear fitting of the exponential growth obtained after amplification of different concentrations of the template using a modified internal standard that allows quantification by comparison of bands or extrapolation in a standard curve. We propose a method using an unmodified internal standard identical to a target molecule that simplifies and improves the methodological criteria. This work demonstrates that AT1 receptor mRNA levels can be studied in human PBMCs. This analysis allows us to study the regulation of this gene in different physiopathologic conditions. It is a very useful tool for examining hypertensive individuals as well as the effect of different antihypertensive treatments. On the other hand, AT2 receptor mRNA was undetectable in the same samples.
Normotensive individuals aged between 35 and 50 years were ACE-typed and 30 of them selected to provide equal numbers (n=10) of the II, ID, and DD genotypes. Fifty hypertensive subjects aged between 36 and 49 years consented to take part in this study (Table 1⇓). Subjects with a history of cardiac or hepatic pathology, creatinine clearance less than 1.33 mL/s, diabetes mellitus, or any other cause of secondary hypertension were excluded. Subjects were considered to be hypertensive if they manifested blood pressure values greater than or equal to 150/90 mm Hg after discontinuation of antihypertensive medication for 15 days. Both normotensive and hypertensive subjects gave informed consent before the study, which was approved by the Ethics Committee at the University Hospital.
After the purpose of the study had been explained to the subjects, their medication was stopped for 15 days and they maintained the same daily standard diet (60 mmol sodium, 90 mmol potassium, 10 mmol calcium, 8400 J daily). All the study subjects were ACE-typed by PCR; 100 μL of peripheral blood was taken by finger puncture for this purpose.
Thirty normotensive and 50 hypertensive subjects were divided into three groups according to their individual genotype (II, ID, or DD). The 50 hypertensive subjects were divided into three groups according to the hypertensive drug to be administered (verapamil HCl: II, n=5; ID, n=7; DD, n=5; enalapril maleate: II, n=5; ID, n=7; DD, n=5; and bisoprolol fumarate: II, n=5; ID, n=6; DD, n=5). Each subject group was studied on no medication before treatment and then after treatment with a single hypertensive drug (verapamil HCl, 240 mg/d; enalapril maleate, 20 mg/d; bisoprolol fumarate, 5 mg/d). After 15 days without treatment and after 15 days with treatment, 20 mL of blood was collected. Subjects were kept half an hour in the supine position, and samples were taken for measurement of biochemical parameters and collection of PBMCs. Pulse rate and blood pressure were also measured after a 5-minute rest while subjects were sitting.
DNA was prepared from whole blood (50 μL) that was mixed with 0.5 mL TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0) and spun for 10 seconds at 13 000g. This procedure was repeated twice more, and the final pellet was resuspended in 100 μL digestion buffer (PCR buffer without gelatin or bovine serum albumin containing 0.5% Tween 20 and 100 mg/L fresh proteinase K). Samples were incubated 45 minutes at 56°C and then 10 minutes at 95°C. For PCR, 10 μL was used. PCR conditions were as previously described by Rigat et al.21
Total RNA Isolation
PBMCs were obtained from 20 mL EDTA-anticoagulated blood. Total RNA from these cells was isolated by the method of Chomczynski and Sacchi.22 For elimination of contaminating DNA, all samples were treated with RNAse-free DNAse.23 RNA was quantified by spectrophotometry at 260 nm and then stored as single-use aliquots at −70°C.
Standard Specific mRNA Isolation
To obtain an internal standard for quantitative RT-PCR, we isolated total RNA as described above. Two hundred micrograms total RNA was used for subsequent specific mRNA isolation. Total RNA was added to a clean tube containing extraction buffer (2 mol/L guanidine thiocyanate, 12.5 mmol/L sodium citrate, 3× SSC, 5 mmol/L Tris [pH 7.4], 0.5 mmol/L Na2EDTA, 0.12% sodium dodecyl sulfate, 1% 2-mercaptoethanol) and a constant concentration of the specific biotinylated oligonucleotide (150 pmol) complementary to the AT1 receptor gene (5′-GGCGGGACTTCATTGGGTGAACAATAGCCAGGTATCGATC-3′, MedProbe) and was incubated at 70°C for 5 minutes. This mixture was centrifuged at 12 000g for 10 minutes at room temperature to precipitate the proteins. Streptavidin paramagnetics particles (2 mg) (Promega Corp) were washed three times with 0.5× SSC and added to the former homogenate. After 2 minutes at room temperature, beads were captured with a magnetic rack, and three stringent washes with 0.5× SSC were done. To elute the specific mRNA, 500 μL RNase-free water was added. Samples were concentrated with 3 mol/L ammonium acetate (0.1 vol), 20 g/L glycogen (0.02 vol) (Boehringer Mannheim), and 2 vol 2-propanol. Pellets were redissolved in 10 μL ribonuclease-free water. To obtain enough of the specific mRNA, we carried out these procedures three times and mixed the pellets together. Free solution capillary electrophoresis with a UV detector (Prime Vision, System IV, Europhor) was carried out with a 75-μm capillary 60 cm in length (450 mm to the detector) and TBE buffer (89 mmol/L Tris, 89 mmol/L boric acid, 2 mmol/L Na2EDTA, pH 8.3). Samples (2.7 nL) were introduced into the capillary by high vacuum for 0.5 second. Separation within the capillary was performed under a constant voltage of 40 V/mm.24 The specific mRNA concentration was calculated by integrating the peak area, and then mRNA samples were stored as single-use aliquots at −70°C.
Specific mRNA was amplified by RT-PCR (see below). A 414-bp PCR product was obtained as expected and confirmed by sequencing. For determination of possible contamination, the interleukin-1α and β-actin genes were amplified by RT-PCR, and no bands were obtained.
Primers were selected with the Oligo program (MedProbe). The specific primers for AT1 were as follows: forward, 5′-ACTGGCTGACTTATGCTTTTTACTG-3′; reverse, 5′-AGAAAAGGAAACAGGAAACCCAGTA-3′. The specific primers for AT2 were as follows: forward, 5′-CCTTTTGGCTACTCTTCCTCTATGG-3′; reverse, 5′-TTGGTCACGGGTTATCCTGTTCTTC-3′.
Isolated total RNA was assayed by RT-PCR in a Perkin-Elmer Cetus Thermocycler. Samples of 800 ng total RNA were duplicated. Captured specific mRNA (0.5 pg) was added to one of the tubes as an internal standard (see below). Samples then were mixed with 40 U RNasin (Promega), 1 mmol/L of each dNTP, 60 U Moloney murine leukemia virus RT (Promega), and 2.5 mmol/L random hexamers in 20 μL RT buffer (10 mmol/L Tris [pH 8.3], 50 mmol/L KCl, 1% Triton X-100, 25 mmol/L MgCl2). Samples were incubated at 42°C for 45 minutes and then denatured at 95°C for 10 minutes. Both aliquots were then subjected to amplification with the addition of 40 pmol of the upper primer, 40 pmol of the lower primer, and 2.5 U AmpliTaq DNA polymerase (Promega) in 80 μL of 1× PCR buffer. The amplification protocol was 60 seconds at 94°C, 60 seconds at 54°C, and 120 seconds at 72°C, 30 cycles, and then 7 minutes at 72°C.
PCR products were electrophoresed on an agarose gel and stained with ethidium bromide; the optical density of the band was measured with a Sun Spark computer station with Visage software (Millipore). Furthermore, 15 μL of each PCR product was quantified by measurement of fluorescence intensity with the use of Hoechst reagent 33258 (Polysciences Inc) as dye (excitation and emission wavelengths of 365 and 460 nm, respectively). AT1 mRNA levels were then determined on the basis of the standard curve.
A standard curve of RNA was obtained by study of the amplification process at different cycles. Serially diluted samples were amplified at different cycles, and the optimal cycle (30 cycles) and seven different amounts (from 5×10−2 to 5×10−8 μg) were used to obtain a standard curve. An amount of 0.5 pg was selected as internal standard for additions.
To evaluate the efficiency of the RT reaction, we studied the molar ratio of the obtained cDNA and the amount of the starting specific mRNA for different concentrations of the specific mRNA. The results indicated that under the conditions used, cDNA first strand synthesis had an efficiency of 67.5±5.5% (n=10). To determine the influence of nonspecific RNA in retrotranscription efficiency, we retrotranscribed different amounts of rRNA (0.1 to 1 μg) with 0.5 pg of the specific mRNA; a higher efficiency was shown when 800 ng total RNA was used.
Blood samples for measurement of PRA, Ang II, and aldosterone were collected (with EDTA as anticoagulant), centrifuged immediately, and kept frozen at −70°C until assayed. Plasma Ang II (Nichols Institute) and aldosterone (DPC) concentrations were determined by radioimmunoassay. PRA was estimated with a radioimmunoassay kit for angiotensin I (Nichols Institute).
Results are reported as mean±SD. Independent Student's t test was used except when data before and after treatment were analyzed. In this case, paired Student's t test was used. Regression analysis was performed by the Pearson correlation coefficient.
AT1 receptor mRNA in PBMCs was detected by RT-PCR, whereas AT2 mRNA was not found in the same samples. With reverse transcription, a PCR product for the AT1 receptor gene was clearly detected as a single band that showed the predicted size of 414 bp. When the RT-PCR procedure was carried out in the absence of the enzyme, neither the band nor other recognizable bands were seen. This indicates that the band originated from cDNA and not from genomic DNA, which was presumably digested by the DNAse treatment. We sequenced the PCR product to confirm that it corresponded to the AT1 receptor gene.
AT1 receptor expression was quantified in 30 normotensive subjects divided into three groups (n=10 for each genotype) and 50 hypertensive individuals who were treated with three different antihypertensive drugs: an ACE inhibitor, a β1-blocker, and a calcium antagonist. Additive quantitative PCR is based on the use of an internal standard added to each sample (Fig 1⇓). The use of an internal standard identical to the target sequence represents a control system for RT-PCR efficiency and allows a continuous validation of accuracy based on recovery.
In normotensive subjects, a significant increase in AT1 expression related to the D allele (P<.01) was observed. These values correlated well with PRA levels (r=.66, P<.05) (Fig 2A⇓). In untreated hypertensive subjects, similar results were obtained (r=.72, P<.01) (Fig 2B⇓), although AT1 expression was higher than in normotensive subjects (Table 2⇓).
ACE inhibitor treatment induced a statistically significant decrease of AT1 receptor expression in the DD and ID genotype groups; this decrease was more significant in the DD group. In the II group, the expression was lower but not significantly. PRA and Ang II plasma levels changed significantly only in the DD group after treatment. This was the only group in which Ang II plasma levels decreased after any antihypertensive therapy. On the other hand, ID subjects did not have a significant decrease in Ang II levels after ACE inhibition. Aldosterone did not show significant variations (Table 3⇓).
A significant decrease was observed in AT1 receptor expression in the II and ID groups after calcium channel antagonist treatment; the decrease was not significant in the DD group. In this case, PRA levels increased significantly in all the groups, but no significant variations in Ang II and aldosterone plasma levels were found (Table 3⇑).
The use of a β1-blocker did not produce any significant variation in the studied parameters (Table 3⇑).
The biological effects of Ang II are initiated by the specific recognition of the hormone by membrane receptors. Analysis of both the number and binding affinity of specific receptors appears to be important in the understanding of the pathophysiological role of the renin-angiotensin system in hypertension.
Because of variation in Ang II receptor expression, it is difficult to relate receptor expression to primary hypertension.25 26 27 28 29 30 31 32 33 One of these difficulties has been to select a target tissue for determination of this variability. Several studies25 26 27 28 31 32 33 have used platelet Ang II receptors, and a few studies27 29 30 31 have examined these receptors in mononuclear leukocytes.16 Since these previous studies measured Ang II receptors at the protein level in human blood cells, the possibility that Ang II is taken up by free fluid endocytosis, especially in mononuclear leukocytes, cannot be excluded.27 31 Therefore, the role of Ang II receptors in hypertension has not yet been clarified.
The present work demonstrates that AT1 receptor mRNA can be studied in human PBMCs. The analysis of AT1 receptor gene expression in these cells allows us to study the regulation in different physiopathologic conditions. However, AT2 receptor mRNA was undetectable in the same samples.
In normotensive and untreated hypertensive subjects, AT1 receptor expression was elevated in relation to the D allele.19 This increment coincided with a moderate increase in plasma Ang II levels, suggesting that expression of the AT1 receptor gene is upregulated by plasma Ang II, as in other tissues. Taking into account that ACE activity increases in the presence of the D allele, it is possible to assume that the increment of AT1 receptor is mediated by Ang II.
The most important decrease in AT1 receptor gene expression was found in subjects with the DD genotype when treated with an ACE inhibitor. This decrease coincided with a normalization of blood pressure levels. A decrease in AT1 receptor expression was also observed in the ID genotype group. No changes were found in either AT1 receptor expression or blood pressure in the II genotype group.34
With respect to calcium channel antagonist treatment, a significant decrease in expression was observed in subjects with the II and ID genotypes. In this case, the decrease of blood pressure in subjects with the II genotype was higher and more stable. A significant increase in PRA levels and nonsignificant decrease in plasma Ang II concentration were also observed in all the genotypes, probably because of the well-documented effect of decreasing intracellular calcium.35
As was expected in hypertensive subjects, nonsignificant decreases in AT1 receptor expression and PRA and plasma Ang II levels were observed after treatment with a β1-blocker, since this molecule directly suppresses renin release by deleting renal β1-adrenergic drive.35
The low number of Ang II receptors in PBMCs led us to use a more sensitive method than binding assays. RT-PCR has been widely used for measurement of gene expression. RT-PCR efficiency and detection signal of PCR products change when an internal standard of different size or structure to the wild-type is used.36 Therefore, if in competitive assays the quantification attempts to be closer to the real value, the obtained data need to be corrected by at least two factors: first, the correction that relates to the efficiencies of the RT step and the efficiency of the PCR itself; and second, the correction of the signal when an internal standard of different size is applied irrespective of the detection system (fluorescence, radioactivity, or optical density). In the method described in the present work, the addition of an identical internal standard has two practical features: It increases the measurable signal to the range of the curve of higher sensitivity, and it controls the accuracy in each single sample. Duplicated aliquots with and without an internal standard can be used for calculation of the recovery of the standard in each sample. Moreover, this protocol is simpler than classic competitive PCR because no standards with different concentrations are used for each assay and there is no unnecessary correction of the efficiency and signal detection.
In conclusion, a more significant decrease of AT1 mRNA expression is observed in subjects with the DD genotype when treated with an ACE inhibitor, whereas in subjects with the II genotype, this decrease is higher when a calcium channel antagonist is used. Furthermore, in a screening of ACE-typed subjects under long-term effective antihypertensive treatment, we found that 72% of those who were taking ACE inhibitors had the DD genotype and 58% of those taking calcium channel antagonists had the II genotype (unpublished data, 1995). Although further studies are needed, we think that knowledge of ACE genotype could help physicians when they are establishing antihypertensive treatment.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1, AT2||=||angiotensin II type 1, type 2|
|PBMC||=||peripheral blood mononuclear cell|
|PCR||=||polymerase chain reaction|
|PRA||=||plasma renin activity|
This work was supported by grants PM91-0127 and SAF95-0804 from Programa Nacional de Salud y Farmacia, CICYT (Ministry of Education and Science in Spain).
Reprint requests to Dr Armando Reyes-Engel, Departamento Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Málaga, 29080-Málaga, Spain. E-mail firstname.lastname@example.org.
- Received January 26, 1996.
- Revision received February 29, 1996.
- Revision received March 13, 1996.
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