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Hypertension. 1995;26:1003-1010

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(Hypertension. 1995;26:1003-1010.)
© 1995 American Heart Association, Inc.

Articles

Gene Expression of Angiotensin II Receptor in Blood Cells of Cushing's Syndrome

Hirotaka Shibata; Hiromichi Suzuki; Tatsuya Maruyama; Takao Saruta

From the Department of Internal Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan.

Correspondence to Takao Saruta, MD, PhD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.


*    Abstract
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 *Abstract
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Abstract The relation between serum cortisol, plasma renin activity, angiotensin II (Ang II), or aldosterone levels and peripheral blood cell (mononuclear leukocytes and platelets) angiotensin II type 1A (AT1A) and 1B (AT1B) receptor mRNA levels was examined in both patients with Cushing's syndrome (seven patients with Cushing's syndrome due to unilateral adrenal cortical adenoma) and control subjects (seven normotensive patients with renal cell carcinoma). Blood was collected from each participant for estimation of plasma renin activity and plasma angiotensin II, aldosterone, and cortisol concentrations and for isolation of mononuclear leukocytes and platelets, which were then used to measure AT1A and AT1B receptor mRNA levels before and after adrenalectomy with the use of reverse transcription–polymerase chain reaction. In patients with Cushing's syndrome, both mononuclear leukocyte and platelet AT1A mRNA levels, which were elevated, were reduced after removal of the adrenal tumors, whereas AT1B receptor mRNA levels of both types of blood cells did not significantly change after adrenalectomy. In contrast, in control subjects, both AT1A and AT1B receptor mRNA levels did not significantly change after unilateral adrenalectomy and nephrectomy. In the adrenal tumors of patients with Cushing's syndrome, gene expression of AT1A receptor was decreased compared with that from adrenals of control subjects. AT1A receptors of the platelets were shown to be upregulated in a manner similar to those of mononuclear leukocytes in patients with Cushing's syndrome. These results suggest that cortisol excess is an important factor upregulating AT1A receptor mRNA levels in human blood cells.


Key Words: receptor, angiotensin II • Cushing's syndrome • glucocorticoid • RNA, messenger • adrenal glands • leukocyte, mononuclear • blood, platelet • blood cells


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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We previously demonstrated that multiple factors contribute to hypertension in patients with CS.1 2 3 Among the factors, the role of the renin-angiotensin system has been considered to be a major factor. For example, the pressor responses to Ang II are enhanced in patients with CS and in experimental animals with glucocorticoid-induced hypertension.1 4 5 Also, glucocorticoid increases Ang II–mediated inositol triphosphate production due to induction of AT1A receptor mRNA and its number in rat cultured vascular smooth muscle cells.6 7 8 These data provide an explanation for the increased sensitivity to Ang II during the glucocorticoid-excess state in rats. However, it has been uncertain how glucocorticoid excess affects the expression of Ang II receptor in humans because it is difficult to access human tissues and methods of evaluating dynamic changes in Ang II receptors. Recently, with the use of the RT-PCR method, characterization and alterations in Ang II receptor mRNA in human platelets and mononuclear leukocytes have been demonstrated in healthy men and in patients with primary or secondary hypertension. In healthy volunteers9 and in patients with primary hypertension,10 plasma Ang II level upregulates AT1A receptor mRNA in mononuclear leukocytes but downregulates the receptor mRNA in platelets, which are used to reflect physiologically important sites such as vascular smooth muscle. In addition to these findings, Ang II receptor gene has been subclassified into AT1A11 12 and AT1B13 14 receptor subtypes in humans as well as in rats and mice. To elucidate the regulation of Ang II receptor in patients with CS, we examined AT1A and AT1B receptor mRNA levels in platelets and mononuclear leukocytes of patients with CS. Also, to compare the levels of Ang II receptor mRNA in blood cells, we determined the levels in adrenal tissues obtained at surgery from patients with CS.


*    Methods
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 up arrowIntroduction
 *Methods
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Patients With CS and Control Subjects
Seven patients with CS due to unilateral adrenal cortical adenoma were studied (Tables 1 and 2). The diagnoses were based on hormonal evaluation and the use of computed tomography and angiography. All patients discontinued their medications, including antihypertensive drugs, for at least 1 week before undergoing adrenalectomy. Preoperative and postoperative blood cell samples (mononuclear leukocytes and platelets) and adrenals (tumor portion and the adjacent adrenals) were used. Preoperative blood cells were collected 1 day before adrenalectomy, and postoperative cells were collected at least 6 months after adrenalectomy. The present study was preapproved by the Human Research Committee, Keio University.1


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  		Table 1. Preoperative Clinical Features of Patients With CS and Control Subjects With Renal Cell Carcinoma


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  		Table 2. Postoperative Clinical Features of Patients With CS and Control Subjects With Renal Cell Carcinoma

Study Protocol
After the nature and purpose of the study were explained and informed consent was obtained, all patients were admitted to the Department of Internal Medicine, Keio University,1 for a period of at least 6 days. Throughout the study, the subjects ingested the same basic diet containing calories (25 kcal/kg body wt), sodium chloride (50 mmol/d), calcium (600 mg/24 h), and potassium (80 mmol/d). All subjects were weighed daily at 7:30 AM, after they had voided and before they had eaten breakfast. Twenty-four–hour urine collection was obtained daily for measurement of creatinine, sodium, and potassium excretion. After the insertion of an indwelling cannula for venous blood sampling and a 60-minute supine rest, blood samples were obtained to determine plasma renin activity; plasma Ang II; aldosterone; cortisol; serum sodium, potassium, and creatinine levels; and hematocrit. Pulse rate and blood pressure were measured after a 5-minute rest, with the participants in the sitting position. A mercury manometer was used by the same observers throughout the study. Korotkoff phase V measurements were accepted as diastolic pressure. Mean arterial pressure was calculated as the sum of diastolic pressure and one third pulse pressure.

Preparation of Mononuclear Leukocytes
Mononuclear leukocytes were prepared from whole blood anticoagulated with heparin. Blood (20 mL) provided sufficient blood cells for performing RT-PCR quantification. The whole blood was centrifuged through a Ficoll/Isopaque solution (specific gravity, 1.077; Pharmacia Biotech AB) for 20 minutes at 200g. The mononuclear/platelet layers were then collected and washed twice with phosphate-buffered saline modified as in previous studies.15 16 The suspensions were then centrifuged for 5 minutes at 100g. When prepared in this way, the blood cells included <2% red blood cells and 90% mononuclear leukocytes with <10% platelets. The cells were finally centrifuged at 1000g for 10 minutes and frozen at -80°C until assay.

Preparation of Platelets
Approximately 30 to 40 mL of blood was drawn and anticoagulated with sodium citrate. Platelet-rich plasma was prepared by centrifugation at 100g for 10 minutes at 22°C. Platelet-rich plasma was removed, washed with 20 vol Medium 199 buffer (GIBCO BRL) containing 5 mmol/L EDTA and 0.2% bovine serum albumin, and centrifuged at 1000g for 10 minutes. Washing and centrifugation were repeated once, and the platelet-poor plasma was aspirated and discarded. There was <0.1% erythrocytes and leukocytes within the platelet preparation. The platelet pellet was frozen in liquid nitrogen and stored at -80°C.15 17 18 19 20 21 22

Preparation of Adrenals
The adrenal glands bearing tumors were bisected and separated into the tumor and the adjacent adrenal and then immediately placed in liquid nitrogen and frozen at -80°C.23 24 25 26 The adrenal glands of control subjects were separated into the core and the capsular layers. For measuring AT1 receptor mRNA, the capsular layers, which are considered to be primarily zona glomerulosa, were used. The histological appearance of adrenal tumors in patients with CS was very typical. Microscopically, all tumors contain cells with pale, lipid-rich cytoplasm similar to that of fasciculata-type cells plus cells with compact eosinophilic cytoplasm resembling that of the zona reticularis. However, the adrenal gland, which was resected with Grawitz tumor, had an apparently normal histological appearance, ie, no invasion, necrosis, or metastasis of renal cell carcinoma in the adrenal gland.

Quantification of mRNA With RT-PCR
Total cellular RNA was extracted from human blood cells and the adrenal glands with the use of guanidinium thiocyanate followed by centrifugation in cesium chloride solutions.27 Total RNA (10 µg) was used for the RT-PCR procedure. To eliminate contaminating genomic DNA, we first treated the prepared total cellular RNA samples with 20 U RNase-free DNase (Stratagene Cloning System) at 37°C for 30 minutes. The reaction was stopped by extraction with phenol/chloroform (1:1 vol/vol), and the RNA samples were precipitated with ethanol, vacuum-dried, and resuspended in RNase-free water. Contamination of genomic DNA was eliminated by subjecting the DNase-treated RNA directly to PCR amplification, in which no significant product was synthesized. RNA from each patient was reverse-transcribed as follows. Each sample was prepared to contain 10 µg total cellular RNA, 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 0.5 mmol/L MgCl2, 10 mmol/L dithiothreitol, 0.5 mmol/L of each dNTP (dATP, dTTP, dGTP, and dCTP), 20 U placental ribonuclease inhibitor (TaKaRa Shuzo Co), 100 pmol random hexamer (TaKaRa), and 200 units of Moloney murine leukemia virus reverse transcriptase in a final volume of 20 µL. After incubation at 37°C for 60 minutes, the samples were heated for 5 minutes at 94°C to terminate the reactions and then stored at -20°C until use. The primers were synthesized with the use of a DNA synthesizer (model 39A, Applied Biosystems, Inc). Oligonucleotide primers were constructed from the published cDNA sequences of AT1A11 12 and AT1B13 14 receptors and GAPDH28 cDNA. The sequences of the AT1A receptor primers were (1) 5'-GGCCAGTGTTTTTCTTTTGAATTTAGCAC-3' (coding sense), corresponding to bases 186 to 214 of the cloned full-length sequence, and (2) 5'-TGAACAATAGCCAGGTATCGA TCAATGC-3' (anticoding sense), which anneals to bases 368 to 395. The sequences of the AT1B receptor primers were (1) 5'-CAGGCAGCAGCGAAGTGAAC-3' (coding sense), corresponding to bases -228 to -209 of the cloned full-length sequence, and (2) 5'-GCGCTCTATGTCGGGTCTAC-3' (anticoding sense), which anneals to bases -86 to -67. The sequences of the GAPDH primers were (1) 5'-CCCATCACCATCTTCCAGGAG-3' (coding sense), corresponding to bases 211 to 231 of the cloned full-length sequence, and (2) 5'-GTTGTCATGGATGACCTTGGC-3' (anticoding sense), which anneals to bases 475 to 495. The predicted sizes of the amplified AT1A and AT1B receptors and GAPDH cDNA products were 210, 162, and 284 bp, respectively. Each sample contained the upstream and downstream primers (0.2 mmol/L of each primer) spanning the given sequence for amplification, 200 µmol/L of each dNTP (dATP, dTTP, dGTP, and dCTP), 50 mmol/L KCl, 10 mmol/L Tris·HCl, pH 8.3, 10 mmol/L MgCl2, 0.01% (wt/vol) gelatin, and 2.5 U Taq DNA polymerase. [{alpha}-32P]dCTP (0.5 µL; 1.85x1014 Bq/mmol; Amersham International) was included in the PCR mixture for autoradiography. The reaction mixture was then overlaid with 3 drops (approximately 60 µL) of mineral oil and amplified in a Perkin-Elmer-Cetus thermal cycler. The amplification profiles consisted of denaturation at 94°C for 1 minute, primer annealing at 65°C for 30 seconds, and extension at 72°C for 1 minute. After completion of RT-PCR, each amplified DNA was electrophoresed through a 4% (wt/vol) polyacrylamide gel. The gels were dried on a filtration paper and quantified with a laser image analyzer (model BAS2000, Fuji Film Co).9 10 29 30 To confirm that the products were AT1 receptor and GAPDH cDNAs, the products were sequenced. PCR products of AT1 receptor and GAPDH from mononuclear leukocytes, platelets, and adrenals were subcloned into pBluescript KS(+) vector (Stratagene), and several clones were then sequenced using the dideoxynucleotide chain-termination reaction described by Sanger et al.31

Northern Hybridization Analysis
The total RNA was resolved electrophoretically on 1% agarose–5.6% formaldehyde gels in a buffer containing 20 mmol/L 3-(N-morpholino)-propanesulfonic acid, 5 mmol/L sodium acetate, and 1 mmol/L EDTA disodium salt. On transfer to nylon membranes (Hybond N+, Amersham International), the blots were prehybridized and then hybridized with AT1A (2.4 kb [a generous gift from Dr T. Inagami, Department of Biochemistry, Vanderbilt University School of Medicine]), AT1B receptor (the 5'-noncoding region of AT1B receptor cDNA -239 to -1 bp),13 14 and GAPDH28 cDNA probe at 65°C in a buffer containing 0.75 mol/L NaCl, 45 mmol/L NaH2PO4·H2O, 5 mmol/L EDTA, 5x Denhardt's [0.1% bovine serum albumin, 0.1% poly(vinyl pyrrolidone), and 0.1% Ficoll], 0.5% sodium dodecyl sulfate, and 20 mg/mL denatured salmon sperm DNA. The hybridized filters were washed for 30 minutes at 65°C in 2x SSPE (1x SSPE contains 150 mmol/L NaCl, 10 mmol/L NaH2PO4, and 1 mmol/L EDTA) and 0.1% sodium dodecyl sulfate and for 30 minutes at 65°C in 0.5x SSPE and 0.1% sodium dodecyl sulfate. They were exposed to a BAS 2000 imaging plate (Fuji Film) and quantified with a BAS 2000 laser image analyzer (Fuji Film).9 10 29 30

Hormone Measurements
Blood samples for measurement of plasma renin activity and plasma Ang II, aldosterone, and serum cortisol concentrations were withdrawn, both with and without EDTA as anticoagulant; centrifuged immediately; and kept frozen at -80°C until assay. The plasma Ang II concentration was determined by radioimmunoassay (ITS Angiotensin II Kit, Nichols Institute). The mean intra-assay and interassay coefficients of variation were 6.9% and 6.8%, respectively. Plasma renin activity was estimated with a radioimmunoassay kit for angiotensin I (Dainabot Ltd). The assay sensitivity was 0.03 mg per tube, and the mean intra-assay and interassay coefficients of variation were 7.5% and 6.2%, respectively. The plasma aldosterone concentration (RIA Kit, Daiichi Radioisotope) was determined by radioimmunoassay. The assay sensitivity was 50 pmol/L, and the mean intra-assay and interassay coefficients of variation were 9.1% and 9.4%, respectively. The serum cortisol concentration (RIA Kit, Daiichi Radioisotope) was estimated by radioimmunoassay. The assay sensitivity was 27.6 nmol/L, and the mean intra-assay and interassay coefficients of variation were 5.1% and 3.0%, respectively.

Statistical Analysis
Results are presented as mean±SEM. Unpaired and paired t tests were used to test for differences. A value of P<.05 was considered to indicate statistical significance.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of RT on Human AT1 Receptor and GAPDH mRNA Amplification
Detection of AT1 receptor mRNA in human blood cells was possible with RT-PCR (Fig 1) but not with Northern blot analysis because AT1 receptor expression may be very low in human blood cells. With RT, we detected a clear single band that had the predicted sizes of 210 and 162 bp for human AT1A and AT1B receptors, respectively, and we detected a clear single band that had the predicted size of 284 bp for human GAPDH. When the PCR procedure was carried out in the absence of RT, the 210-, 162-, and 284-bp bands were not seen, and there was no other recognizable band. This indicated that the 210-, 162-, and 284-bp bands originated from mRNA and not from genomic DNA, which presumably was digested by the DNase treatment. To confirm that the PCR products were authentic AT1 receptor and GAPDH cDNAs, the PCR products were sequenced. Fragments of PCR products were gel purified and inserted into pBluescript II KS(+) (Stratagene), and subcloned clones were analyzed with an automated fluorescence-based sequencing system (GENESIS TM 2000, Applied Biosystems, Inc) with a fluorescent sequencing technique.



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  		Figure 1. Blots showing human AT1A and AT1B receptors and GAPDH PCR products with and without RT. Left, PCR carried out without RT. Right, PCR carried out with RT.

Relation Between Quantity of Starting Material and That of Amplification Product for Human AT1A and AT1B Receptors and GAPDH mRNA in Human Mononuclear Leukocytes and Platelets
Fig 2 shows data from an experiment in which we performed an analysis of AT1A and AT1B receptors and GAPDH cDNA with semilogarithmic plots. If PCR was used as a template of human mononuclear leukocyte or platelet cDNA, a linear relation between PCR cycle number and AT1A or AT1B receptor cDNA as PCR products could be obtained from 28 to 31 cycles, and we selected 30 cycles of PCR to analyze the AT1 receptor in human mononuclear leukocytes and platelets. In a similar manner, a linear relation between the PCR cycle number and GAPDH cDNA as PCR product was obtained from 17 to 23 cycles in human mononuclear leukocytes and platelets, and we selected 20 cycles to analyze the GAPDH in human blood cells. At the selected PCR cycles, linear regression analysis revealed strong correlations between the PCR product and quantity of human mononuclear leukocyte total RNA (r=.99). GAPDH primers were added to the reaction tubes after 10 cycles, and amplification was continued for 20 additional cycles. The 30 cycle products within the linear logarithmic phase of the amplification curve were analyzed. GAPDH was used as an internal standard, and the reaction was performed in the same tube as the specific AT1A or AT1B reaction. For platelet AT1 receptor and GAPDH mRNA, similar strong correlations between the AT1 receptor or GAPDH PCR product and quantity of platelet total RNA was obtained (r=.99) (data not shown).



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  		Figure 2. Line graphs showing cycle- and dose-dependent amplification of AT1A receptor, AT1B receptor, and GAPDH mRNA in human mononuclear leukocytes. The y axes show semilogarithmic plots of RT-PCR products of (a) AT1A receptor, (b) AT1B receptor, and (c) GAPDH mRNA, which were measured with a laser image analyzer. d through f, Linear relation between the input of total RNA quantity and the resultant RT-PCR product. cDNA was synthesized from 1000, 750, 600, 500, 250, and 100 ng of total RNA obtained from human mononuclear leukocytes, and AT1A or AT1B receptor cDNA was amplified at 30 PCR cycles (d and e). Similarly, cDNA was synthesized from 100, 75, 50, 25, and 10 ng total RNA obtained from human mononuclear leukocytes, and GAPDH cDNA was amplified at 20 cycles (f). a: {circ}, 100 ng RNA; {bullet}, 1000 ng RNA. b: {square}, 100 ng RNA; {blacksquare}, 1000 ng RNA. c: {triangleup}, 10 ng RNA; {blacktriangleup}, 100 ng RNA. d: r=.99. e: r=.99. f: r=.99. PSL indicates photostimulated luminescence.

Measurement of AT1A and AT1B Receptor mRNA Levels in Human Blood Cells of Patients With CS
The clinical profiles of patients with CS and control subjects with renal cell carcinoma are shown in Tables 1 and 2. Levels of AT1A and AT1B receptor mRNAs were examined before and after adrenalectomy in the mononuclear leukocytes and in platelets of patients with CS and control subjects. Both mononuclear leukocyte and platelet AT1A but not AT1B receptor mRNA levels were significantly higher in patients with CS than in control subjects (Fig 3). Furthermore, the receptor mRNA levels returned to control levels after adrenalectomy in the same individuals with CS (P<.01). The mRNA levels did not significantly change after nephrectomy and adrenalectomy in the control subjects (Fig 4). However, mRNA levels of AT1A and AT1B receptors in both types of blood cells partially correlated with blood pressure levels in patients with CS (data not shown).



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  		Figure 3. Blots showing gene expression of AT1A, AT1B, and GAPDH in human mononuclear leukocytes (MNL) and platelets (PLT) of patients with CS (Cushing) and control subjects (Control) with renal cell carcinoma with the use of RT-PCR. Control: Lane 1, preoperative MNL and PLT of control patient 1; lane 2, postoperative MNL and PLT of control patient 1; lane 3, preoperative MNL and PLT of control patient 2; and lane 4, postoperative MNL and PLT of control patient 2. Cushing: Lane 1, preoperative MNL and PLT of patient 1 with CS; lane 2, postoperative MNL and PLT of patient 1 with CS; lane 3, preoperative MNL and PLT of patient 2 with CS; and lane 4, postoperative MNL and PLT of patient 2 with CS.



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  		Figure 4. Line graphs showing serial changes in AT1A and AT1B receptors: GAPDH mRNA ratio between adrenalectomy in ({circ}) mononuclear leukocytes and ({bullet}) platelets of patients with CS (Cushing's syndrome) and control subjects (Control) with renal cell carcinoma. *P<.01, values are significantly different between adrenalectomy.

Measurements of AT1A and AT1B Receptor mRNA Levels in Adrenals of Patients With CS and in Control Subjects With Renal Cell Carcinoma
The gene expression of human AT1A and AT1B receptors in cortisol-producing adrenal cortical adenomas and normal control adrenals as determined with Northern blot analysis is illustrated in Fig 5. The results show that gene expression of AT1A receptor in the tumor portions of cortisol-producing adenomas was significantly lower than in the control adrenals (by approximately one third). The gene expression of AT1B receptor was detected in cortisol-producing adenomas as well as in control adrenals; however, the levels were not significantly changed.



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  		Figure 5. Blots showing Northern blot analysis of AT1A and AT1B receptors and GAPDH in adrenals of CS (Cushing) and normal adrenals with renal cell carcinoma (Control). Control: Lanes 1, 2, and 3, control subjects 1, 2, and 3. Cushing: Lanes 1, 2, and 3, CS patients 1, 2, and 3 with adrenal tumors.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we investigated gene expression of AT1A and AT1B receptors in platelets and mononuclear leukocytes of patients with CS and of control subjects with renal cell carcinoma. The results demonstrated that AT1A but not AT1B receptor mRNA levels were significantly higher in preoperative platelets and mononuclear leukocytes of patients with CS than in those of control subjects. Furthermore, AT1A receptor mRNA levels in both blood cell types of patients with CS decreased after adrenalectomy to control levels, whereas those of control subjects did not change significantly. These results strongly suggest that a humoral factor other than Ang II, in particular chronic cortisol excess, plays an important role in upregulation of AT1A receptor in blood cells of patients with CS.

Regulation of Ang II receptor in human blood cells has been reported. Several studies have used platelet Ang II receptor,15 17 18 19 20 21 22 and a few studies have examined the receptor of mononuclear leukocytes.15 16 32 33 These studies measured Ang II binding in human blood cells indicating possibly that Ang II is taken up by free fluid endocytosis.15 33 In the present study, AT1 receptor mRNA was detected in platelets and mononuclear leukocytes, suggesting that the AT1 receptor levels that we measured reflect de novo synthesized receptors that were not taken up by endocytosis.

To examine Ang II receptor expression in situ, we measured the receptor mRNA levels with the use of RT-PCR in human blood cells. In healthy volunteers9 and in primary hypertensive patients,10 plasma Ang II level might be one of the determinant factors of Ang II receptor expression in human blood cells, ie, plasma Ang II level downregulates AT1 receptor mRNA in platelets, whereas it upregulates the receptor in mononuclear leukocytes.

In patients with primary aldosteronism and renovascular hypertension, both platelet and mononuclear leukocyte AT1 receptor mRNA levels were upregulated, and the levels were reduced after removal of adrenal tumor or correction of the renal artery stenosis.10 These results suggest that a humoral factor other than plasma Ang II, aldosterone excess in particular, might contribute to upregulation of AT1 receptor. However, the effects of glucocorticoids on the regulation of human AT1 receptor are not yet known.

CS is caused by excessive glucocorticoid production in adrenal tumor or hyperplasia and is characterized by factors such as truncal obesity, hirsutism in women, plethora, red striae, and impaired glucose tolerance. Hypertension is present in {approx}80% of patients with CS. Multiple mechanisms might contribute to the pathogenesis of this hypertension1 3 4 5 34 35 36 37 ; it has been demonstrated that the increased levels and activity of the renin-angiotensin system are most important among the mechanisms.1 Dalakos et al38 reported that the Ang II receptor antagonist salarasin produced a marked fall in blood pressure in patients with CS. In our previous reports,1 4 infusion of salarasin and oral administration of the Ang I–converting enzyme inhibitor captopril reduced blood pressure in dexamethasone-treated rats. In humans, however, only captopril reduced blood pressure of patients with CS; the Ang II antagonist was ineffective. Taken together, hypertension in CS is known to be at least in part responsive to renin-angiotensin inhibition. The regulation of Ang II receptor expression in CS is not well known.

Human platelets have been used to study regulation of Ang II receptor in humans and are considered to reflect the vascular Ang II receptor, which is a physiologically important site.17 18 21 In seven patients with CS, AT1A receptor mRNA levels in preoperative platelets and mononuclear leukocytes were significantly higher than levels in control subjects. Furthermore, after adrenalectomy, the AT1A receptor mRNA levels in both blood cell types significantly decreased to control levels in the same individual, suggesting that chronic glucocorticoid excess upregulated AT1A receptor expression.

In the adrenal tumors of patients with CS, gene expression of AT1A receptor was significantly lower by approximately one third compared with that in adrenals of control subjects. Takayanagi et al12 demonstrated that AT1 receptor mRNA levels were significantly higher in a CS patient with adrenal tumor compared with levels in the adrenals of control subjects. However, Opocher et al39 reported that AT1 receptor number decreased in three CS patients with adrenal tumors compared with that in the adrenals of control subjects. Therefore, the expression of AT1 receptor in adrenal tumors of CS patients is controversial. How cortisol-producing adenoma responds to Ang II in vitro and in vivo remains to be elucidated. Our results were consistent with those of Opocher et al, but the reason for downregulation of AT1A receptor is unknown. Downregulation of AT1A receptor in the adrenal tumors could be explained by tissue-specific regulation by glucocorticoid excess. Alternatively, as we previously reported, cytochromes P-45017{alpha} and P-450C21 were overexpressed in the adrenal tumors of CS patients,24 26 so downregulation of AT1A receptor in the tumors might be related to the tumorigenic process.

There are several points to be addressed in the present study. First, what is the physiological significance of Ang II receptors in human blood cells? The role of platelet and mononuclear leukocyte Ang II receptors is not well established. The platelet Ang II receptor concentration is approximately 10-fold lower than that of the adrenal glomerulosa receptor; however, potentiation of epinephrine-induced platelet aggregation by Ang II has been reported.40 On the other hand, Keidar et al41 demonstrated that Ang II might enhance lipid peroxidation of low-density lipoprotein and foam-cell formation acting on mononuclear cell Ang II receptor. The physiological function of Ang II receptor in human blood cells remains to be elucidated.

Second, several investigators have found only a single AT1 receptor in humans that differs from that in rodents. Very recently, a novel subtype of human AT1 receptor (AT1B) was cloned by Konishi et al.13 14 According to the cloning sequence of AT1B receptor, high nucleotide sequence homology was noted in the open reading frame (98.1%) and the 3'-untranslated region (98.5%) between AT1A and AT1B receptor cDNAs; however, no significant sequence homology (52.0%) was noted in the 5'-untranslated region. Although the idea of two AT1 receptor subtypes existing in the human is controversial, we tried to examine AT1B receptor mRNA levels with primers corresponding to the 5'-untranslated region. Both AT1A and AT1B receptor mRNAs can be detected in platelets and mononuclear leukocytes; however, only AT1A receptor expression was dynamically changed after adrenalectomy. It is, however, necessary to further investigate the pathophysiological role of the novel subtype of AT1 receptor.

Third, to detect a small amount of mRNA, RT-PCR was used. This method is widely used in many areas; however, there are some problems in identification and quantification. To adjust the interassay variabilities, target and GAPDH internal standard cDNAs were coamplified within the same tube. We compared AT1 receptor expression as a ratio of AT1 receptor to GAPDH. This method has been shown to be as quantitative as the competitive mutant template PCR method.42 43 44

Finally, although our preparation of mononuclear cells is accepted in the literature,15 16 glucocorticoids may cause some artifact through compositional change of unfractionated mononuclear cells. Therefore, the effects of glucocorticoids on AT1 receptor expression in fractionated mononuclear cells remain to be elucidated.

In summary, (1) gene expression of AT1A receptor is upregulated in human blood cells of patients with CS and (2) the receptor levels decreased after adrenalectomy in the same individuals. These data suggest that chronic glucocorticoid excess plays a more important role than the renin-angiotensin system in the upregulation of AT1A receptor in human blood cells.


*    Selected Abbreviations and Acronyms
 
Ang II = angiotensin II
AT1A = angiotensin II type 1A
AT1B = angiotensin II type 1B
CS = Cushing's syndrome
GAPDH = glyceraldehyde-3-phosphate dehydrogenase
PCR = polymerase chain reaction
RT = reverse transcription


*    Acknowledgments
 
This work was supported in part by grants-in-aid for Disorders of Adrenal Hormones from the Ministry of Health and Welfare of Japan and for Encouragement of Young Scientists from the Ministry of Education, Science and Culture of Japan. We thank Tadashi Inagami (Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn) for the generous gift of AT1 receptor cDNA probe.

Received March 2, 1995; first decision March 30, 1995; accepted August 3, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Saruta T, Suzuki H, Handa M, Igarashi Y, Kondo K, Senba S. Multiple factors contribute to the pathogenesis of hypertension in Cushing's syndrome. J Clin Endocrinol Metab. 1986;62:275-279. [Abstract]
  2. Okuno T, Suzuki H, Saruta T. Dexamethasone hypertension in rats. Clin Exp Hypertens A. 1981;3:1075-1086.
  3. Whitworth J. Mechanisms of glucocorticoid-induced hypertension in rats. Kidney Int. 1987;31:1213-1224. [Medline] [Order article via Infotrieve]
  4. Suzuki H, Handa M, Kondo K, Saruta T. Role of renin-angiotensin system in glucocorticoid-induced hypertension. Am J Physiol. 1982;243:E48-E51. [Abstract/Free Full Text]
  5. Nakamoto H, Suzuki H, Kageyama Y, Ohishi A, Murakami M, Naito M, Saruta T. Characterization of alterations of hemodynamics and neuroendocrine hormones in dexamethasone induced hypertension in dogs. Clin Exp Hypertens A. 1991;13:587-606. [Medline] [Order article via Infotrieve]
  6. Sato A, Suzuki H, Iwaita Y, Nakazoto Y, Kato H, Saruta T. Potentiation of inositol trisphosphate production by dexamethasone. Hypertension. 1992;19:109-115. [Abstract/Free Full Text]
  7. Sato A, Suzuki H, Murakami M, Nakazato Y, Iwaita Y, Saruta T. Glucocorticoid increases angiotensin II type 1 receptor and its gene expression. Hypertension. 1994;23:25-30. [Abstract/Free Full Text]
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