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25 Glucocorticoid Increases Angiotensin II Type 1 Receptor and Its Gene Expression Atsuhisa Sato, Hiromichi Suzuki, Marohito Murakami, Yuichi Nakazato, Yasushi Iwaita, Takao Saruta Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Abstract Recently, we demonstrated that glucocorticoid potentiates inositol trisphosphate production evoked by angiotensin II in vascular smooth muscle cells. To clarify this mechanism, we investigated the effects of dexamethasone on the modulation of angiotensin II type 1 receptor and on postreceptor mechanisms in vascular smooth muscle cells. The number of angiotensin II type 1 receptors began to increase after 12 hours' incubation with dexamethasone. After 48 hours, the B m value reached 27±3 fmol/mg protein in dexamethasone-treated cells and 15±3 fmol/mg protein in control cells. However, binding affinity did not change. A glucocorticoid antagonist, RU 38486, completely blocked these effects of dexamethasone. Also, to elucidate the effects of dexamethasone on postreceptor mechanisms, GTP analogue-induced inositol trisphosphate production in permeabilized cells was examined. Pretreatment with 1 ^tmol/L dexamethasone for 48 hours did not affect these inositol trisphosphate productions. Moreover, dexamethasone had no effect on the level of G,^ protein. Furthermore, steady-state levels of angiotensin II type 1 receptor messenger RNA were increased 2.2±0.3-fold after 30 minutes' exposure to 1 /imol/L dexamethasone and 7.8±0.4-fold after 24 hours. We conclude that glucocorticoid induced expression of the angiotensin II type 1 receptor gene and resulted in an increase in the number of angiotensin II type 1 receptors through the glucocorticoid-specific receptor, without significant effect on postreceptor systems in vascular smooth muscle cells. (Hypertension. 1994;23:25-3©.) Key Words • dexamethasone • postreceptor signaling systems • RNA, messenger • receptors, angiotensin • muscle, smooth, vascular H modulates the function of G proteins in several other kinds of cells.11'3 As to Ang II receptors, the development of nonpeptide Ang II antagonists has allowed the identification of at least two different subtypes, Ang II type 1 (AT,) receptor and Ang II type 2 (AT2) receptor.14-15 Recently, Murphy et al16 succeeded in complementary DNA (cDNA) cloning of AT, receptor from rat VSMCs. These significant advances have revealed that the actions of Ang II on VSMCs are mediated primarily through the AT! receptor. In the present study, to clarify the mechanism for glucocorticoid-enhanced Ang II receptor-mediated responses in VSMCs, the effect of glucocorticoid on AT, receptor expression and the transcription of AT, receptor gene in VSMCs were examined. Furthermore, the effect of glucocorticoid on postreceptor signaling systems in VSMCs was studied. ypertension induced by glucocorticoid hormone is not uncommon in clinical practice.1 Although the exact mechanisms of glucocorticoid-induced hypertension are still uncertain, enhancement of vascular responsiveness has been considered one of the major contributing factors.2-3 Previously, we demonstrated that the pressor responses to angiotensin II (Ang II) and riorepinephrine are enhanced in patients with Cushing's syndrome4 and experimental models with glucocorticoid-induced hypertension.55 Very recently, we found that glucocorticoid potentiates inositol trisphosphate (IP3) production evoked by Ang II in cultured vascular smooth muscle cells (VSMCs),7 and these results showed that glucocorticoid treatment would increase sensitivity to Ang II at the cellular level. In that study, the action of glucocorticoid was, in part, through prostaglandin synthesis inhibition; however, the possibility that glucocorticoid directly modulates Ang II receptor-mediated signaling pathways in VSMCs is suggested. This, together with other experimental data, makes it conceivable that glucocorticoid increases transcription and expression of a1Badrenergic receptors8 and ft-adrenergic receptors in smooth muscle cells910 and that glucocorticoid directly Received March 26, 1993; accepted in revised form September 13, 1993. From the Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan. Presented in part at the Council for High Blood Pressure Research 46th Annual Fall Conference and Scientific Sessions in Cleveland, Ohio, September 29-October 2, 1992, and published in abstract form in Hypertension (1992;20:430). Correspondence to Takao Saruta, MD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160, Japan. Methods Materials Reagents for these studies were purchased as follows: dexamethasone; the peptidase inhibitors antipain, phosphoramidon, leupeptin, pepstatin, bestatin, amastatin, and bacitracin; and bovine serum albumin (BSA) were from Sigma Chemical Co, St Louis, Mo. The nonpeptide AT, receptor antagonist DuP 753 was a gift from Dr Smith (Du Pont de Nemours Co, Wilmington, Del). PD 123319, an AT2 receptor antagonist, was a gift from Dr Taylor (Parke-Davis Pharmaceutical Research, Warner-Lambert Co, Ann Arbor, Mich). Ang II was from the Peptide Institute, Osaka, Japan. 125I-Ang II and anti-G^ protein were from Du Pont NEN Research Products, Boston, Mass. Fetal calf serum (FCS) was purchased from GIBCO Laboratories, Grand Island, NY. Dulbecco's modified Eagle medium (DMEM) and phosphate-buffered saline (PBS) were from Nissui Pharmaceutical Co, Ltd, Tokyo, Japan. RU 38486, a glucocorticoid antagonist, was kindly provided by Roussel Uclaf, Paris, France. 26 Hypertension Vol 23, No 1 January 1994 Cell Culture VSMCs were prepared from the thoracic aorta of 6-week-old Wistar-Kyoto rats by an enzyme method previously described7 and were cultured in DMEM containing 10% FCS, 100 U/mL penicillin, and 100 /ig/mL streptomycin. The cultures were kept at 37°C in a humid atmosphere consisting of 5% CO2 in air, and the medium was changed every 3 or 4 days. After the cells reached confluence, the medium was changed to culture media containing various doses of dexamethasone or ethanol vehicle as a control for various periods of time before the experiments. Cells between the second and fourth passages were used for the experiments. The cells had typical smooth muscle morphology, including the presence of hills and valleys seen under a phasecontrast microscope. Cell viability was estimated by trypan blue exclusion to be approximately 90%. We used dexamethasone because of its more potent and pure glucocorticoid action than the endogenous glucocorticoid. Radioligand Receptor Binding Assay Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 The specific binding of 1HI-Ang II to cell membrane receptors was determined by use of intact VSMCs. Confluent monolayers of VSMCs grown in 24-well plates were rinsed twice with PBS, pH 7.4. Equilibrium binding studies were performed at room temperature for 60 minutes in PBS, pH 7.4, containing increasing concentrations of 125I-Ang II, 1.6% heatinactivated BSA, and the peptidase inhibitors antipain, phosphoramidon, leupeptin, pepstatin, bestatin, and amastatin, each at 1 Mg/mL, and bacitracin at 100 ftg/mL. Preliminary experiments demonstrated that under these conditions, equilibrium was reached. After incubation, the cells were washed three times with ice-cold PBS, pH 7.4. The attached cells were dissolved with 1.0 mL of 0.2N NaOH and transferred to polypropylene tubes. The dishes were rinsed with 1.0 mL of distilled water, and the rinses were combined with the original samples. Radioactivity in the dissolved cells was determined in an autowell gamma system (Aloka Co, Tokyo, Japan). A portion of the dissolved cells was used for determining the protein content. Nonspecific binding was determined in the presence of 10 /tmol/L unlabeled Ang II. Receptor density (B,^ values) and dissociation constants (K,,) for 125I-Ang II were calculated from Scatchard plots. Furthermore, to characterize Ang II receptor subtypes in VSMCs, monolayers of VSMCs were also incubated with the fixed 12!I-Ang II and increasing concentrations of Ang II, AT! receptor antagonist DuP 753, and AT2 receptor antagonist PD 123319. Isolation of Total Cellular RNA The cells were washed three times with PBS and then lysed in 4 mol/L guanidine isothiocyanate. Total cellular RNA was isolated by the acid guanidinium/phenol/chlorofonn method. After extraction with phenol/chloroform and precipitation with ethanol, the RNA pellet was resuspended in 50 fiL of TE buffer (Tris-HCl, EDTA), pH 7.4, and quantified by absorbance measurements at 260 nm. The integrity of the purified RNA was determined by visualization of the 28S and 18S ribosomal RNA bands after electrophoresis of each RNA sample through a 1% agarose gel. Northern Blot Hybridization For Northern blot analysis, RNA (total cellular RNA, 20 /ig) was denatured with formamide and Formalin at 65°C for 10 minutes and fractionated by electrophoresis through a 1% agarose gel. The RNA was transferred to a nylon filter (blotting membrane Hybond-N was purchased from Amersham International, Ltd, Buckinghamshire, England). Prehybridization was conducted at 65°C for 2 hours in 5 x SSPE (1 x SSPE=0.18 mol/L sodium chloride, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA, pH 7.7), 5 x Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), and heat-denatured salmon sperm DNA (1 mg/mL). A Kpn ISac I fragment that encodes the rat AT, receptor, kindly provided by Dr Inagami (Vanderbilt University, Nashville, Tenn), was used as a probe for hybridization. The filters were then hybridized at 65°C for at least 12 hours to the probe that was radiolabeled with [a-^PJdCTP by the random primer synthesis method (Random Primer DNA Labeling Kit, TaKaRa Shuzo Co, Ltd, Kyoto, Japan). After hybridization, the filter was washed twice in 2x SSC (sodium chloride and sodium citrate buffer) and 0.1% SDS at 65°C and successively in l x SSC and 0.1% SDS once and in 0.5 x SSC and 0.1% SDS once. The autoradiograms were scanned with a BAS 2000 (Fuji Film, Tokyo, Japan) and quantified.17'18 Measurement of Inositol 1,4,5Trisphosphate Production Production of IP3 in VSMCs was measured with a radioimmunoassay kit (TRK1000, Amersham), as previously described.7 To examine the effects of dexamethasone on postreceptor signaling systems, the accumulation of IP3 was determined in permeabilized VSMCs by use of the nonhydrolyzable GTP analogue guanosine-5'-O-(3'-thiotriphosphate) (GTP-yS) in dexamethasone-treated (1 /imol/L for 48 hours) or nontreated cells. VSMCs were exposed to 200 U/mL a-toxin (Life Technologies, Inc, Gaithersburg, Md), a transmembrane pore-making exoprotein produced by Staphylococcus aureus,19 for 20 minutes at 37°C in buffer containing (mmol/L) glutamate potassium salt, 150; EGTA, 5; magnesium chloride, 1; and 1,4-piperazine diethanesulfonic acid (PIPES), 10; pH 7.2, adjusted with KOH. The cells were then rinsed with the above medium and incubated with 100 fimo\fL GTP-yS for 20 minutes. IP3 production was measured with a competitive binding assay that used a bovine-derived adrenal binding protein specific for IP3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblotting The cells were washed three times with ice-cold PBS and added sample buffer (60 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.1% bromophenol blue) and boiled for 5 minutes before resolution by SDSpolyacrylamide gel electrophoresis on gels containing 12% acrylamide. The protein was loaded in 150-^tg aliquots to achieve optimal blots. Low molecular weight protein standards (Bio-Rad Laboratories, Hercules) were run in parallel with the samples under study. After electrophoresis, proteins were transferred to nitrocellulose membrane (TEF Co, Nagano, Japan), which was then blocked overnight in blocking solution (5% BSA in buffer comprising 150 mmol/L NaCl in 20 mmol/L Tris-HQ, pH 7.5). After washing, the blot was incubated for 2 hours with the first antiserum (anti-Gqo) at a 1:1000 dilution in 0.05% Tween 20 in blocking solution. After a further wash, the blot was incubated for 2 hours with the second antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG, BioRad Laboratories). After the final washes, the detection was performed by the immunoblot assay procedure (Bio-Rad Laboratories). Antiserum G,,, was raised against the C-terminal sequence of G^, and recognized the 42-kD forms of G,,,. Protein Determination The protein levels in VSMCs were determined by the method of Pierce (Pierce Chemical Co, Rockford, III) using BSA as a standard. Statistical Analysis The results are expressed as mean±SEM. Statistical significance was assessed by Student's t test. Values of P<£)5 were considered to be significant. Results Effects of Dexamethasone on Angiotensin II Type 1 Receptor Fig 1 shows the competition curves with Ang II, DuP 753, and PD 123319 in 1 /imol/L dexamethasone- Sato et al 100 o o Glucocorticoid and Angiotensin II Type 1 Receptor 27 2.0- 80 60 40 ""5 in 0 20 i0" l J 10" 1 l 10" 1 0 10" 9 10" 10"7 10"6 10'5 Concentration (mol/l) Fo 1. Competition curves with angiotensin II (o), DuP 753 (•), and PD 123319 (•) In dexamethasone-treated (—) or untreated cells ( — ) . Trie data are based on the average of three independent (—) experiments. Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 treated (48 hours) and untreated cells. In VSMCs, Ang II inhibited the l23I-Ang II binding with an IQo of 2.0 nmol/L. DuP 753 produced a single displacement curve (IQo=0.25 ^mol/L), but PD 123319 did not. These findings clearly suggested that the AT, receptor was dominantly present in VSMCs and are consistent with earlier observations.15'20 The displacement curve in dexamethasone-treated cells, expressed by percentage of control, showed that the characteristics of the Ang II receptor were similar to those in the control cells. Next, to investigate the effect of dexamethasone on AT! receptor, VSMCs were incubated with 1 /xmol/L dexamethasone for various periods before the cells were harvested. AT, receptor number, measured with the ligand 125 I-Ang II, was unchanged during the first several hours of exposure to dexamethasone. The receptor number was found to be increased after 12 hours' exposure to dexamethasone and progressively increased to 185.6±9.8% of control at 48 hours (Fig 2). Fig 3 shows the typical binding curves (A) and Scatchard plot (B) for l25I-Ang II binding in dexamethasone-treated and control cells at 48 hours. The B,,,,, value was 27±3 fmol/mg protein in dexamethasone-treated cells and 15 ±3 fmol/mg protein in control cells. Conversely, the K,, of 125I-Ang n did not change significantly (dexamethasone-treated cells, 1.2±0.3 nmol/L; control cells, 1.1 ±0.2 nmol/L). As shown in Fig 4, VSMCs treated with different concentrations of dexamethasone exhibited a dose-dependent increase in AT, receptor number. The increase in AT, receptor density became apparent at as low as 10 nmol/L dexamethasone and became maximal at 1 ^mol/L dexamethasone. To demonstrate whether the effect of dexamethasone was through the glucocorticoid receptor, VSMCs were incubated with 10 ^mol/L RU 38486, a glucocorticoid receptor-specific antagonist,21 with or without 1 /xmol/L dexamethasone for 48 hours. Although RU 38486 alone had no effect on the number of AT, receptors, it completely blocked the effect of 1 /xmol/L dexamethasone (Fig 5). These results suggested that the effect of dexamethasone on AT, receptor was through the glucocorticoid-specific receptor. 2 6 12 24 Time (hours) 48 FIG 2. Bar graph shows time course for the effect of dexamethasone on relative Increase in numbers of angiotensin II type 1 (AT,) receptors. Vascular smooth muscle cells were Incubated for the Indicated time periods with 1 ^mol/L dexamethasone. AT, receptor density was calculated from Scatchard plots. Data represent mean±SEM of three independent experiments. *P<.05 vs the value in the control experiment. Effect of Dexamethasone on Angiotensin II Type 1 Receptor Messenger RNA Level The steady-state AT, receptor mRNA level was measured by Northern blot analysis. Fig 6A shows Northern blot analysis of total RNA from rat VSMCs. A single band of 2.3 kb was observed in both dexamethasonetreated cells and control cells. The size of this mRNA was consistent with the previous report.16 Fig 6B shows the ethidium bromide staining of total RNA used for Northern blot in Fig 6A. Both 28S and 18S ribosomal RNA were present in similar amounts throughout the gel. Fig 7 shows the relative increase in AT, receptor mRNA calculated by the BAS 2000 system. After 30 minutes' treatment with dexamethasone, the abundance of AT, receptor mRNA was 2.2±0.3 times control, and it was 7.8 ±0.4 times control after 24 hours' exposure to dexamethasone. Effect of Dexamethasone on GTP Analogue-Induced IP., Production in Permeabilized Cells To examine the effect of dexamethasone on Ang H-mediated postreceptor signaling systems in VSMCs, we first directly activated G proteins by GTPyS in permeabilized cells. GTP-yS has been shown to stimulate IP3 accumulation and potentiate contraction of permeabilized smooth muscle.1922 The basal intracellular IP3 content was 4.5±0.5 pmol/106 cells, consistent with the previous study.7 After treatment with 1 /xmol/L dexamethasone or ethanol vehicle for 48 hours, the cells were stimulated with GTP-yS (100 /xmol/L for 20 minutes). As shown in Fig 8, increased accumulation of IP3 occurred in both dexamethasone-treated and control cells. However, there was no significant difference of IP3 production between dexamethasone-treated cells and control cells. It should be noted that in the experiments conducted in the absence of a-toxin treatment, there was no rise in IP3 levels in response to GTP-yS in both groups (data not shown). Effect of Dexamethasone on Gq Protein a-Subunit Protein Level To examine the effect of dexamethasone on Ang II-mediated postreceptor signaling systems in VSMCs, we next examined the effect of dexamethasone on Gq protein a-subunit protein level by Western blot analysis. 28 Hypertension Vol 23, No 1 January 1994 B FIG 3. A typical binding curve (A) and its Scatchard plot (B) for 128l-angiotensin II binding to vascular smooth muscle cells. The cells were exposed to 1 ^mol/L dexamethasone (•) or ethano) vehicle (o) for 48 hours. The maximum binding for control was 16.5 fmol/mg protein with a Kd of 1.15 nmol/L, and the maximum binding for dexamethasone-treated cells was 30.5 fmol/mg protein with a Kd of 1.10 nmol/L ' I ] Anglotensin II. n m d / l r a l ] Angiotensin II bound, fmol/mg. protein Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Fig 9 shows the results of Western blot analysis of the proteins in 1 ^mol/L dexamethasone-treated cells and control cells for various periods of time. No differences were seen in Gqa protein level between dexamethasonetreated cells and control cells. Effect of Dexamethasone on Cell Numbers and Cell Protein Dexamethasone had no significant effects on cell numbers and cell protein in VSMCs incubated for 48 hours compared with control. Discussion In the present study, we investigated the effects of glucocorticoid on AT, receptor, its gene expression, and postreceptor signaling systems in VSMCs. Dexamethasone induced the gene expression of AT, receptor and increased AT, receptor number through the glucocorticoid-specific receptor, without significant effect on postreceptor systems in VSMCs. These findings could potentially explain how glucocorticoid induces vascular hypersensitivity to Ang II and are quite important in elucidating the mechanism of increased vascular sensitivity that is found in patients with Cushing's syndrome.4 The radioligand binding study using the specific receptor antagonists DuP 753 and PD 123319 disclosed that the actions of Ang II on VSMCs were mediated primarily through the AT, receptor. Moreover, treatment with dexamethasone did not alter the characteristics of Ang II receptor subtypes. From the results of the time course study, the number of AT, receptors was increased after 12 hours' exposure to dexamethasone and maintained its levels at 48 hours. This relatively 100 X3 If C long duration might account for protein synthesis, and this behavior is consistent with the general mode of action of steroid hormones. Furthermore, dexamethasone exhibited a dose-related increase in AT, receptor numbers. The effects of glucocorticoid on the regulation of Ang II receptor have been demonstrated in in vivo and in vitro studies. Douglas23 demonstrated that dexamethasone infusion produced downregulation of glomerular Ang II receptor obtained from sodium-loaded rats. Those results contradict our present findings. Since glomerular mesangium and vascular smooth muscle share many properties, similar actions of dexamethasone on Ang II receptor are expected. Although the exact reasons for the difference between these studies and our present findings remain to be determined, several possible explanations can be suggested. The dose of dexamethasone infused in sodium-loaded rats was lower than in our in vitro study. In our experiment, low doses of dexamethasone did not induce any significant changes in the number of AT, receptor. Second, other factors, such as low levels of circulating Ang II, may modulate the effects of glucocorticoid in sodium-loaded rats. Furthermore, it is also possible that glucocorticoid has tissue- and cell-specific regulation. Indeed, Chappell et al24 showed that glucocorticoid downregulated both AT, and AT2 receptors in pancreatic acinar cells. Several recent studies have investigated the effects of glucocorticoid on the mRNA of seven transmembrane- i 80 60 40 20 0 -10 -9 -8 -7 -6 Log [dexamethasone] (moll) FIG 4. Bar graph shows effect of various doses of dexamethasone on relative Increase in numbers of angiotensin II type 1 (AT,) receptors. Vascular smooth musde cells were exposed to dexamethasone for 48 hours. Data are the average of two independent experiments. CONTROL RUQMEIOumd/l DX 1jJmd/l DX+RU38M6 Fra 5. Bar graph shows effect of RU 38486 on relative increase in numbers of angiotensin II type 1 (AT,) receptors. Vascular smooth muscle cells were incubated with 10 /imol/L RU 38486 with or without 1 timoi/L dexamethasone (DX) for 48 hours. Data represent mean±SEM of three Independent experiments. *P<.05 vs the value in the control experiment. Sato et al Glucocorticoid and Angiotensin II Type 1 Receptor A. 1 2 3 4 5 6 29 %of basal IP3 -28S -I8S Alter Stimulation 200 0) To sz a. 150 itol t:risp V) O B. 100 50 w o -rRNA 28S c 0 Baut -rRNA I8S Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 FIG 6. A, Northern Wot analysis of tot il RNA from cultured vascular smooth muscle cells. The cells v ere incubated with or without 1 nJnctf/L dexamethasone for the indicated time periods. The 2.3-kb receptor messenger RNA is indicated by the arrow. Each lane contains 20 fig total RNA. Lane 1, dexamethasone 30 minutes; lane 2, dexamethasone 1 hour; lane 3, dexamethasone 6 hours; lane 4, dexamethasone 12 hours; lane 5, dexamethasone 24 hours; lane 6, dexamethasone 0 hours (control). B, Ethidlum bromide staining of total RNA used for Northern blot In A; 28S and 18S ribosomal RNA (rRNA) are present in similar amounts throughout the gel. type receptors. Sakaue and Hoffman8 have clearly demonstrated that glucocorticoid regulated expression of a,B-receptors by increasing the rate of transcription of this gene. The pattern of change in expression of AT, receptor mRNA by dexamethasone was similar to that of the a1B-adrenergic receptor mRNA. Moreover, there was a good correlation between the increase in the expression of AT! receptor and the change in the abundance of the receptor's mRNA, as observed in a1B-receptors. Compared with these similarities, the reported pattern of change in /E^-adrenergic receptor gene is different.10 ft-Adrenergic receptor mRNA increased very rapidly, within 1 to 2 hours, and returned slowly, by 10 hours, in the continued presence of glucocorticoid. These different DX1ymol/l GIFTS lOOj/md/l GTPyS+DX FIG 8. Bar graph shows the effect of dexamethasone (DX) on guanosine-5'-0-(3'-thlotriphosphate) (GTP-ySHnduced Inositol trisphosphate (IP3) production in permeaWlized cells. Vascular smooth muscle cells were incubated with or without 1 jimol/1. dexamethasone for 48 hours. IP3 was measured after 20 minutes of stimulation with 100 ^mol/L GTP-yS. Data represent mean±SEM of three independent experiments. *P<.05 vs the value in the control experiment. regulatory mechanisms among AT,, a1B-, and ft-adrenergic receptor mRNA may be important, because these receptors are closely involved in cardiovascular regulation in glucocorticoid-induced hypertension. From our present study, it still remains unclear whether dexamethasone elevated transcription rate or increased the stability of ATi receptor gene product. Our data only presented the increased accumulation of AT, receptor mRNA. For most steroid-regulated genes, gene expression is effected primarily at the transcription rate.25 Glucocorticoid has been shown, however, to have effects on mRNA stability such as insulin mRNA in insulinoma cells26 and interleukin-1/3 mRNA in monocytic cell lines.27 Further studies will be needed. Control O 0.5 2 4 8 12 24 /Hours 8 12 24 /Hours 97.4 66 45 -• 31 DX 0.5 1 97.4 66 0C< 0 0.5 1 6 12 Time (hours) FIG 7. Bar graph shows the time course of changes in the abundance of angiotensin II type 1 (AT,) receptor messenger RNA (mRNA). The cells were incubated with or without 1 jtmol/L dexamethasone for the indicated time periods. Data represent mean±SEM of three Independent experiments. *P<.05 vs the value in the control experiment. 2 4 m » 45 31 FIG 9. Western Wot analysis. The protein was prepared from vascular smooth muscle cells incubated without (top) or with (bottom) 1 fimoVL dexamethasone (DX) for the time shown. Proteins were analyzed by Western blotting with rabbit antl-G^ and alkaline phosphatase-conjugated goat anti-rabbit IgG. Molecular size markers, in kilodaltons, are shown In the left lane. 30 Hypertension Vol 23, No 1 January 1994 Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 We previously reported marked elevation of Ang IIinduced IP3 production in response to dexamethasone exposure in VSMCs.7 These changes (threefold) were significantly greater than could be explained by modest increases in receptor density (twofold) and led us to test the hypothesis that augmented signal transduction might be partially explained by changes in postreceptor systems. Recent advances in the investigation of G protein have demonstrated that the Ang II-linked G protein concerned in IP3 production in VSMCs is G,.2829 First, therefore, o-toxin-permeabilized VSMCs were used. In these cells, GTP-yS induced an increase in IP3 production without Ang II, and 48 hours' exposure to dexamethasone did not affect the IP3 production induced by GTP-yS. It is therefore less likely that dexamethasone exerts its action on IP3 production through postreceptor signaling systems. However, GTP-yS might have the capability of stimulating all kinds of G proteins other than Gq in VSMCs. Then, we investigated the effect of dexamethasone on G,,, protein level, and no significant changes in Gqa protein level were found. The classification "G protein" is now used to refer to a family of G proteins. In Gq protein, the interaction and regulation with other G proteins, such as G,, G,, and so on, were not fully understood. Therefore, it is clear that before we conclude that there is no modulatory role of dexamethasone in postreceptor signaling systems, further precise study would be needed. In conclusion, we have first demonstrated that glucocorticoid induced the expression of the AT[ receptor gene and resulted in an increase in the number of AT! receptors through the glucocorticoid-specific receptor in VSMCs. Moreover, we also demonstrated that glucocorticoid did not exert significant action on postreceptor signaling systems. Taken together with the results of our recent study,7 these results indicate that glucocorticoid elevates AT, receptor numbers and also enhances receptor-mediated response, that is, Ang II-stimulated IP3 production. This evidence makes clear the mechanisms of vascular hypersensitivity to Ang II in glucocorticoid-induced hypertension. Acknowledgments We would like to thank Dr Ronald D. Smith (Du Pont de Nemours) and Dr Taylor (Parke-Davis Pharmaceutical Research) for their generous gift of the nonpeptide Ang II antagonists DuP 753 and PD 123319 and Dr Inagami (Vanderbilt University) for his generous gift of rat AT, receptor cDNA. References 1. Whitworth JA. Mechanisms of glucocorticoid-induced hypertension. Kidney Int. 1987;31:1213-1224. 2. Mendlowitz M, Gitlow S, Naftchi N. Work of digital vasoconstriction produced by infused norepinephrine in Cushing's syndrome. J Appl Physiol. 1958;13:252-256. 3. Grunfeld JP, Eloy L. Glucocorticoids modulate vascular reactivity in the rat. Hypertension. 1987;10:608-618. 4. 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 Chn Endocrinol Metab. 1986;62:275-279. 5. Handa M, Kondo K, Suzuki H, Saruta T. Dexamethasone hypertension in rats: role of prostaglandins and pressor sensitivity to norepinephrine. Hypertension. 1984;6:236-241. 6. Nakamoto H, Suzuki H, Kageyama Y, Ohishi A, Murakami M, Naito M, Saruta T. Characterization of alterations of hemody- namics and neuroendocrine hormones in dexamethasone induced hypertension in dogs. Clin Exp Hypertens A. 1991;13:587-606. 7. Sato A, Suzuki H, Iwaita Y, Nakazato Y, Kato H, Saruta T. Potentiation of inositol trisphosphate production by dexamethasone. Hypertension. 1992;19:109-115. 8. Sakaue M, Hoffman BB. Glucocorticoids induce transcription and expression of the alB adrenergic receptor gene in DTT1 MF-2 smooth muscle cells. / Chn Invest. 1991;88:385-389. 9. Jazayeri A, Meyer WJ III. Glucocorticoid modulation of ^-adrenergic receptors of cultured rat arterial smooth muscle cells. Hypertension. 1988;12:393-398. 10. Collins S, Caron MG, Lefkowitz RJ. /32-Adrenergic receptors in hamster smooth muscle cells are transcriptionalry regulated by glucocorticoids. J Biol Chem. 1988;263:9067-9070. 11. Johnson GS, Jaworski CJ. Glucocorticoids increase GTPdependent adenylate cyclase activity in cultured fibroblasts. Mol Pharmacol. 1983;23:648-652. 12. Chang FH, Bourne HR. Dexamethasone increases adenylyl cyclase activity and expression of the a-subunit of Gs in GH3 cells. Endocrinology. 1987;121:1711-1715. 13. Holztman EJ, Soper BW, Stow JL, Ausiello DA, Ercolani U Regulation of the G-protein cn.2 subunit gene in LLC-PK, renal cells and isolation of porcine genomic clones encoding the gene promoter. J Biol Chem. 1991;266:1763-1771. 14. Whitebread S, Mele M, Kamber B, de Gasparo M. Preliminary biological characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;163:284-291. 15. Chiu AT, Herblin WF, MaCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;165:196-203. 16. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. 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A Sato, H Suzuki, M Murakami, Y Nakazato, Y Iwaita and T Saruta Hypertension. 1994;23:25-30 doi: 10.1161/01.HYP.23.1.25 Downloaded from http://hyper.ahajournals.org/ by guest on August 3, 2017 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1994 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://hyper.ahajournals.org/content/23/1/25 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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