0013-7227/90/1262-0963102.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Glucocorticoid Regulation of Rat Diencephalon Angiotensinogen Production C. F. DESCHEPPER* AND M. FLAXMAN Department of Physiology, University of California, San Francisco, California 94143-0444
ABSTRACT. To investigate whether glucocorticoids can stimulate rat brain angiotensinogen production directly, we have studied the effect of dexamethasone on angiotensinogen secretion and angiotensinogen mRNA concentration in primary astroglial cultures from rat diencephalon. Dexamethasone stimulated angiotensinogen secretion by astroglial cells in a doserelated fashion. The half-maximally effective concentration was 11 nM, and the effect was blocked by RU 486, an antagonist of type II glucocorticoid receptors. This was similar to what was observed in rat hepatoma H4IIEC cells, where the half-maximally effective concentration of dexamethasone on angiotensinogen secretion was 10 nM. At maximal concentrations, dexamethasone increased angiotensinogen secretion and angiotensinogen mRNA concentration 2-fold in astroglial cells. In the
hepatoma cells, however, the increase in angiotensinogen secretion was 5-fold. The in vivo diencephalon angiotensinogen mRNA concentration was decreased after adrenalectomy. Dexamethasone restored those levels to normal and induced a modest increase when the animals were killed 6 h after drug administration. In contrast, dexamethasone induced a robust increase in liver angiotensinogen mRNA concentration in the same animals. These results indicate that glucocorticoids increase angiotensinogen production through a direct receptor-mediated mechanism in both liver and brain. However, the angiotensinogen gene appears much more responsive to the action of glucocorticoids in liver than in brain. {Endocrinology 126: 963-970, 1990)
A
NGIOTENSINOGEN is the glycoprotein precursor of angiotensin-II. Liver is the main source of angiotensinogen in plasma (1) and contains the highest concentration of angiotensinogen mRNA (2), but other tissues also produce angiotensinogen, including the brain (2-4). In vivo, numerous hormones increase the production of liver angiotensinogen and its concentration in plasma; these include glucocorticoids (5, 6), thyroid hormones (7), and estrogens (8). Recently, the availability of well defined culture systems has made it possible to investigate at the cellular level the mode of action of these hormones. In particular, the effect of glucocorticoids on angiotensinogen mRNA concentration has been studied in a rat hepatoma cell line (9) and in rat freshly dispersed hepatocytes (10). Other investigators have reported that high doses of hydrocortisone increase angiotensinogen protein secretion by freshly dispersed hepatocytes, but have not determined whether the effect is dose related (11, 12). In vivo studies investigating the effect of adrenalectomy and glucocorticoid administration suggest that glucocorticoids also regulate rat brain angiotensinogen synthesis (13-16). However, the mechanisms underlying those effects have not been investigated in detail. In the
rat brain, astrocytes appear to be the main site of angiotensinogen production (17-19). We have recently shown that rat diencephalon astroglial primary cultures also produce angiotensinogen (19). The purpose of the present study was to determine if glucocorticoids can stimulate angiotensinogen production by rat astroglial cells by a direct mechanism and to compare those results to some of the effects observed in vivo. Materials and Methods Materials and reagents Adult male and pregnant female Sprague-Dawley rats were obtained from Bantin and Klingman (Fremont, CA); tissue culture plates from Falcon (Oxnard, CA); poly-D-lysine (mol wt, 452,000), insulin, transferrin, selenium, deoxyribonucleaseI, and porcine renin from Sigma (St. Louis, MO); dexamethasone sodium phosphate from Elkins-Sinn (Cherry Hill, NJ); reagents for the Bradford protein assay from Bio-Rad (Burlingame, CA); fetal calf serum from Hyclone (Logan, UT); 32P deoxycytidine triphosphate from Amersham (Arlington Heights, IL); the RU486 compound from Roussel-UCLAF, (Romainville, France; gift from M. Moguilewsky); the random primed DNA labeling kit from Boehringer Mannheim (Indianapolis, IN); and the BA85 nitrocellulose membrane and Minifold II slot blot apparatus from Schleicher and Schuell (Keene, NH). The pRagl6 rat angiotensinogen cDNA was a generous gift from S. Nakanishi.
Received August 31,1989. * Supported by USPHS Grants HL-29714 and HL-38774. To whom requests for reprints should be addressed.
963
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GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN
964
Endo • 1990 Voll26-No2
Rat astroglial primary cultures
In vivo experiments
Rat diencephalon astroglial primary cultures were prepared essentially as described previously, with slight modifications (19). Briefly, the thalami and hypothalami from the brains of 1-day-old Sprague-Dawley rats were dissociated with trypsin (0.25%) and plated at a density of 1 X 107 cells/100-mm diameter poly-D-lysine-coated culture dish. The cells were grown in DH16-F12 medium containing 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 Mg/ml), and fungizone (250 ng/ml). After 10 days in an atmosphere of 95% air and 5% CO2 at 37 C, the cells were redissociated with trypsin (0.25%) and replated in 6-well tissue culture plates at a density of 200,000 cells/well. The cells were grown for an additional 10 days under similar conditions. Each well was then rinsed 3 times with 2 ml PBS to remove any trace of fetal calf serum, and 1.5 ml serum-free supplemented medium [DH16-F12 containing HEPES (20 mM), insulin (5 Mg/ml), transferrin (5 ng/ ml), selenium (5 ng/ml), penicillin, streptomycin, and fungizone and the appropriate experimental hormone concentration was added to each well. The cells were incubated for an additional 3 days. The entire incubation medium was removed from each well, dried under a stream of air, and resuspended in 100 ^1 assay buffer for the angiotensinogen assay. The cells were dissolved in 0.5 ml 1 N sodium hydroxide for determination of total protein content. For RNA experiments, the cells were replated at the same density in 100-mm culture dishes instead of six-well plates and grown under the same conditions described above. Cells from pairs of plates were pooled for RNA extraction.
Exp 1. Twelve adult male rats were used. Five rats were adrenalectomized, and the other seven were sham operated. The adrenalectomized rats received a 5 g/liter solution of sodium chloride instead of drinking water. The animals were killed by decapitation 12 days after the operation. Blood was collected on EDTA for the angiotensinogen and renin assays. A block of cerebral tissue made up of hypothalamus and thalamus was removed, using the following landmarks: anteriorly, the optic chiasma; posteriorly, the mamillary bodies; dorsally, the corpus callosum; and laterally, 3 mm on each side of the sagittal line. This block was essentially the diencephalon and was similar to what was dissected from the neonatal rats for preparation of the astroglial cultures.
Rat hepatoma cell culture conditions
Tissues from rats or cultured astroglial cells were disrupted in 5 M guanidium monothiocyanate with 10% /3-mercaptoethanol and 1% AMauroyloarcosine using a Brinkmann tissue homogenizer (Brinkmann Instruments, Westbury, NY). RNA was extracted by ultracentrifugation on a cushion of 5.7 M cesium chloride. After precipitation in ethanol, RNA was resuspended in buffer containing 10 mM Tris-HCl, pH 7.6, and 1 mM EDTA and quantitated by light absorption at 260 nm. Quantification of angiotensinogen mRNA was adapted from the method of Heinrich et al. (23). Total RNA from each experimental sample (either rat tissue or pairs of plates of astroglial cells) was aliquoted in six increasing concentrations, from 0.25-1.5 ng. The RNA was denatured in formaldehyde and applied to a nitrocellulose membrane with a Minifold II slot blot system. The membrane was baked under vacuum at 80 C and hybridized at 45 C to the AccI fragment of the pRagl6 rat angiotensinogen cDNA (24). The probe was labeled by random primed DNA labeling with [32P]deoxycytidine triphosphate to a specific activity of about 109 cpm//xg DNA. After hybridization, the blot was washed sequentially in 2 X SSC (1 X SSC = 0.15 M NaCl plus 0.15 M sodium citrate) plus 0.1% sodium dodecyl sulfate (SDS) at room temperature for 10 min, in 0.1 X SSC plus 0.1% SDS at room temperature for 10 min, and finally in 0.1 x SSC plus 0.1% SDS at 55 C twice for 30 min each time. The blot was air dried and exposed to x-ray film in a light-tight cassette. An example of such an autoradiogram is shown in Fig. 1 (upper part). The autoradiograms were
The H4IIEC rat hepatoma clonal cell line, originally from the American Tissue Culture Collection (Rockville, MD) was obtained from K. Yamamoto (San Francisco, CA). The cells were grown in six-well tissue culture plates in DH-16 Modified Eagle's Medium with 5% fetal calf serum and no antibiotics. When the cells reached approximately 80% confluence, they were rinsed three times with 2 ml PBS. The cells were then incubated 24 h in serum-free supplemented medium (SFM) prepared as described above except that no antibiotics were used. The medium was replaced with SFM containing different concentrations of dexamethasone. After 16 h of incubation, 100-fA aliquots were taken and used directly in the angiotensinogen assay. The cells were dissolved in 1 ml 1 N NaOH for total protein determination. Assays The angiotensinogen concentration in cell incubation medium and rat plasma was determined by incubating the samples with excess hog renin and measuring the angiotensin-I generated by RIA. Details of the procedure have been reported previously (19, 20). The plasma renin concentration and PRA were also determined as previously described (20), according to the method of Menard and Catt (21). Total protein assays were performed according to the method of Bradford (22).
Exp 2. Sixteen male adult rats were used. Eight were adrenalectomized, and eight were sham operated. The adrenalectomized rats were maintained on 5 g/liter sodium chloride. All rats were killed by decapitation 12 days after surgery. Six hours before death, the animals received a sc injection of either dexamethasone (1 mg/kg BW) or saline. Blood was collected on EDTA. Slices of liver tissue and the diencephalon were dissected from each animal, frozen in liquid nitrogen, and stored at —70 C until RNA extraction. Exp 3. The protocol was similar to that of Exp 2, but the rats received the injection of either dexamethasone or saline 16 h before death. RNA preparation and analysis
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:52 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN Control
Adx+Dex
Dex
965
ple. The results presented in the following figures and table have been obtained by the second method of calculation. Data analysis To analyze the response of angiotensinogen secretion to glucocorticoid administration, the amounts of angiotensinogen measured in the incubation medium were plotted against the log (concentration) of dexamethasone in the medium. Using the equation described by Leppard et al. (25), the data were fitted to a sigmoid curve with the Enzfitter program (ElsevierBiosoft, Cambridge, United Kingdom) run on an IBM-PC compatible personal computer. After successive iterations, the program determined the curve that fit the data best and calculated the half-maximally effective dose (ED50). To compare mean values in experiments with only two experimental groups, t tests were performed. In experiments with more than two groups, significance was tested by one-way analysis of variance, followed by a Newman-Keul multiple comparison test.
0.00
0.S0
1.00
1.60
2.00
Results
CON ADX+DEX DEX
meg of total RNA
FIG. 1. Example of quantitation of the liver angiotensinogen mRNA concentration. The data are from the in vivo Exp 2 (see Materials and Methods). Their numerical value and statistical differences are also listed in one column of Table 1. Upper part, Autoradiograms of the slot blot RNA analysis of three experimental groups: sham-operated (control), adrenalectomy plus dexamethasone administration (Adx + Dex), and sham-operated plus dexamethasone administration (Dex). The amounts of liver total RNA applied on the membrane are, from top to bottom: 1.5, 1.25, 1, 0.75, 0.5, and 0.25 ng. Lower left, Regression lines for all the density values from each experimental group. The error bars are SEs. Lower right, Histogram comparing the mean values of the corrected slopes in the three experimental groups. The error bars are
The effect of dexamethasone on angiotensinogen secretion by rat astroglial cultures is shown in Fig. 2. The maximally effective concentration of dexamethasone was between 10~7-10~6 M, at which angiotensinogen secretion was increased approximately 2.5-fold compared to the control value. The calculated ED50 of dexamethasone was 1.1 X 10~8 M. Identical patterns of response were O
a
1.25
"5 a
10
SES.
scanned with an Ultroscan XL laser densitometer (LKB, Gaithersburg, MD). The integration program of the densitometer provided optical density values for each RNA aliquot. To quantity the angiotensinogen mRNA concentrations in each group, two slightly different calculation methods were used. The first method is illustrated in Fig. 1 (lower left). The optical density values from all aliquots within a group were plotted us. the amount of total RNA applied to the membrane. Linear regression curves were fitted to the data points, and differences were evaluated by comparing the slopes of the lines of each experimental group. The second method is illustrated in Fig. 1 (lower right). Regression lines were fitted to each set of six optical density values to calculate slope values for each experimental sample. For convenience, the slope values were multiplied by 100 to eliminate two decimal points. The means of the corrected slopes were calculated for each group, and statistical differences between groups were tested as described below. While there was no essential difference between the two methods of calculation, the second method had the advantage of generating individual values for each experimental sample and could be compared to other measurements performed on the same sam-
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LOQ CONC OF DEX FIG. 2. Effect of dexamethasone on angiotensinogen secretion by rat diencephalon astroglial cultures. Each point represents the mean from six wells. The error bars are SEs.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:52 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1990 Voll26»No2
GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN
966
observed in three other replication experiments (data not shown). The effect of dexamethasone on angiotensinogen mRNA concentration is shown in Fig. 3. When incubated with dexamethasone at 10~7 M, the angiotensinogen mRNA concentration in astroglial cells was approximately 2 times higher than the control level (Fig. 3). The effect of dexamethasone on angiotensinogen secretion by H4IIEC cells is shown in Fig. 4. As in astroglial cells, a maximal effect was observed between 10~7-10~6 M. The calculated ED50 was 1 x 10~8 M. However, the amount of secreted angiotensinogen in stimulated cells was approximately 5 times higher than the control value. To determine whether the effect of dexamethasone is mediated through a mineralocorticoid-preferring (type I) receptor or a glucocorticoid-preferring (type II) receptor, we tested the effect of dexamethasone against the background of a higher concentration of RU486, which specifically blocks the type II receptor. As shown in Fig. 5, dexamethasone (10~6 and 10~7 M) did not significantly increase angiotensinogen secretion when administered in the presence of RU486 (10~5 M). To determine whether glucocorticoids participate in vivo in the basal expression of angiotensinogen in rat brain, we studied the effect of adrenalectomy on the angiotensinogen mRNA concentration in the dienceph-
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Vol. 126, No. 2 Printed in U.S.A.
Glucocorticoid Regulation of Rat Diencephalon Angiotensinogen Production C. F. DESCHEPPER* AND M. FLAXMAN Department of Physiology, University of California, San Francisco, California 94143-0444
ABSTRACT. To investigate whether glucocorticoids can stimulate rat brain angiotensinogen production directly, we have studied the effect of dexamethasone on angiotensinogen secretion and angiotensinogen mRNA concentration in primary astroglial cultures from rat diencephalon. Dexamethasone stimulated angiotensinogen secretion by astroglial cells in a doserelated fashion. The half-maximally effective concentration was 11 nM, and the effect was blocked by RU 486, an antagonist of type II glucocorticoid receptors. This was similar to what was observed in rat hepatoma H4IIEC cells, where the half-maximally effective concentration of dexamethasone on angiotensinogen secretion was 10 nM. At maximal concentrations, dexamethasone increased angiotensinogen secretion and angiotensinogen mRNA concentration 2-fold in astroglial cells. In the
hepatoma cells, however, the increase in angiotensinogen secretion was 5-fold. The in vivo diencephalon angiotensinogen mRNA concentration was decreased after adrenalectomy. Dexamethasone restored those levels to normal and induced a modest increase when the animals were killed 6 h after drug administration. In contrast, dexamethasone induced a robust increase in liver angiotensinogen mRNA concentration in the same animals. These results indicate that glucocorticoids increase angiotensinogen production through a direct receptor-mediated mechanism in both liver and brain. However, the angiotensinogen gene appears much more responsive to the action of glucocorticoids in liver than in brain. {Endocrinology 126: 963-970, 1990)
A
NGIOTENSINOGEN is the glycoprotein precursor of angiotensin-II. Liver is the main source of angiotensinogen in plasma (1) and contains the highest concentration of angiotensinogen mRNA (2), but other tissues also produce angiotensinogen, including the brain (2-4). In vivo, numerous hormones increase the production of liver angiotensinogen and its concentration in plasma; these include glucocorticoids (5, 6), thyroid hormones (7), and estrogens (8). Recently, the availability of well defined culture systems has made it possible to investigate at the cellular level the mode of action of these hormones. In particular, the effect of glucocorticoids on angiotensinogen mRNA concentration has been studied in a rat hepatoma cell line (9) and in rat freshly dispersed hepatocytes (10). Other investigators have reported that high doses of hydrocortisone increase angiotensinogen protein secretion by freshly dispersed hepatocytes, but have not determined whether the effect is dose related (11, 12). In vivo studies investigating the effect of adrenalectomy and glucocorticoid administration suggest that glucocorticoids also regulate rat brain angiotensinogen synthesis (13-16). However, the mechanisms underlying those effects have not been investigated in detail. In the
rat brain, astrocytes appear to be the main site of angiotensinogen production (17-19). We have recently shown that rat diencephalon astroglial primary cultures also produce angiotensinogen (19). The purpose of the present study was to determine if glucocorticoids can stimulate angiotensinogen production by rat astroglial cells by a direct mechanism and to compare those results to some of the effects observed in vivo. Materials and Methods Materials and reagents Adult male and pregnant female Sprague-Dawley rats were obtained from Bantin and Klingman (Fremont, CA); tissue culture plates from Falcon (Oxnard, CA); poly-D-lysine (mol wt, 452,000), insulin, transferrin, selenium, deoxyribonucleaseI, and porcine renin from Sigma (St. Louis, MO); dexamethasone sodium phosphate from Elkins-Sinn (Cherry Hill, NJ); reagents for the Bradford protein assay from Bio-Rad (Burlingame, CA); fetal calf serum from Hyclone (Logan, UT); 32P deoxycytidine triphosphate from Amersham (Arlington Heights, IL); the RU486 compound from Roussel-UCLAF, (Romainville, France; gift from M. Moguilewsky); the random primed DNA labeling kit from Boehringer Mannheim (Indianapolis, IN); and the BA85 nitrocellulose membrane and Minifold II slot blot apparatus from Schleicher and Schuell (Keene, NH). The pRagl6 rat angiotensinogen cDNA was a generous gift from S. Nakanishi.
Received August 31,1989. * Supported by USPHS Grants HL-29714 and HL-38774. To whom requests for reprints should be addressed.
963
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:52 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN
964
Endo • 1990 Voll26-No2
Rat astroglial primary cultures
In vivo experiments
Rat diencephalon astroglial primary cultures were prepared essentially as described previously, with slight modifications (19). Briefly, the thalami and hypothalami from the brains of 1-day-old Sprague-Dawley rats were dissociated with trypsin (0.25%) and plated at a density of 1 X 107 cells/100-mm diameter poly-D-lysine-coated culture dish. The cells were grown in DH16-F12 medium containing 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 Mg/ml), and fungizone (250 ng/ml). After 10 days in an atmosphere of 95% air and 5% CO2 at 37 C, the cells were redissociated with trypsin (0.25%) and replated in 6-well tissue culture plates at a density of 200,000 cells/well. The cells were grown for an additional 10 days under similar conditions. Each well was then rinsed 3 times with 2 ml PBS to remove any trace of fetal calf serum, and 1.5 ml serum-free supplemented medium [DH16-F12 containing HEPES (20 mM), insulin (5 Mg/ml), transferrin (5 ng/ ml), selenium (5 ng/ml), penicillin, streptomycin, and fungizone and the appropriate experimental hormone concentration was added to each well. The cells were incubated for an additional 3 days. The entire incubation medium was removed from each well, dried under a stream of air, and resuspended in 100 ^1 assay buffer for the angiotensinogen assay. The cells were dissolved in 0.5 ml 1 N sodium hydroxide for determination of total protein content. For RNA experiments, the cells were replated at the same density in 100-mm culture dishes instead of six-well plates and grown under the same conditions described above. Cells from pairs of plates were pooled for RNA extraction.
Exp 1. Twelve adult male rats were used. Five rats were adrenalectomized, and the other seven were sham operated. The adrenalectomized rats received a 5 g/liter solution of sodium chloride instead of drinking water. The animals were killed by decapitation 12 days after the operation. Blood was collected on EDTA for the angiotensinogen and renin assays. A block of cerebral tissue made up of hypothalamus and thalamus was removed, using the following landmarks: anteriorly, the optic chiasma; posteriorly, the mamillary bodies; dorsally, the corpus callosum; and laterally, 3 mm on each side of the sagittal line. This block was essentially the diencephalon and was similar to what was dissected from the neonatal rats for preparation of the astroglial cultures.
Rat hepatoma cell culture conditions
Tissues from rats or cultured astroglial cells were disrupted in 5 M guanidium monothiocyanate with 10% /3-mercaptoethanol and 1% AMauroyloarcosine using a Brinkmann tissue homogenizer (Brinkmann Instruments, Westbury, NY). RNA was extracted by ultracentrifugation on a cushion of 5.7 M cesium chloride. After precipitation in ethanol, RNA was resuspended in buffer containing 10 mM Tris-HCl, pH 7.6, and 1 mM EDTA and quantitated by light absorption at 260 nm. Quantification of angiotensinogen mRNA was adapted from the method of Heinrich et al. (23). Total RNA from each experimental sample (either rat tissue or pairs of plates of astroglial cells) was aliquoted in six increasing concentrations, from 0.25-1.5 ng. The RNA was denatured in formaldehyde and applied to a nitrocellulose membrane with a Minifold II slot blot system. The membrane was baked under vacuum at 80 C and hybridized at 45 C to the AccI fragment of the pRagl6 rat angiotensinogen cDNA (24). The probe was labeled by random primed DNA labeling with [32P]deoxycytidine triphosphate to a specific activity of about 109 cpm//xg DNA. After hybridization, the blot was washed sequentially in 2 X SSC (1 X SSC = 0.15 M NaCl plus 0.15 M sodium citrate) plus 0.1% sodium dodecyl sulfate (SDS) at room temperature for 10 min, in 0.1 X SSC plus 0.1% SDS at room temperature for 10 min, and finally in 0.1 x SSC plus 0.1% SDS at 55 C twice for 30 min each time. The blot was air dried and exposed to x-ray film in a light-tight cassette. An example of such an autoradiogram is shown in Fig. 1 (upper part). The autoradiograms were
The H4IIEC rat hepatoma clonal cell line, originally from the American Tissue Culture Collection (Rockville, MD) was obtained from K. Yamamoto (San Francisco, CA). The cells were grown in six-well tissue culture plates in DH-16 Modified Eagle's Medium with 5% fetal calf serum and no antibiotics. When the cells reached approximately 80% confluence, they were rinsed three times with 2 ml PBS. The cells were then incubated 24 h in serum-free supplemented medium (SFM) prepared as described above except that no antibiotics were used. The medium was replaced with SFM containing different concentrations of dexamethasone. After 16 h of incubation, 100-fA aliquots were taken and used directly in the angiotensinogen assay. The cells were dissolved in 1 ml 1 N NaOH for total protein determination. Assays The angiotensinogen concentration in cell incubation medium and rat plasma was determined by incubating the samples with excess hog renin and measuring the angiotensin-I generated by RIA. Details of the procedure have been reported previously (19, 20). The plasma renin concentration and PRA were also determined as previously described (20), according to the method of Menard and Catt (21). Total protein assays were performed according to the method of Bradford (22).
Exp 2. Sixteen male adult rats were used. Eight were adrenalectomized, and eight were sham operated. The adrenalectomized rats were maintained on 5 g/liter sodium chloride. All rats were killed by decapitation 12 days after surgery. Six hours before death, the animals received a sc injection of either dexamethasone (1 mg/kg BW) or saline. Blood was collected on EDTA. Slices of liver tissue and the diencephalon were dissected from each animal, frozen in liquid nitrogen, and stored at —70 C until RNA extraction. Exp 3. The protocol was similar to that of Exp 2, but the rats received the injection of either dexamethasone or saline 16 h before death. RNA preparation and analysis
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:52 For personal use only. No other uses without permission. . All rights reserved.
GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN Control
Adx+Dex
Dex
965
ple. The results presented in the following figures and table have been obtained by the second method of calculation. Data analysis To analyze the response of angiotensinogen secretion to glucocorticoid administration, the amounts of angiotensinogen measured in the incubation medium were plotted against the log (concentration) of dexamethasone in the medium. Using the equation described by Leppard et al. (25), the data were fitted to a sigmoid curve with the Enzfitter program (ElsevierBiosoft, Cambridge, United Kingdom) run on an IBM-PC compatible personal computer. After successive iterations, the program determined the curve that fit the data best and calculated the half-maximally effective dose (ED50). To compare mean values in experiments with only two experimental groups, t tests were performed. In experiments with more than two groups, significance was tested by one-way analysis of variance, followed by a Newman-Keul multiple comparison test.
0.00
0.S0
1.00
1.60
2.00
Results
CON ADX+DEX DEX
meg of total RNA
FIG. 1. Example of quantitation of the liver angiotensinogen mRNA concentration. The data are from the in vivo Exp 2 (see Materials and Methods). Their numerical value and statistical differences are also listed in one column of Table 1. Upper part, Autoradiograms of the slot blot RNA analysis of three experimental groups: sham-operated (control), adrenalectomy plus dexamethasone administration (Adx + Dex), and sham-operated plus dexamethasone administration (Dex). The amounts of liver total RNA applied on the membrane are, from top to bottom: 1.5, 1.25, 1, 0.75, 0.5, and 0.25 ng. Lower left, Regression lines for all the density values from each experimental group. The error bars are SEs. Lower right, Histogram comparing the mean values of the corrected slopes in the three experimental groups. The error bars are
The effect of dexamethasone on angiotensinogen secretion by rat astroglial cultures is shown in Fig. 2. The maximally effective concentration of dexamethasone was between 10~7-10~6 M, at which angiotensinogen secretion was increased approximately 2.5-fold compared to the control value. The calculated ED50 of dexamethasone was 1.1 X 10~8 M. Identical patterns of response were O
a
1.25
"5 a
10
SES.
scanned with an Ultroscan XL laser densitometer (LKB, Gaithersburg, MD). The integration program of the densitometer provided optical density values for each RNA aliquot. To quantity the angiotensinogen mRNA concentrations in each group, two slightly different calculation methods were used. The first method is illustrated in Fig. 1 (lower left). The optical density values from all aliquots within a group were plotted us. the amount of total RNA applied to the membrane. Linear regression curves were fitted to the data points, and differences were evaluated by comparing the slopes of the lines of each experimental group. The second method is illustrated in Fig. 1 (lower right). Regression lines were fitted to each set of six optical density values to calculate slope values for each experimental sample. For convenience, the slope values were multiplied by 100 to eliminate two decimal points. The means of the corrected slopes were calculated for each group, and statistical differences between groups were tested as described below. While there was no essential difference between the two methods of calculation, the second method had the advantage of generating individual values for each experimental sample and could be compared to other measurements performed on the same sam-
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LOQ CONC OF DEX FIG. 2. Effect of dexamethasone on angiotensinogen secretion by rat diencephalon astroglial cultures. Each point represents the mean from six wells. The error bars are SEs.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:52 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1990 Voll26»No2
GLUCOCORTICOIDS AND BRAIN ANGIOTENSINOGEN
966
observed in three other replication experiments (data not shown). The effect of dexamethasone on angiotensinogen mRNA concentration is shown in Fig. 3. When incubated with dexamethasone at 10~7 M, the angiotensinogen mRNA concentration in astroglial cells was approximately 2 times higher than the control level (Fig. 3). The effect of dexamethasone on angiotensinogen secretion by H4IIEC cells is shown in Fig. 4. As in astroglial cells, a maximal effect was observed between 10~7-10~6 M. The calculated ED50 was 1 x 10~8 M. However, the amount of secreted angiotensinogen in stimulated cells was approximately 5 times higher than the control value. To determine whether the effect of dexamethasone is mediated through a mineralocorticoid-preferring (type I) receptor or a glucocorticoid-preferring (type II) receptor, we tested the effect of dexamethasone against the background of a higher concentration of RU486, which specifically blocks the type II receptor. As shown in Fig. 5, dexamethasone (10~6 and 10~7 M) did not significantly increase angiotensinogen secretion when administered in the presence of RU486 (10~5 M). To determine whether glucocorticoids participate in vivo in the basal expression of angiotensinogen in rat brain, we studied the effect of adrenalectomy on the angiotensinogen mRNA concentration in the dienceph-
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