Corticoid secretion
regulation of atria1 natriuretic and gene expression
factor
JAMIE DANANBERG AND ROGER J. GREKIN Division of Endocrinology, University of Michigan, Ann Arbor 48109; and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105 Dananberg, Jamie, and Roger J. Grekin. Corticoid regulation of atria1 natriuretic factor secretion and gene expression. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1377-H1381, 1992.-This study investigated the acute effects of glucocorticoids and mineralocorticoids on atria1 natriuretic factor (ANF) biosynthesis in vivo. Groups of male Sprague-Dawley rats were injected with 1 mg dexamethasone (Dex), 10 mg deoxycorticosterone acetate (DOCA) or vehicle alone. Different groups were studied after periods of 30 min to 8 h. Plasma and left atria1 ANF concentrations and ANF mRNA levels were measured 2-8 h after corticoid injection. From 30 min to 2 h after injection, ANF mRNA was analyzed by quantitative and qualitative assessments. There was a two- to threefold increase in plasma levels of ANF in Dex-treated rats compared with controls at all time periods (P < 0.05). Although ANF plasma levels increased over time following DOCA treatment, they were not significantly different from control values. Dex treatment also increased normalized ANF mRNA levels 77% above control levels during the first 4 h after injection (P < 0.05). Thereafter there was a return of mRNA levels to that seen in controls. There was no qualitative difference in the ANF mRNA at any time as assessed by Northern hybridization. In contrast, DOCA increased ANF mRNA levels only after 8 h (P < 0.05). No significant changes in left atria1 ANF content were noted during this study. In a separate study, Dex was administered to isolated left atria in vitro in a superfusion system. Superfusion with 2 x lo-” M Dex produced a 40% increase in ANF secretory rate within 20 min (P = 0.036). We conclude that Dex induces a direct rapid increase in ANF mRNA levels and ANF secretion in rats. DOCA increases ANF secretion but the effect is delayed and is likely to be mediated through a primary effect on volume expansion. atria1 natriuretic peptides; dexamethasone; one; messenger ribonucleic acid
deoxycorticoster-
ADRENOCORTICAL STEROIDS are known to affect volume homeostasis in intact organisms. Accumulating evidence suggests that atria1 natriuretic factor (ANF) may also play a central role in salt and volume balance. Over the past several years, the role of glucocorticoids and mineralocorticoids in the regulation of ANF secretion and gene expression has been evaluated; however, a complete picture of the interactions between these agents has not been fully determined. A number of investigators have examined the chronic effects of deoxycorticosterone acetate (DOCA) and dexamethasone (Dex) on ANF biosynthesis. When evaluated over a period of 12 h to several days, we (13) and others (2,14,20, 23,24) have shown that DOCA administration causes an increase in plasma levels of ANF. In vivo studies have shown a decrease in atria1 ANF content (20, 23) and an increase in ANF mRNA levels (2, 11) over this time period. In contrast, no effect of DOCA was found on ANF secretion rates or on ANF mRNA levels in primary myocyte cultures (10). Glucocorticoids have been found to raise plasma ANF
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levels in humans (26), intact rats (9, 11,16), and isolated rat atria1 myocytes (10, 19) although at least one study has shown a decrease in plasma ANF after 4 wk of low-dose treatment with Dex (25). Changes in intraatria1 ANF following glucocorticoid administration have been less well studied but there is some evidence that atria1 content of ANF is diminished (9). Several investigators have shown that chronic administration of dexamethasone causes an increase in ANF mRNA levels (10, 1l), but this effect may occur primarily in ventricular myocardium (7, 18). No study has examined the effect of these agents over a short time course. Evaluating the effects of corticoids acutely may help determine whether effects of steroids are primarily mediated through changes in secretion or increases in new hormone synthesis. To this end, we treated rats with DOCA, Dex, or vehicle injections and obtained simultaneous samples for the measurement of plasma ANF, intra-atria1 ANF content, and ANF mRNA over a period of 30 min to 8 h following the intervention. MATERIALS
AND METHODS
Treatment of rats and sample collection. Three-month-old male Sprague-Dawley rats were obtained from Charles River Breeders (Portage, MI). Rats were divided into three groups according to treatment with Dex (Sigma, St. Louis, MO), DOCA (Sigma), or vehicle control. The Dex-treated and control rats were maintained on normal rat chow and had free access to water. The DOCA-treated rats were maintained on sodium-free rat chow (sodium-deficient diet, rat modified; ICN Biochemicals, Cleveland, OH) and given 1% NaCl solution as the sole source of drinking water starting 1 wk before and continuing through the experimental period. Polyethylene glycol (1 ml) was used as the vehicle for delivery of corticoids. On the morning of the study, rats were injected subcutaneously with 1 mg Dex, 10 mg DOCA, or vehicle alone. During the course of the study, Dex and control rats were given access to water only, whereas DOCA-treated animals were given access to 1% NaCl solution and sodium-free chow. Groups of six rats from each group were killed by decapitation 2, 4, 6, and 8 h after injections. Trunk blood was collected into EDTA tubes on ice immediately after decapitation. The left atria were rapidly removed, frozen in liquid nitrogen, and kept at -70°C until processing. In a separate experiment, animals were injected with Dex or vehicle and were killed 30, 60, or 120 min following injection. The atria from these animals were frozen in liquid nitrogen and then were immediately used for extraction of total RNA. Isolation and quantification of RNA and isolation of atria1 ANF. Frozen atria were placed in denaturing solution (4 M guanidine isothiocyanate, 0.75 M sodium citrate, 10% N-lauroylsarcosine 0.72% vol/vol ,&mercaptoethanol) and homogenized in a Polytron tissue homogenizer (Kinematica, Lucerne, Switzerland). After centrifugation, an aliquot of supernatant was removed and diluted 1:50 in 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.4, and stored at -20°C until ANF
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H1378
CORTICOIDS
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radioimmunoassay. Cellular RNA was isolated from the remaining supernatant by phenol-isoamyl alcohol-chloroform (25:24:1 vol/vol) extraction followed by cold isopropanol precipitation. The RNA was quantified in a model 1001 Plus Spectrophotometer (Milton Roy, Columbus, OH). A total of 2 pg of total RNA was used to quantify specific ANF mRNA by dot-blot or Northern hybridization. In the dot-blot procedure, RNA samples were denatured in 7.4% formaldehyde, 6~ SSC (0.9 M sodium chloride, 0.09 M sodium citrate) and heated at 65°C. Samples were applied to nylon membranes (Magnagraph, Micron Separations, Westborough, MA) using a minifold apparatus (Schleicher & Schuell, Keene, NH), and fixed by baking at 70°C for 15 min. During the Northern hybridizations, RNA samples were denatured as above, electrophoretically separated in an agaroseformaldehyde gel, and transferred to nylon membranes overnight by capillary action. In all cases, membranes were prehybridized in 5~ Denhardt’s solution (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, Oil% Ficoll), 6~ SSC, 50% formamide, 0.1% lauryl sulfate, and salmon sperm DNA, 100 pg/ml, for 90 min at 42°C. Hybridization was then carried out for 16-24 h in solution identical to the prehybridization solution except for the presence of 10% dextran and the radiolabeled cDNA probe. After hybridization with the ANF cDNA probe, filters were washed with the final wash conditions at 65°C with 0.1~ SSC, 1% lauryl sulfate. After hybridization, specifically bound counts were measured using a Betascope model 603 Blot Analyzer (Betagen, Waltham, MA), which detects and directly quantifies P-particles. Before additional probing with control cDNAs, bound counts were stripped from the filters by washing in 5 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.5% pyrophosphate, 0.1~ Denhardt’s for 90 min at 65°C and rinsed in 1X SSC. The quantification of ANF mRNA was normalized to the quantification of actin mRNA. To control for the possibility that adrenocortical steroids may also affect actin mRNA concentrations, the filters were probed again with a cDNA clone encoding the rat cyclophilin mRNA, which is expressed in large amounts in nearly all rat tissues (6). The final wash conditions were adjusted following hybridization with the actin and cyclophilin cDNAs to 45”C, 0.1X SSC, 1% lauryl sulfate. Complementary DNA probes. The ANF cDNA was kindly given to us by Dr. Howard Dene (8), who has previously reported its isolation. The probe encodes for all of the coding region and portions of the 5’ and 3’ noncoding regions of the ANF mRNA. After denaturation, the probes were labeled with deoxycytidine 5’- [ &3ZP] triphosphate ([ cy:“P]dCTP; 3,000 Ci/ mmol; Amersham, Arlington Heights, IL) to a specific activity of 10” dpm/pg using a random hexanucleotide primer kit (Prime-A-Gene System, Promega, Madison, WI). Unincorporated labeled nucleotides were removed by Sephadex centrifugation (G-50 Spin Columns, Boehringer Mannheim, Indianapolis, IN). In vitro superfusion system. Male Sprague-Dawley rats weighing 300-350 g on an ad libitum sodium diet with free access to water were killed by decapitation. The hearts were rapidly removed and placed in iced medium 199 (GIBCO Laboratories, Grand Island, NY) gassed with 95% O,-5% CO,. Left atria were carefully dissected from the hearts and were suspended between two modified aneurysm clips, one of which served as a unipolar electrode. The other was suspended from a force transducer. The suspended atria were put into a superfusion chamber with gassed medium 199 at 37°C as described by Schiebinger et al. (21) with an initial tension of 0.7 g. The atria were paced with an electric stimulator (model S88, Grass Instruments, Quincy, MA). The force transducer was connected to a direct current power amplifier and pre-amplifier (models 7DAG and 7PIG, respectively, Grass Instruments), and the signal was sent to a paper chart recorder (model 7S1225, Grass Instruments). The atria underwent a stabilization period in
GENE
EXPRESSION
which they were paced at 1 Hz for 30 min and 3 Hz for 35 min during superfusion with medium 199 at 2 ml/min. After this stabilization period, superfusate was collected at 2-min intervals. After a lo-min basal collection period, atria were superfused with Dex (2 x lo-” M) for 120 min. Measurement of ANF. Plasma ANF concentration, atria1 ANF content, and ANF secretion rates from superfused atria were measured by radioimmunoassay (22). Plasma ANF is expressed as picomoles per liter. Extracted atria1 ANF was diluted 1:22,050 during the assay. Denaturing solution diluted to these levels did not interfere with the assay standard curve. Total ANF in these samples was normalized to the measurement of total protein as measured in a calorimetric assay based on the Bradford dye-binding procedure (3; Bio-Rad Protein Assay, Bio-Rad, Richmond, CA) and is expressed as picograms per milligram of protein. ANF measured from superfusion collections in medium 199 is expressed as picograms per minute. Statistical analysis. ANF plasma levels, atria1 content, and mRNA concentrations were analyzed using a factorial analysis of variance model. ANF secretion during the in vitro experiments was analyzed using a repeated measures analysis of variance model. RESULTS
The plasma levels of ANF in animals treated with Dex, DOCA, or vehicle control are shown in Fig. 1. ANF plasma concentrations were substantially elevated 2 h after Dex administration compared with vehicle-injected animals or DOCA-treated animals (130 t 20 vs. 59 -+ 33 pmol/l, control; 22 t 7, DOCA; P < 0.05). This effect was also noted at 4,6, and 8 h after Dex treatment (185 t 22, 196 t 40, and 186 t 11 pmol/l, respectively; P < 0.05 at all points compared with control levels). There was a tendency for DOCA-treated animals to have an increase in plasma levels of ANF (27.7 t 11, 81 t 24, and 95.0 t 39.0 pmol/l at 4, 6, and 8 h); however, these values were not significantly different from those measured in control animals at the same time intervals (29.2 t 12.5, 43.3 t 15.3, and 54.8 t 9.2 pmol/l). The changes in atria1 ANF content are shown in Fig. 2. The content of ANF in left atria taken from control rats remained constant throughout the study period. Although there was a steady rise in the atria1 ANF content of animals treated with DOCA during 6 h following injection, the effect was not significant. This change was not noted at 8 h. Similarly, ANF content in atria from Dex-treated rats was not significantly different from that 250 [ n
Dex
2
* 1
4 6 Hours After lnter-ventlon
8
Fig. 1. Effect of corticoids on plasma levels of atria1 natriuretic factor (ANF) in rats treated with a single injection of dexamethasone (Dex; 1 acetate (DOCA; 10 mg), or vehicle control; n md deoxycorticosterone = 6 for each group. * P c 0.05compared with control. 9
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CORTICOIDS
AND
ANF
of controls throughout the study. The effects of the corticoids on ANF mRNA concentrations are noted in Fig. 3. There was a high degree of concordance between the actin and cyclophilin cDNA hybridization experiments ( rrL = 0.646, P = 0.0001). For this reason, only the ANF mRNA data normalized to actin are reported. Control levels of ANF mRNA did not change throughout the study period. Dex significantly increased ANF mRNA levels within 2 h of treatment when compared with either DOCA-treated or control animals [DOCA 9.1 t 2.6 vs. 4.1 t 0.4 and control 5.1 t 0.5 counts/min (cpm) of specifically bound ANF cDNA relative units normalized to cpm of specifically bound ,&actin cDNA; P < 0.051. The increase in ANF mRNA was transient in that the mRNA levels were not different from those of control animals at 6 and 8 h. In sharp contrast to the Dex effect, DOCA increased ANF mRNA levels only after 8 h (DOCA 8.0 t 0.6 vs. control 4.9 t 0.2, relative units, P < 0.05). To better assess the short-term effect of Dex on ANF gene expression, the experiment was repeated to examine the responses during a 2-h time period. To determine whether Dex caused the transcription of a lengthened or shortened ANF mRNA species, a Northern hybridization was undertaken. The results are shown in Fig. 4. At 30 min there was a 40% increase in ANF mRNA in atria of Dex-treated animals compared with those of controls. By 120 min, there was approximately a twofold increase in mRNA levels, comparable to that noted in the previous experiment. In all cases, the ANF cDNA detected an g
Dex
a
DOCA
l-l
Control
2
4
Hours
6
0
After lnterventlon
Fig. 2. Effect of corticoids on left atria1 ANF content in rats treated a single injection of Dex (1 mg), DOCA (10 mg), or vehicle control; 6 for each group. P = not significant for all comparisons.
H
Dex
q
DOCA
with n =
a Hours
After lnterventton
Fig. 3. Effect of corticoids on left atria1 ANF mRNA concentrations in rats treated with a single injection of Dex (1 mg), DOCA (10 mg), or vehicle control; n = 6 for each group. * P < 0.05 compared with control.
GENE
EXPRESSION
H1379
mRNA which was - 1,075 nucleotides in length. The results of the in vitro study are shown in Fig. 5. Very early in this protocol, mean ANF secretory rates were higher in the Dex-treated group than in the control atria (P = 0.036). This enhancement was seen for a period of 90 min. The maximal increase in ANF secretion rate was -40%. DISCUSSION
This study evaluated the effects of Dex and DOCA on ANF plasma concentrations, left atria1 ANF content, and ANF mRNA levels over a period of 8 h. Additionally, the in vitro effect of Dex on left atria1 ANF secretion rates was assessed. Several previous studies have examined the in vivo effects of glucocorticoids on ANF biosynthesis. Garcia et al. (9) found a marked increase in plasma and atria1 levels of ANF in rats following corticoid replacement after adrenalectomy. Gardner and co-workers (11) noted an increase in these levels after Dex at 48 h but not at 24 h in both intact and adrenalectomized rats. LaChance et al. (16) found that atria from rats pretreated with Dex released higher amounts of ANF after l-4 h of in vitro incubation than atria did from DOCA-treated animals. The present study demonstrated a marked increase in plasma concentrations of ANF within 2 h of Dex administration. This effect was maintained for 8 h after injection. To determine whether this increase in ANF plasma levels was due to an increase in secretion or a decrease in clearance, we evaluated the effect of Dex administered in vitro in a superfusion system. We found a significant increase in ANF secretory rates following Dex administration. This increase was on the order of 30-40% over basal rates and was maintained for a period of 90 min. These data suggest that Dex stimulates ANF secretion directly, although this finding does not fully account for the threefold increase in ANF levels following Dex treatment in vivo. This difference may be explained by a decrease in ANF clearance in intact animals or by the appearance of an intermediate stimulus which further enhances ANF production. Expression of ANF receptors on vascular endothelial cells, which are primarily of the clearance type (4), has been shown to be glucocorticoid dependent (17). No known study has examined whether clearance receptors are increased above basal expression levels by glucocorticoids. A decrease in ANF receptormediated guanosine 3’,5’-cyclic monophosphate (cGMP) production following treatment with Dex has been shown (27). The effects of glucocorticoids on ANF endopeptidases, the other probable mechanism of ANF clearance (12), have not been reported. The present study showed a rapid increase in levels of ANF mRNA following Dex administration. The mRNA species was not different in size from that found in control rats, although the resolution of Northern hybridization does not exclude the possibility that small changes in mRNA size occurred. This finding suggests that a substantial change in the size of the ANF mRNA with an associated change in the stability of the messenger RNA does not explain the mechanism by which Dex raises transcript levels. Gardner et al. (10) have shown that Dex
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H1380
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30 Minutes
AND ANF GENE EXPRESSION
60 Minutes
120 Minutes Fig. 4. Northern hybridization showing effect of glucocorticoids on left atria1 ANF mRNA concentration in rats treated with a single injection of Dex (1 mg). Lane 8: sample with known low concentration of total cellular RNA. All other lanes were loaded with 2 pg total RNA. Dex and control values were normalized to j3-actin hybridization. Ratios of these values are shown for each pair.
980 nt
-
DEXKontrol
1.40
1.40
2.87
1.47
increases both transcriptional activity and ANF mRNA half-life from 17 to 30 hours. In our study, however, the disappearance rate of the ANF mRNA after the initial 4-h rise suggests that there is a substantially shorter halflife during this first phase of stimulation of the mRNA. It also suggests that a significant portion of the rise is due to an increase in transcriptional activity. A second, more prolonged increase in ANF mRNA has been reported to occur after 24-48 h (10,ll) and may, to a larger degree, be due to an increase in mRNA half-life. Whereas a number of studies have shown that, over a period of 12 h to several weeks, DOCA increases plasma ANF concentrations (2, 13, 14, 20, 23, 24) and ANF mRNA levels (2, ll), only limited data are available on the acute effects of DOCA upon these parameters. The present study showed a trend toward an increase in plasma ANF concentrations in rats treated with DOCA; however these changes were not significant when compared with those of control rats. This study did not find a significant change in the atria1 content of ANF over the study period. In contrast, we were able to demonstrate a significant rise in ANF mRNA levels 8 h after stimulation with DOCA. These data are consistent with the findings of Ballerman and co-workers (2) in which DOCA 175
I
Basal
Dexamethasone
1
1.74
stimulated increases in plasma ANF and ANF mRNA were noted at 12 h after a single injection of DOCA. Based on these findings, we postulate that the effect of mineralocorticoids on ANF biosynthesis is an indirect one, whereas the glucocorticoid influence is more direct. The finding that Dex is able to alter ANF plasma concentration within 2 h and ANF mRNA levels within 30 min is in sharp contrast to the later response seen with DOCA. It is possible that the primary effect of DOCA is to cause volume expansion, which directly mediates ANF secretion, most likely by causing atria1 distension (1, 5). An additional explanation suggests that other intermediates need to be generated before the DOCA effect is apparent. This would explain the relatively longer period of time elapsed before the appearance of increases in plasma ANF. This hypothesis is further substantiated by the fact that DOCA does not directly stimulate ANF secretion from myocardial cells in vitro (10). Glucocorticoids administered in high doses typically cause a hypertensive response (15). These data suggest that direct glucocorticoid stimulation of ANF biosynthesis modulates this hypertensive effect. It is possible that without the stimulus to ANF secretion, the elevation in blood pressure might be even greater. In contrast, the ANF secretory response to mineralocorticoid stimulation occurs secondarily and is more likely part of an overall mechanism to maintain hemodynamic homeostasis in the face of volume expansion and hypertension. This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-18575 and by grants from the American Heart Association and its Indiana affiliate. J. Dananberg performed this work during the tenure of a ClinicianScientist Award from the American Heart Association. Address for reprint requests: J. Dananberg, Div. of Endocrinology, Univ. of Michigan Medical Ctr., 5570 MSRB-II Box 0678, Ann Arbor, MI 48109-0678. Received 16 December 1991; accepted in final form 12 June 1992.
50, t
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30 40 50 Time (mln)
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Fig. 5. Effect of Dex (squares; n = 10) or superfusion medium alone (circles; n = 7) on left atria1 ANF secretion rate in an in vitro superfusion system. Atria were superfused with gassed medium 199 at 2 ml/ min. Results expressed as a percent of basal secretory rates, means + SE. ANF secretion by Dex-treated atria is significantly greater than that from atria exposed to superfusion medium alone (P < 0.05; repeated measures analysis of variance).
REFERENCES 1. Anderson, J. V., J. Donckier, W. J. McKenna, and S. R. Bloom. The plasma release of atria1 natriuretic peptide in man. Clin. Sci. 71: 151-155, 1986. 2. Ballermann, B. J., K. D. Bloch, J. G. Seidman, and B. M. Brenner. Atria1 natriuretic peptide transcription, secretion, and glomerular receptor activity during mineralocorticoid escape in 1986. the rat. J. Clin. Invest. 78: 840-843,
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3. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 4. Chevalier, R. L., M. Garmey, R. M. Scarborough, J. Linden, R. A. Gomez, M. J. Peach, and R. M. Carey. Inhibition of ANP clearance receptors and endopeptidase 24.11 in maturing rats. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R1218-R1228, 1991. 5. Dananberg, J., B. M. Egan, E. R. Bates, and R. J. Grekin. Sustained saline-induced secretion of atria1 natriuretic hormone is not maintained by atria1 stretch. J. Clin. Endocrinol. Metab. 68: 735-739, 1989. 6. Danielson, P. E., S. Forss-Petter, M. A. Brow, L. Calavetta, J. Douglass, R. J. Milner, and J. G. Sutcliffe. plBl5: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7: 261-267, 1988. 7. Day, M. L., D. Schwartz, R. C. Wiegand, P. T. Stockman, S. R. Brunnert, H. E. Tolunay, M. G. Currie, D. G. Standaert, and P. Needleman. Ventricular atriopeptin. Unmasking of messenger RNA and peptide synthesis by hypertrophy or dexamethasone. Hypertension Dallas 9: 485-491, 1987. 8. Dene, H., and J. P. Rapp. Quantification of messenger ribonucleic acid for atria1 natriuretic factor in atria and ventricles of Dahl salt-sensitive and salt-resistant rats. Mol. Endocrinol. 1: 614-620, 1987. R., W. Debinski, J. Gutkowska, 0. Kuchel, G. 9. Garcia, Thibault, J. Genest, and M. Cantin. Gluco- and mineralocorticoids may regulate the natriuretic effect and the synthesis and release of atria1 natriuretic factor by the rat atria in vivo. Biochem. Biophys. Res. Commun. 131: 806-814, 1985. D. G., B. J. Gertz, C. F. Deschepper, and D. Y. 10. Gardner, Kim. Gene for the rat atria1 natriuretic peptide is regulated by glucocorticoids in vitro. J. Clin. Invest. 82: 1275-1281, 1988. D. G., S. Hane, D. Trachewsky, D. Schenk, and 11. Gardner, J. D. Baxter. Atria1 natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem. Biophys. Res. Commun. 139: 1047-1054, 1986. 12. Gerbes, A. L., and A. M. Vollmar. Degradation and clearance of atria1 natriuretic factors (ANF). Life Sci. 47: 1173-l 180, 1990. 13. Grekin, R. J., W. D. Ling, Y. Shenker, and D. F. Bohr. Immunoreactive atria1 natriuretic hormone levels increase in deoxycorticosterone acetate-treated pigs. Hypertension Dallas, Suppl. II 8: 11-16-11-20, 1986. 14. Itoh, H., K. Nakao, M. Mukoyama, H. Arai, T. Yamada, Y. Saito, S. Shiono, K. Hosoda, G. Shirakami, and S. Suga. Pathophysiological role of augmented atria1 natriuretic polypeptide gene expression in DOCA-salt hypertension. Effects of atria1 natriuretic polypeptide monoclonal antibody. Am. J. Hypertens. 4: 39-44, 1991.
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15. Krakoff, L. R. Glucocorticoid excess syndromes causing hypertension. Cardiol. Clin. 6: 537-545, 1988. 16. Lachance, D., R. Garcia, J. Gutkowska, M. Cantin, and G. Thibault. Mechanisms of release of atria1 natriuretic factor. I. Effect of several agonists and steroids on its release by atria1 minces. Biochem. Biophys. Res. Commun. 135: 1090-1098, 1986. 17. Lanier-Smith, K. L., and M. G. Currie. Effect of glucocorticoids on the binding of atria1 natriuretic peptide to endothelial cells. Eur. J. Pharmacol. 178: 105-109, 1990. 18. Matsubara, H., Y. Hirata, H. Yoshimi, S. Takata, Y. Takagi, T. Iida, Y. Yamane, Y. Urneda, M. Nishikawa, and M. Inada. Effects of steroid and thyroid hormones on synthesis of atria1 natriuretic peptide by cultured atria1 myocytes of rat. Biothem. Biophys. Res. Commun. 145: 336-343, 1987. 19. Matsubara, H., Y. Hirata, H. Yoshimi, S. Takata, Y. Takagi, Y. Yamane, Y. Umeda, M. Nishikawa, and M. Inada. Ventricular myocytes from neonatal rats are more responsive to dexamethasone than atria1 myocytes in synthesis of atria1 natriuretic peptide. Biochem. Biophys. Res. Commun. 148: 1030-1038, 1987. Y., Y. Ito, K. Ando, E. Ogata, and T. Fujita. Phasic 20. Sato, plasma atria1 natriuretic polypeptide changes in DOCA-salt hypertensive rats. Jpn. Heart J. 31: 703-711, 1990. R. J., and J. Linden. Effect of atria1 contraction 21. Schiebinger, frequency on atria1 natriuretic peptide secretion. Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H1095-H1099, 1986. Y., R. S. Sider, E. A. Ostafin, and R. J. Grekin. 22. Shenker, Plasma levels of immunoreactive atria1 natriuretic factor in healthy subjects and in patients with edema. J. Clin. Invest. 76: 1684-1687, 1985. T., M. Ishii, Y. Hirata, H. Matsuoka, A. Miyata, 23. Sugimoto, T. Toshimori, H. Masuda, K. Kangawa, and H. Matsuo. Increased release of atria1 natriuretic polypeptides in rats with DOCA-salt hypertension. Life Sci. 38: 1351-1358, 1986. 24. Tikkanen, T., I. Tikkanen, and F. Fyhrquist. Plasma atria1 natriuretic peptide in DOCA-NaCl-treated rats. Acta Physiol. Stand. 129: 151-155, 1987. 25 Tonolo, G., R. Fraser, J. M. Connell, and C. J. Kenyon. Chronic low-dose infusions of dexamethasone in rats: effects on blood pressure, body weight and plasma atria1 natriuretic peptide. J. Hypertens. 6: 25-31, 1988. 26 Weidmann, P., D. R. Matter, E. E. Matter, M. P. Gnadinger, D. E. Uehlinger, S. Shaw, and C. Hess. Glucocorticoid and mineralocorticoid stimulation of atria1 natriuretic peptide release in man. J. Clin. Endocrinol. Metab. 66: 1233-1239, 1988. 27 Yasunari, K., M. Kohno, K. Murakawa, K. Yokokawa, and T. Takeda. Glucocorticoids and atria1 natriuretic factor receptors on vascular smooth muscle. Hypertension Dallas 16: 581-586, 1990.
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regulation of atria1 natriuretic and gene expression
factor
JAMIE DANANBERG AND ROGER J. GREKIN Division of Endocrinology, University of Michigan, Ann Arbor 48109; and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105 Dananberg, Jamie, and Roger J. Grekin. Corticoid regulation of atria1 natriuretic factor secretion and gene expression. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1377-H1381, 1992.-This study investigated the acute effects of glucocorticoids and mineralocorticoids on atria1 natriuretic factor (ANF) biosynthesis in vivo. Groups of male Sprague-Dawley rats were injected with 1 mg dexamethasone (Dex), 10 mg deoxycorticosterone acetate (DOCA) or vehicle alone. Different groups were studied after periods of 30 min to 8 h. Plasma and left atria1 ANF concentrations and ANF mRNA levels were measured 2-8 h after corticoid injection. From 30 min to 2 h after injection, ANF mRNA was analyzed by quantitative and qualitative assessments. There was a two- to threefold increase in plasma levels of ANF in Dex-treated rats compared with controls at all time periods (P < 0.05). Although ANF plasma levels increased over time following DOCA treatment, they were not significantly different from control values. Dex treatment also increased normalized ANF mRNA levels 77% above control levels during the first 4 h after injection (P < 0.05). Thereafter there was a return of mRNA levels to that seen in controls. There was no qualitative difference in the ANF mRNA at any time as assessed by Northern hybridization. In contrast, DOCA increased ANF mRNA levels only after 8 h (P < 0.05). No significant changes in left atria1 ANF content were noted during this study. In a separate study, Dex was administered to isolated left atria in vitro in a superfusion system. Superfusion with 2 x lo-” M Dex produced a 40% increase in ANF secretory rate within 20 min (P = 0.036). We conclude that Dex induces a direct rapid increase in ANF mRNA levels and ANF secretion in rats. DOCA increases ANF secretion but the effect is delayed and is likely to be mediated through a primary effect on volume expansion. atria1 natriuretic peptides; dexamethasone; one; messenger ribonucleic acid
deoxycorticoster-
ADRENOCORTICAL STEROIDS are known to affect volume homeostasis in intact organisms. Accumulating evidence suggests that atria1 natriuretic factor (ANF) may also play a central role in salt and volume balance. Over the past several years, the role of glucocorticoids and mineralocorticoids in the regulation of ANF secretion and gene expression has been evaluated; however, a complete picture of the interactions between these agents has not been fully determined. A number of investigators have examined the chronic effects of deoxycorticosterone acetate (DOCA) and dexamethasone (Dex) on ANF biosynthesis. When evaluated over a period of 12 h to several days, we (13) and others (2,14,20, 23,24) have shown that DOCA administration causes an increase in plasma levels of ANF. In vivo studies have shown a decrease in atria1 ANF content (20, 23) and an increase in ANF mRNA levels (2, 11) over this time period. In contrast, no effect of DOCA was found on ANF secretion rates or on ANF mRNA levels in primary myocyte cultures (10). Glucocorticoids have been found to raise plasma ANF
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levels in humans (26), intact rats (9, 11,16), and isolated rat atria1 myocytes (10, 19) although at least one study has shown a decrease in plasma ANF after 4 wk of low-dose treatment with Dex (25). Changes in intraatria1 ANF following glucocorticoid administration have been less well studied but there is some evidence that atria1 content of ANF is diminished (9). Several investigators have shown that chronic administration of dexamethasone causes an increase in ANF mRNA levels (10, 1l), but this effect may occur primarily in ventricular myocardium (7, 18). No study has examined the effect of these agents over a short time course. Evaluating the effects of corticoids acutely may help determine whether effects of steroids are primarily mediated through changes in secretion or increases in new hormone synthesis. To this end, we treated rats with DOCA, Dex, or vehicle injections and obtained simultaneous samples for the measurement of plasma ANF, intra-atria1 ANF content, and ANF mRNA over a period of 30 min to 8 h following the intervention. MATERIALS
AND METHODS
Treatment of rats and sample collection. Three-month-old male Sprague-Dawley rats were obtained from Charles River Breeders (Portage, MI). Rats were divided into three groups according to treatment with Dex (Sigma, St. Louis, MO), DOCA (Sigma), or vehicle control. The Dex-treated and control rats were maintained on normal rat chow and had free access to water. The DOCA-treated rats were maintained on sodium-free rat chow (sodium-deficient diet, rat modified; ICN Biochemicals, Cleveland, OH) and given 1% NaCl solution as the sole source of drinking water starting 1 wk before and continuing through the experimental period. Polyethylene glycol (1 ml) was used as the vehicle for delivery of corticoids. On the morning of the study, rats were injected subcutaneously with 1 mg Dex, 10 mg DOCA, or vehicle alone. During the course of the study, Dex and control rats were given access to water only, whereas DOCA-treated animals were given access to 1% NaCl solution and sodium-free chow. Groups of six rats from each group were killed by decapitation 2, 4, 6, and 8 h after injections. Trunk blood was collected into EDTA tubes on ice immediately after decapitation. The left atria were rapidly removed, frozen in liquid nitrogen, and kept at -70°C until processing. In a separate experiment, animals were injected with Dex or vehicle and were killed 30, 60, or 120 min following injection. The atria from these animals were frozen in liquid nitrogen and then were immediately used for extraction of total RNA. Isolation and quantification of RNA and isolation of atria1 ANF. Frozen atria were placed in denaturing solution (4 M guanidine isothiocyanate, 0.75 M sodium citrate, 10% N-lauroylsarcosine 0.72% vol/vol ,&mercaptoethanol) and homogenized in a Polytron tissue homogenizer (Kinematica, Lucerne, Switzerland). After centrifugation, an aliquot of supernatant was removed and diluted 1:50 in 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.4, and stored at -20°C until ANF
0 1992 the American
Physiological
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H1377
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H1378
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radioimmunoassay. Cellular RNA was isolated from the remaining supernatant by phenol-isoamyl alcohol-chloroform (25:24:1 vol/vol) extraction followed by cold isopropanol precipitation. The RNA was quantified in a model 1001 Plus Spectrophotometer (Milton Roy, Columbus, OH). A total of 2 pg of total RNA was used to quantify specific ANF mRNA by dot-blot or Northern hybridization. In the dot-blot procedure, RNA samples were denatured in 7.4% formaldehyde, 6~ SSC (0.9 M sodium chloride, 0.09 M sodium citrate) and heated at 65°C. Samples were applied to nylon membranes (Magnagraph, Micron Separations, Westborough, MA) using a minifold apparatus (Schleicher & Schuell, Keene, NH), and fixed by baking at 70°C for 15 min. During the Northern hybridizations, RNA samples were denatured as above, electrophoretically separated in an agaroseformaldehyde gel, and transferred to nylon membranes overnight by capillary action. In all cases, membranes were prehybridized in 5~ Denhardt’s solution (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, Oil% Ficoll), 6~ SSC, 50% formamide, 0.1% lauryl sulfate, and salmon sperm DNA, 100 pg/ml, for 90 min at 42°C. Hybridization was then carried out for 16-24 h in solution identical to the prehybridization solution except for the presence of 10% dextran and the radiolabeled cDNA probe. After hybridization with the ANF cDNA probe, filters were washed with the final wash conditions at 65°C with 0.1~ SSC, 1% lauryl sulfate. After hybridization, specifically bound counts were measured using a Betascope model 603 Blot Analyzer (Betagen, Waltham, MA), which detects and directly quantifies P-particles. Before additional probing with control cDNAs, bound counts were stripped from the filters by washing in 5 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.5% pyrophosphate, 0.1~ Denhardt’s for 90 min at 65°C and rinsed in 1X SSC. The quantification of ANF mRNA was normalized to the quantification of actin mRNA. To control for the possibility that adrenocortical steroids may also affect actin mRNA concentrations, the filters were probed again with a cDNA clone encoding the rat cyclophilin mRNA, which is expressed in large amounts in nearly all rat tissues (6). The final wash conditions were adjusted following hybridization with the actin and cyclophilin cDNAs to 45”C, 0.1X SSC, 1% lauryl sulfate. Complementary DNA probes. The ANF cDNA was kindly given to us by Dr. Howard Dene (8), who has previously reported its isolation. The probe encodes for all of the coding region and portions of the 5’ and 3’ noncoding regions of the ANF mRNA. After denaturation, the probes were labeled with deoxycytidine 5’- [ &3ZP] triphosphate ([ cy:“P]dCTP; 3,000 Ci/ mmol; Amersham, Arlington Heights, IL) to a specific activity of 10” dpm/pg using a random hexanucleotide primer kit (Prime-A-Gene System, Promega, Madison, WI). Unincorporated labeled nucleotides were removed by Sephadex centrifugation (G-50 Spin Columns, Boehringer Mannheim, Indianapolis, IN). In vitro superfusion system. Male Sprague-Dawley rats weighing 300-350 g on an ad libitum sodium diet with free access to water were killed by decapitation. The hearts were rapidly removed and placed in iced medium 199 (GIBCO Laboratories, Grand Island, NY) gassed with 95% O,-5% CO,. Left atria were carefully dissected from the hearts and were suspended between two modified aneurysm clips, one of which served as a unipolar electrode. The other was suspended from a force transducer. The suspended atria were put into a superfusion chamber with gassed medium 199 at 37°C as described by Schiebinger et al. (21) with an initial tension of 0.7 g. The atria were paced with an electric stimulator (model S88, Grass Instruments, Quincy, MA). The force transducer was connected to a direct current power amplifier and pre-amplifier (models 7DAG and 7PIG, respectively, Grass Instruments), and the signal was sent to a paper chart recorder (model 7S1225, Grass Instruments). The atria underwent a stabilization period in
GENE
EXPRESSION
which they were paced at 1 Hz for 30 min and 3 Hz for 35 min during superfusion with medium 199 at 2 ml/min. After this stabilization period, superfusate was collected at 2-min intervals. After a lo-min basal collection period, atria were superfused with Dex (2 x lo-” M) for 120 min. Measurement of ANF. Plasma ANF concentration, atria1 ANF content, and ANF secretion rates from superfused atria were measured by radioimmunoassay (22). Plasma ANF is expressed as picomoles per liter. Extracted atria1 ANF was diluted 1:22,050 during the assay. Denaturing solution diluted to these levels did not interfere with the assay standard curve. Total ANF in these samples was normalized to the measurement of total protein as measured in a calorimetric assay based on the Bradford dye-binding procedure (3; Bio-Rad Protein Assay, Bio-Rad, Richmond, CA) and is expressed as picograms per milligram of protein. ANF measured from superfusion collections in medium 199 is expressed as picograms per minute. Statistical analysis. ANF plasma levels, atria1 content, and mRNA concentrations were analyzed using a factorial analysis of variance model. ANF secretion during the in vitro experiments was analyzed using a repeated measures analysis of variance model. RESULTS
The plasma levels of ANF in animals treated with Dex, DOCA, or vehicle control are shown in Fig. 1. ANF plasma concentrations were substantially elevated 2 h after Dex administration compared with vehicle-injected animals or DOCA-treated animals (130 t 20 vs. 59 -+ 33 pmol/l, control; 22 t 7, DOCA; P < 0.05). This effect was also noted at 4,6, and 8 h after Dex treatment (185 t 22, 196 t 40, and 186 t 11 pmol/l, respectively; P < 0.05 at all points compared with control levels). There was a tendency for DOCA-treated animals to have an increase in plasma levels of ANF (27.7 t 11, 81 t 24, and 95.0 t 39.0 pmol/l at 4, 6, and 8 h); however, these values were not significantly different from those measured in control animals at the same time intervals (29.2 t 12.5, 43.3 t 15.3, and 54.8 t 9.2 pmol/l). The changes in atria1 ANF content are shown in Fig. 2. The content of ANF in left atria taken from control rats remained constant throughout the study period. Although there was a steady rise in the atria1 ANF content of animals treated with DOCA during 6 h following injection, the effect was not significant. This change was not noted at 8 h. Similarly, ANF content in atria from Dex-treated rats was not significantly different from that 250 [ n
Dex
2
* 1
4 6 Hours After lnter-ventlon
8
Fig. 1. Effect of corticoids on plasma levels of atria1 natriuretic factor (ANF) in rats treated with a single injection of dexamethasone (Dex; 1 acetate (DOCA; 10 mg), or vehicle control; n md deoxycorticosterone = 6 for each group. * P c 0.05compared with control. 9
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of controls throughout the study. The effects of the corticoids on ANF mRNA concentrations are noted in Fig. 3. There was a high degree of concordance between the actin and cyclophilin cDNA hybridization experiments ( rrL = 0.646, P = 0.0001). For this reason, only the ANF mRNA data normalized to actin are reported. Control levels of ANF mRNA did not change throughout the study period. Dex significantly increased ANF mRNA levels within 2 h of treatment when compared with either DOCA-treated or control animals [DOCA 9.1 t 2.6 vs. 4.1 t 0.4 and control 5.1 t 0.5 counts/min (cpm) of specifically bound ANF cDNA relative units normalized to cpm of specifically bound ,&actin cDNA; P < 0.051. The increase in ANF mRNA was transient in that the mRNA levels were not different from those of control animals at 6 and 8 h. In sharp contrast to the Dex effect, DOCA increased ANF mRNA levels only after 8 h (DOCA 8.0 t 0.6 vs. control 4.9 t 0.2, relative units, P < 0.05). To better assess the short-term effect of Dex on ANF gene expression, the experiment was repeated to examine the responses during a 2-h time period. To determine whether Dex caused the transcription of a lengthened or shortened ANF mRNA species, a Northern hybridization was undertaken. The results are shown in Fig. 4. At 30 min there was a 40% increase in ANF mRNA in atria of Dex-treated animals compared with those of controls. By 120 min, there was approximately a twofold increase in mRNA levels, comparable to that noted in the previous experiment. In all cases, the ANF cDNA detected an g
Dex
a
DOCA
l-l
Control
2
4
Hours
6
0
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Fig. 2. Effect of corticoids on left atria1 ANF content in rats treated a single injection of Dex (1 mg), DOCA (10 mg), or vehicle control; 6 for each group. P = not significant for all comparisons.
H
Dex
q
DOCA
with n =
a Hours
After lnterventton
Fig. 3. Effect of corticoids on left atria1 ANF mRNA concentrations in rats treated with a single injection of Dex (1 mg), DOCA (10 mg), or vehicle control; n = 6 for each group. * P < 0.05 compared with control.
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EXPRESSION
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mRNA which was - 1,075 nucleotides in length. The results of the in vitro study are shown in Fig. 5. Very early in this protocol, mean ANF secretory rates were higher in the Dex-treated group than in the control atria (P = 0.036). This enhancement was seen for a period of 90 min. The maximal increase in ANF secretion rate was -40%. DISCUSSION
This study evaluated the effects of Dex and DOCA on ANF plasma concentrations, left atria1 ANF content, and ANF mRNA levels over a period of 8 h. Additionally, the in vitro effect of Dex on left atria1 ANF secretion rates was assessed. Several previous studies have examined the in vivo effects of glucocorticoids on ANF biosynthesis. Garcia et al. (9) found a marked increase in plasma and atria1 levels of ANF in rats following corticoid replacement after adrenalectomy. Gardner and co-workers (11) noted an increase in these levels after Dex at 48 h but not at 24 h in both intact and adrenalectomized rats. LaChance et al. (16) found that atria from rats pretreated with Dex released higher amounts of ANF after l-4 h of in vitro incubation than atria did from DOCA-treated animals. The present study demonstrated a marked increase in plasma concentrations of ANF within 2 h of Dex administration. This effect was maintained for 8 h after injection. To determine whether this increase in ANF plasma levels was due to an increase in secretion or a decrease in clearance, we evaluated the effect of Dex administered in vitro in a superfusion system. We found a significant increase in ANF secretory rates following Dex administration. This increase was on the order of 30-40% over basal rates and was maintained for a period of 90 min. These data suggest that Dex stimulates ANF secretion directly, although this finding does not fully account for the threefold increase in ANF levels following Dex treatment in vivo. This difference may be explained by a decrease in ANF clearance in intact animals or by the appearance of an intermediate stimulus which further enhances ANF production. Expression of ANF receptors on vascular endothelial cells, which are primarily of the clearance type (4), has been shown to be glucocorticoid dependent (17). No known study has examined whether clearance receptors are increased above basal expression levels by glucocorticoids. A decrease in ANF receptormediated guanosine 3’,5’-cyclic monophosphate (cGMP) production following treatment with Dex has been shown (27). The effects of glucocorticoids on ANF endopeptidases, the other probable mechanism of ANF clearance (12), have not been reported. The present study showed a rapid increase in levels of ANF mRNA following Dex administration. The mRNA species was not different in size from that found in control rats, although the resolution of Northern hybridization does not exclude the possibility that small changes in mRNA size occurred. This finding suggests that a substantial change in the size of the ANF mRNA with an associated change in the stability of the messenger RNA does not explain the mechanism by which Dex raises transcript levels. Gardner et al. (10) have shown that Dex
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H1380
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30 Minutes
AND ANF GENE EXPRESSION
60 Minutes
120 Minutes Fig. 4. Northern hybridization showing effect of glucocorticoids on left atria1 ANF mRNA concentration in rats treated with a single injection of Dex (1 mg). Lane 8: sample with known low concentration of total cellular RNA. All other lanes were loaded with 2 pg total RNA. Dex and control values were normalized to j3-actin hybridization. Ratios of these values are shown for each pair.
980 nt
-
DEXKontrol
1.40
1.40
2.87
1.47
increases both transcriptional activity and ANF mRNA half-life from 17 to 30 hours. In our study, however, the disappearance rate of the ANF mRNA after the initial 4-h rise suggests that there is a substantially shorter halflife during this first phase of stimulation of the mRNA. It also suggests that a significant portion of the rise is due to an increase in transcriptional activity. A second, more prolonged increase in ANF mRNA has been reported to occur after 24-48 h (10,ll) and may, to a larger degree, be due to an increase in mRNA half-life. Whereas a number of studies have shown that, over a period of 12 h to several weeks, DOCA increases plasma ANF concentrations (2, 13, 14, 20, 23, 24) and ANF mRNA levels (2, ll), only limited data are available on the acute effects of DOCA upon these parameters. The present study showed a trend toward an increase in plasma ANF concentrations in rats treated with DOCA; however these changes were not significant when compared with those of control rats. This study did not find a significant change in the atria1 content of ANF over the study period. In contrast, we were able to demonstrate a significant rise in ANF mRNA levels 8 h after stimulation with DOCA. These data are consistent with the findings of Ballerman and co-workers (2) in which DOCA 175
I
Basal
Dexamethasone
1
1.74
stimulated increases in plasma ANF and ANF mRNA were noted at 12 h after a single injection of DOCA. Based on these findings, we postulate that the effect of mineralocorticoids on ANF biosynthesis is an indirect one, whereas the glucocorticoid influence is more direct. The finding that Dex is able to alter ANF plasma concentration within 2 h and ANF mRNA levels within 30 min is in sharp contrast to the later response seen with DOCA. It is possible that the primary effect of DOCA is to cause volume expansion, which directly mediates ANF secretion, most likely by causing atria1 distension (1, 5). An additional explanation suggests that other intermediates need to be generated before the DOCA effect is apparent. This would explain the relatively longer period of time elapsed before the appearance of increases in plasma ANF. This hypothesis is further substantiated by the fact that DOCA does not directly stimulate ANF secretion from myocardial cells in vitro (10). Glucocorticoids administered in high doses typically cause a hypertensive response (15). These data suggest that direct glucocorticoid stimulation of ANF biosynthesis modulates this hypertensive effect. It is possible that without the stimulus to ANF secretion, the elevation in blood pressure might be even greater. In contrast, the ANF secretory response to mineralocorticoid stimulation occurs secondarily and is more likely part of an overall mechanism to maintain hemodynamic homeostasis in the face of volume expansion and hypertension. This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-18575 and by grants from the American Heart Association and its Indiana affiliate. J. Dananberg performed this work during the tenure of a ClinicianScientist Award from the American Heart Association. Address for reprint requests: J. Dananberg, Div. of Endocrinology, Univ. of Michigan Medical Ctr., 5570 MSRB-II Box 0678, Ann Arbor, MI 48109-0678. Received 16 December 1991; accepted in final form 12 June 1992.
50, t
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5
, 10
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30 40 50 Time (mln)
,
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70
, 60
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Fig. 5. Effect of Dex (squares; n = 10) or superfusion medium alone (circles; n = 7) on left atria1 ANF secretion rate in an in vitro superfusion system. Atria were superfused with gassed medium 199 at 2 ml/ min. Results expressed as a percent of basal secretory rates, means + SE. ANF secretion by Dex-treated atria is significantly greater than that from atria exposed to superfusion medium alone (P < 0.05; repeated measures analysis of variance).
REFERENCES 1. Anderson, J. V., J. Donckier, W. J. McKenna, and S. R. Bloom. The plasma release of atria1 natriuretic peptide in man. Clin. Sci. 71: 151-155, 1986. 2. Ballermann, B. J., K. D. Bloch, J. G. Seidman, and B. M. Brenner. Atria1 natriuretic peptide transcription, secretion, and glomerular receptor activity during mineralocorticoid escape in 1986. the rat. J. Clin. Invest. 78: 840-843,
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3. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 4. Chevalier, R. L., M. Garmey, R. M. Scarborough, J. Linden, R. A. Gomez, M. J. Peach, and R. M. Carey. Inhibition of ANP clearance receptors and endopeptidase 24.11 in maturing rats. Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29): R1218-R1228, 1991. 5. Dananberg, J., B. M. Egan, E. R. Bates, and R. J. Grekin. Sustained saline-induced secretion of atria1 natriuretic hormone is not maintained by atria1 stretch. J. Clin. Endocrinol. Metab. 68: 735-739, 1989. 6. Danielson, P. E., S. Forss-Petter, M. A. Brow, L. Calavetta, J. Douglass, R. J. Milner, and J. G. Sutcliffe. plBl5: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7: 261-267, 1988. 7. Day, M. L., D. Schwartz, R. C. Wiegand, P. T. Stockman, S. R. Brunnert, H. E. Tolunay, M. G. Currie, D. G. Standaert, and P. Needleman. Ventricular atriopeptin. Unmasking of messenger RNA and peptide synthesis by hypertrophy or dexamethasone. Hypertension Dallas 9: 485-491, 1987. 8. Dene, H., and J. P. Rapp. Quantification of messenger ribonucleic acid for atria1 natriuretic factor in atria and ventricles of Dahl salt-sensitive and salt-resistant rats. Mol. Endocrinol. 1: 614-620, 1987. R., W. Debinski, J. Gutkowska, 0. Kuchel, G. 9. Garcia, Thibault, J. Genest, and M. Cantin. Gluco- and mineralocorticoids may regulate the natriuretic effect and the synthesis and release of atria1 natriuretic factor by the rat atria in vivo. Biochem. Biophys. Res. Commun. 131: 806-814, 1985. D. G., B. J. Gertz, C. F. Deschepper, and D. Y. 10. Gardner, Kim. Gene for the rat atria1 natriuretic peptide is regulated by glucocorticoids in vitro. J. Clin. Invest. 82: 1275-1281, 1988. D. G., S. Hane, D. Trachewsky, D. Schenk, and 11. Gardner, J. D. Baxter. Atria1 natriuretic peptide mRNA is regulated by glucocorticoids in vivo. Biochem. Biophys. Res. Commun. 139: 1047-1054, 1986. 12. Gerbes, A. L., and A. M. Vollmar. Degradation and clearance of atria1 natriuretic factors (ANF). Life Sci. 47: 1173-l 180, 1990. 13. Grekin, R. J., W. D. Ling, Y. Shenker, and D. F. Bohr. Immunoreactive atria1 natriuretic hormone levels increase in deoxycorticosterone acetate-treated pigs. Hypertension Dallas, Suppl. II 8: 11-16-11-20, 1986. 14. Itoh, H., K. Nakao, M. Mukoyama, H. Arai, T. Yamada, Y. Saito, S. Shiono, K. Hosoda, G. Shirakami, and S. Suga. Pathophysiological role of augmented atria1 natriuretic polypeptide gene expression in DOCA-salt hypertension. Effects of atria1 natriuretic polypeptide monoclonal antibody. Am. J. Hypertens. 4: 39-44, 1991.
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15. Krakoff, L. R. Glucocorticoid excess syndromes causing hypertension. Cardiol. Clin. 6: 537-545, 1988. 16. Lachance, D., R. Garcia, J. Gutkowska, M. Cantin, and G. Thibault. Mechanisms of release of atria1 natriuretic factor. I. Effect of several agonists and steroids on its release by atria1 minces. Biochem. Biophys. Res. Commun. 135: 1090-1098, 1986. 17. Lanier-Smith, K. L., and M. G. Currie. Effect of glucocorticoids on the binding of atria1 natriuretic peptide to endothelial cells. Eur. J. Pharmacol. 178: 105-109, 1990. 18. Matsubara, H., Y. Hirata, H. Yoshimi, S. Takata, Y. Takagi, T. Iida, Y. Yamane, Y. Urneda, M. Nishikawa, and M. Inada. Effects of steroid and thyroid hormones on synthesis of atria1 natriuretic peptide by cultured atria1 myocytes of rat. Biothem. Biophys. Res. Commun. 145: 336-343, 1987. 19. Matsubara, H., Y. Hirata, H. Yoshimi, S. Takata, Y. Takagi, Y. Yamane, Y. Umeda, M. Nishikawa, and M. Inada. Ventricular myocytes from neonatal rats are more responsive to dexamethasone than atria1 myocytes in synthesis of atria1 natriuretic peptide. Biochem. Biophys. Res. Commun. 148: 1030-1038, 1987. Y., Y. Ito, K. Ando, E. Ogata, and T. Fujita. Phasic 20. Sato, plasma atria1 natriuretic polypeptide changes in DOCA-salt hypertensive rats. Jpn. Heart J. 31: 703-711, 1990. R. J., and J. Linden. Effect of atria1 contraction 21. Schiebinger, frequency on atria1 natriuretic peptide secretion. Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H1095-H1099, 1986. Y., R. S. Sider, E. A. Ostafin, and R. J. Grekin. 22. Shenker, Plasma levels of immunoreactive atria1 natriuretic factor in healthy subjects and in patients with edema. J. Clin. Invest. 76: 1684-1687, 1985. T., M. Ishii, Y. Hirata, H. Matsuoka, A. Miyata, 23. Sugimoto, T. Toshimori, H. Masuda, K. Kangawa, and H. Matsuo. Increased release of atria1 natriuretic polypeptides in rats with DOCA-salt hypertension. Life Sci. 38: 1351-1358, 1986. 24. Tikkanen, T., I. Tikkanen, and F. Fyhrquist. Plasma atria1 natriuretic peptide in DOCA-NaCl-treated rats. Acta Physiol. Stand. 129: 151-155, 1987. 25 Tonolo, G., R. Fraser, J. M. Connell, and C. J. Kenyon. Chronic low-dose infusions of dexamethasone in rats: effects on blood pressure, body weight and plasma atria1 natriuretic peptide. J. Hypertens. 6: 25-31, 1988. 26 Weidmann, P., D. R. Matter, E. E. Matter, M. P. Gnadinger, D. E. Uehlinger, S. Shaw, and C. Hess. Glucocorticoid and mineralocorticoid stimulation of atria1 natriuretic peptide release in man. J. Clin. Endocrinol. Metab. 66: 1233-1239, 1988. 27 Yasunari, K., M. Kohno, K. Murakawa, K. Yokokawa, and T. Takeda. Glucocorticoids and atria1 natriuretic factor receptors on vascular smooth muscle. Hypertension Dallas 16: 581-586, 1990.
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