The Role of Development and Adrenal Steroids in the Regulation of the Mineralocorticoid Receptor Messenger RNA Judith E. Kalinyak, Joyce G. Bradshaw and A. J. Perlman Department of Medicine, Stanford University School of Medicine, Stanford, California, U. S. A.
The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 2 5 30 % of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney. Key words Mineralocorticoid — Glucocorticoid — Receptor —mRNA—Adrenalectomy—Brain—Kidney
Introduction Adrenal steroids have a diverse range of physiologic actions in the central nervous system. Two functionally distinct (Type I and Type II) adrenocorticosteroid receptors have been characterized in the brain (Funder and Shepard 1987). In vitro studies have shown that the Type I receptor is mineralocorticoid receptor (MR)-like in its binding properties. Patel, Sherman, Goldman and Watson (1989) cloned the rat hippocampal Type I receptor and showed that it is highly homologous to the human renal MR {Arriza, Weinberger, Cerelli, Glaser, Handelin, Housman and Evans 1987). The Type II, or glucocorticoid receptor (GR), is more widely distributed in the brain and is found in both neurons and glial cells (Reul and DeKloet 1986). Previous studies of the ontogeny of brain corticosteroid receptors were performed using radioligand binding assays {Clayton, Grosser and Stevens 1977; Sarrieau, Vial, McEwen, Broer, Dussaillant, Philibert, Moguilewsky and Rostene 1986; Rosenfeld, Sutano, Levine and
Horm.metab.Res.24(1992) 106-109 © Georg Thieme Verlag Stuttgart • New York
DeKloet 1988). Using a specific cRNA probe for the MR gene, the present study was undertaken to examine the developmental and adrenocorticosteroid regulation of MR gene expression in both the brain and the kidney. Methods Animals Male Sprague-Dawley rats (Simenson, Gilroy, CA) were maintained according to Stanford University guidelines on ad lib rat chow and tap water with a 12 h light/dark cycle. Animals were killed by decapitation, and the tissues were frozen in liquid nitrogen and stored at — 80 °C. Fetal tissues were obtained from timed-pregnant rats. Three-12 organs at each age were pooled prior to RNA isolation. Six week old rats were adrenalectomized (ADX) under chloral hydrate (6.25%, 0.5 ml/100 g) anesthesia and maintained on 0.9% saline instead of water for two weeks before use. Synthesis
of hybridization
probes
The GR cRNA probe was the cRNA transcript of the 3' nontranslated, 2.2-Kb Xba-Pst I fragment of pRM16 rat GR clone inserted into pSP65 Riboprobe in the anti-sense orientation (a kind gift of K. Yamamoto, University of California, San Francisco) {Miesfield, Okret, Wilkstrom, Wrange, Gustafsson and Yamamoto 1984). The MR cRNA probe was the cRNA transcript from the Eco RI fragment of the human MR clone hkl 800 (a kind gift of J. Arriza, Salk Institute, San Diego) {Arriza et al. 1987). With Sp6 RNA polymerase, the average specific activity of these probes was ~ 1 x 109 cpm/|xg. A fi-actin clone (a kind gift of L. Kedes, University of Southern California, Los Angeles) was nick-translated to high specific activity {Maniatis, Fritsch and Sambrook 1982) and was hybridized to duplicate filters. mRNA
quantification
Total cellular RNA was isolated from frozen tissue as previously described {Kalinyak and Perlman 1987). RNA was quantitated by UV absorbance and its integrity examined by agarose gel electrophoresis followed by ethidium bromide staining. MR, GR and actin mRNA were quantitated by slot blot hybridization {Kalinyak, Dorin, Hoffman and Perlman 1987). Prehybridizations and hybridizations were performed at 60 °C (MR), 65 °C (GR) and 42 °C (actin) in 50% formamide, 3 x SSC, 10 x Denhardt's solution, 20 mM Tris (pH 7.6), 10 mM EDTA (pH 8.0), 0.2% SDS, and 200 ug/ml sheared denatured salmon sperm DNA with a hybridization buffer containing 3 x 106 cpm/ml of the radiolabeled probe. Following hybridization, the filters were sequentially washed at 65 °C (GR), 60 °C (MR) and 42 °C (actin) in 2 x SSC, 0.2% SDS and 0.2 x SSC, 0.2% SDS. Autoradiographs were scanned with a laser densitometer (LKB 2202 Ultro Scan, Piscataway, NJ, U. S. A.).
Received: 15 May 1991
Accepted: 23 May 1991
Downloaded by: University of British Columbia. Copyrighted material.
Summary
Fig. 1 Ontogenic regulation of MR mRNA in the brain and kidney. Figure 1 depicts the developmental regulation of the MR mRNA in the brain and kidney of newborn, adult and aged rats. The plots were not corrected for actin because brain actin decreases in the brain with rat development and this would artifactually result in an increase in MR mRNA levels.
Fig. 2 Brain MR and GR mRNA levels. Total RNA was isolated from the following pooled (10 rats) brain regions: cortex (C); midbrain (MB); brain stem (BS); hippocampus (HIP); caudate (CAUD); hypothalamus (HYPO); septum (S); and pituitary (P); of control (white bars) or ADX (stippled bars) rats: The relative abundance of MR or GR mRNA was subsequently determined by slot blot hybridization, and laser densitometric scanning of the resulting autoradiographs.
Table 1 Dexamethasone regulation of mineralocorticoid receptor mRNA. Age
Control
Dexamethasone
Brain
1 week 6 weeks
18.24 ±0.54 23.67 ±2.88
17.35 ±1.32 21.09 ±2.17
P = NS P = NS
Kidney
1 week 6 weeks
31.88 ±1.72 26.00 ±4.08
11.77 ±4.36 16.10 ±2.55
P < 0.05 P = NS
One and 6 week old rats received a single i. p. injection of diluent or dexamethasone (7 mg/kg), stippled bars) (n = 3, ±SEM). mRNA was quantitated in arbitrary scanner units. NS = not significant.
Results The relative abundance of brain MR mRNA remained relatively constant throughout development (Fig. 1). The specificity of the probe was verified by the lack of significant hybridization to total liver RNA, an organ with abundant GR receptor mRNA (Kalinyak, Griffin, Hamilton, Bradshaw, Perlman and Hoffman 1989) (data not shown). The amount of actin m R N A decreased during development (data not shown). A different developmental pattern of MR gene expression was seen in the kidneys. High levels of kidney MR mRNA were found in the early neonatal period, but over the next 6 weeks, these levels decreased, reaching a nadir of 25 — 30 % of the neonatal concentrations (Fig. 1). The actin mRNA levels in the kidney did not change during development (data not shown).
To investigate the impact adrenal steroids had on the regulation of the MR mRNA, total brain and kidney RNA from pooled control or ADX 6 - 8 week old animals was obtained. Following adrenalectomy, the levels of MR mRNA increased by 35 % in the whole brain and by 97 % in the kidney (data not shown). Marked differences were found in the relative distribution of the MR and G R mRNA throughout the brain (Fig. 2). The MR mRNA was most abundant in the hippocampus and pituitary, while G R m R N A was more diffusely distributed. After adrenalectomy, the MR mRNA increased in the hippocampus (32 %) and cortex (35 %) but decreased in the caudate (40%), spetum (40%) and pituitary (50%), while the GR mRNA increased by 10 - < 200 % throughout all brain regions.
107
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Horm. metab. Res. 24 (1992)
Mineralocorticoid Receptor mRNA
Horm. metab. Res. 24 (1992) To investigate whether glucocorticoids could modulate the MR gene expression in vivo, rats received a single intraperitoneal injection of dexamethasone (7 mg/kg) six hours prior to sacrifice. In 1 week old but not in six week old rats, high dose glucocorticoid treatment reduced the MR mRNA levels significantly in the kidney. In contrast to the kidney, high dose dexamethasone administration did not alter total brain MR mRNA levels (Table 1).
Judith E. Kalinyak, Joyce G. Bradshaw and A. J. Perlman In conclusion, we have demonstrated tissuespecific developmental patterns of MR mRNA accumulation in brain and kidney. Adrenalectomy caused an increase in MR mRNA in the hippocampus, in the cortex of the brain, and in the kidney, and dexamethasone decreased the relative abundance of renal MR mRNA in neonatal rats without influencing brain MR mRNA levels. Acknowledgements
In the brain, the expression of the MR gene differs from that of the G R gene in its ontogeny, regional distribution, and its regulation by adrenal steroids. While brain GR mRNA levels progressively increase during the first 3 weeks of life {Kalinyak et al. 1989), brain MR m R N A concentration did not vary substantially throughout the lifespan of the rat. The GR mRNA levels were widely distributed throughout the CNS, but the presence of MR mRNA was most abundant in the hippocampus (Arriza et al. 1987; Herman, Patel, Akil and Watson 1989; Chao, Choo and McEwen 1989). Using radioligand studies, Sarrieau, Sharma and Meany (1988) reported an increase in hippocampal GR but not MR number in the neonatal rat, and Rosenfeld et al. (1988) confirmed the divergent ontogenic regulation of the MR and GR in the hipocampus. They demonstrated an increase in GR receptors during the first 3 weeks of life, but did not detect any MR receptors until 8 days of age. We found that the brain M R mRNA is present as early as fetal day 15 and persists at this level after birth, a time when no MR was detected by binding assays. The difference between our findings and those of Rosenfeld et al. (1988) may reflect a greater sensitivity of our methods or ontogenic differences in the translation of the MR mRNA. In contrast to the brain, renal MR mRNA was under pronounced ontogenic control, with levels falling dramatically after 3 weeks of age. Adrenalectomy caused a 95 % increase in renal MR mRNA levels. The increase in brain MR mRNA was restricted to the hippocampus and cortical regions. Reul, van den Bosch and DeKloet (1987) showed that the MR receptor was localized almost exclusively to the hippocampus and that both the M R and G R increased after adrenalectomy. In contrast, Luttge, Rupp and Day da (1989) reported an increase in both type I and type II receptors soon after adrenalectomyovariectomy, but receptor levels, as measured by radioligand binding studies, returned to near baseline levels 16 days after surgery. Reul, Pearce, Funder and Krozowski (1989) demonstrated only a transient increase in hippocampal MR mRNA following adrenalectomy, while Herman et al. (1989) reported an increase in MR mRNA in a single subfield of the hippocampus 8 days after adrenalectomy. Chao, Choo and McEwen (1989) reported an increase in binding to the type II hippocampal receptor after adrenalectomy, but found no change in local MR mRNA abundance. Pharmacologic doses of dexamethasone caused a significant decrease in MR mRNA in the kidneys of 1 week old rats. The lack of down-regulation of the MR mRNA in the brain following dexamethasone treatment agrees with previous studies (Herman et al. 1989; Luttge, Rupp and Davda 1989).
This research was supported by grants from the NIH (HL 35351) and the American Heart Association (86N139A). JEK was a recipient of an American Heart Association (California Affiliate) Fellowship (87534A) and a Daland Fellowship from the American Philosophical Society.
References Arriza, J. L., C. Weinberger, G. Cerelli, T. M. Glaser, R. L. Handelin, D. E. Housman, R. M. Evans: Cloning of human mineralocorticoid receptor complementary DNAL. Structural and functional kinship with the glucocorticoid receptor. Science 237: 268—275 (1987) Chao, H. M., P. H. Choo, B. S. McEwen: Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain. Neuroendocrinology 50:365-371 (1989) Clayton, C. J., B. I. Grosser, W. Stevens: The ontogeny of corticosterone and dexamethasone receptors in rat brain. Brain Res. 134:445-453(1977) Funder, J. W., K. Shepard: Adrenocortical steroids and the brain. Ann. Rev. Physiol. 49:397-411(1987) Herman, J. P., P. D. Patel, H. Akil, S. J. Watson: Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat. Mol. Endocrinol. 3:1886-1894 (1989) Kalinyak, J. E., A. J. Perlman: Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone. J. Biol. Chem. 262:460-464(1987) Kalinyak, J. E., C. A. Griffin, R. W. Hamilton, J. G. Bradshaw, A. J. Perlman, A. R. Hoffman: Developmental and hormonal regulation of glucocorticoid receptor mRNA in the rat. J. Clin. Invest. 84: 1843-1848(1989) Kalinyak, J. E., R. I. Dorin, A. R. Hoffman, A. J. Perlman: Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone. J. Biol. Chem. 262:10441 -10444 (1987) Luttge, W. G, M. E. Rupp, M. M. Davda: Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors. Endocrinology 125:817-824(1989) Maniatis, R., E. F. Fritsch, J. K. Sambrook: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1982) Miesfleld, R., S. Okret, A.-C Wilkstrom, O. Wrange, J.-A. Gustafsson, K. R. Yamamoto: Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312: 779781 (1984) Patel, P. D., T. G. Sherman, D. J. Goldman, S. J. Watson: Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus. Mol. Endocrinol. 3: 1877—1885 (1989) Reul, J. M. H. M., F. R. van den Bosch, E. R. De Kloet: Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrin. 115:459-467 (1987) Reul, J. M. H. M., P. T. Pearce, J. W. Funder, Z. S. Krozowski: Type I and type II corticosteroid receptor gene expression in the rat: Effect of adrenalectomy and dexamethasone administration. Mol. Endo. 3:1674-1680(1989) Reul, J. M. H. M., E. R. De Kloet: Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradio-
Downloaded by: University of British Columbia. Copyrighted material.
Discussion
Mineralocorticoid Receptor mRNA
Horm. metab. Res. 24 (1992)
109
Downloaded by: University of British Columbia. Copyrighted material.
graphy and computerized image analysis. J. Steroid Biochem. 25: Requests for reprints should be addressed to: 269-272(1986) Rosenfeld, P., W. Sutanto, S. Levine, E. R. De Kloet: Ontogeny of typeJudith Kalinyak, M. D., Ph. D. I and type II corticosteroid receptors in the rat hippocampus. Dev. Division of Endocrinology Brainres.42:113-118(1988) Building 100, Room 286 Sarrieau, A., M. Vial, B. McEwen, Y. Broer, M. Dussaillant, D. Phil-San Francisco General Hospital ibert, M. Moguilewsky, W. Rostene: Corticosteroid receptors in rat San Francisco, CA 94110 (U. S. A.) hippocampal sections: Effect of adrenalectomy and corticosterone replacement. J. Steroid Biochem. 24:721 -724 (1986) Sarrieau, A., S. Sharma, M. J. Meany: Postnatal development and environmental regulation of hippocampal glucocorticoid and mineralocorticoid receptors. Dev. Brain Res.43:158-162(1988)
The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 2 5 30 % of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney. Key words Mineralocorticoid — Glucocorticoid — Receptor —mRNA—Adrenalectomy—Brain—Kidney
Introduction Adrenal steroids have a diverse range of physiologic actions in the central nervous system. Two functionally distinct (Type I and Type II) adrenocorticosteroid receptors have been characterized in the brain (Funder and Shepard 1987). In vitro studies have shown that the Type I receptor is mineralocorticoid receptor (MR)-like in its binding properties. Patel, Sherman, Goldman and Watson (1989) cloned the rat hippocampal Type I receptor and showed that it is highly homologous to the human renal MR {Arriza, Weinberger, Cerelli, Glaser, Handelin, Housman and Evans 1987). The Type II, or glucocorticoid receptor (GR), is more widely distributed in the brain and is found in both neurons and glial cells (Reul and DeKloet 1986). Previous studies of the ontogeny of brain corticosteroid receptors were performed using radioligand binding assays {Clayton, Grosser and Stevens 1977; Sarrieau, Vial, McEwen, Broer, Dussaillant, Philibert, Moguilewsky and Rostene 1986; Rosenfeld, Sutano, Levine and
Horm.metab.Res.24(1992) 106-109 © Georg Thieme Verlag Stuttgart • New York
DeKloet 1988). Using a specific cRNA probe for the MR gene, the present study was undertaken to examine the developmental and adrenocorticosteroid regulation of MR gene expression in both the brain and the kidney. Methods Animals Male Sprague-Dawley rats (Simenson, Gilroy, CA) were maintained according to Stanford University guidelines on ad lib rat chow and tap water with a 12 h light/dark cycle. Animals were killed by decapitation, and the tissues were frozen in liquid nitrogen and stored at — 80 °C. Fetal tissues were obtained from timed-pregnant rats. Three-12 organs at each age were pooled prior to RNA isolation. Six week old rats were adrenalectomized (ADX) under chloral hydrate (6.25%, 0.5 ml/100 g) anesthesia and maintained on 0.9% saline instead of water for two weeks before use. Synthesis
of hybridization
probes
The GR cRNA probe was the cRNA transcript of the 3' nontranslated, 2.2-Kb Xba-Pst I fragment of pRM16 rat GR clone inserted into pSP65 Riboprobe in the anti-sense orientation (a kind gift of K. Yamamoto, University of California, San Francisco) {Miesfield, Okret, Wilkstrom, Wrange, Gustafsson and Yamamoto 1984). The MR cRNA probe was the cRNA transcript from the Eco RI fragment of the human MR clone hkl 800 (a kind gift of J. Arriza, Salk Institute, San Diego) {Arriza et al. 1987). With Sp6 RNA polymerase, the average specific activity of these probes was ~ 1 x 109 cpm/|xg. A fi-actin clone (a kind gift of L. Kedes, University of Southern California, Los Angeles) was nick-translated to high specific activity {Maniatis, Fritsch and Sambrook 1982) and was hybridized to duplicate filters. mRNA
quantification
Total cellular RNA was isolated from frozen tissue as previously described {Kalinyak and Perlman 1987). RNA was quantitated by UV absorbance and its integrity examined by agarose gel electrophoresis followed by ethidium bromide staining. MR, GR and actin mRNA were quantitated by slot blot hybridization {Kalinyak, Dorin, Hoffman and Perlman 1987). Prehybridizations and hybridizations were performed at 60 °C (MR), 65 °C (GR) and 42 °C (actin) in 50% formamide, 3 x SSC, 10 x Denhardt's solution, 20 mM Tris (pH 7.6), 10 mM EDTA (pH 8.0), 0.2% SDS, and 200 ug/ml sheared denatured salmon sperm DNA with a hybridization buffer containing 3 x 106 cpm/ml of the radiolabeled probe. Following hybridization, the filters were sequentially washed at 65 °C (GR), 60 °C (MR) and 42 °C (actin) in 2 x SSC, 0.2% SDS and 0.2 x SSC, 0.2% SDS. Autoradiographs were scanned with a laser densitometer (LKB 2202 Ultro Scan, Piscataway, NJ, U. S. A.).
Received: 15 May 1991
Accepted: 23 May 1991
Downloaded by: University of British Columbia. Copyrighted material.
Summary
Fig. 1 Ontogenic regulation of MR mRNA in the brain and kidney. Figure 1 depicts the developmental regulation of the MR mRNA in the brain and kidney of newborn, adult and aged rats. The plots were not corrected for actin because brain actin decreases in the brain with rat development and this would artifactually result in an increase in MR mRNA levels.
Fig. 2 Brain MR and GR mRNA levels. Total RNA was isolated from the following pooled (10 rats) brain regions: cortex (C); midbrain (MB); brain stem (BS); hippocampus (HIP); caudate (CAUD); hypothalamus (HYPO); septum (S); and pituitary (P); of control (white bars) or ADX (stippled bars) rats: The relative abundance of MR or GR mRNA was subsequently determined by slot blot hybridization, and laser densitometric scanning of the resulting autoradiographs.
Table 1 Dexamethasone regulation of mineralocorticoid receptor mRNA. Age
Control
Dexamethasone
Brain
1 week 6 weeks
18.24 ±0.54 23.67 ±2.88
17.35 ±1.32 21.09 ±2.17
P = NS P = NS
Kidney
1 week 6 weeks
31.88 ±1.72 26.00 ±4.08
11.77 ±4.36 16.10 ±2.55
P < 0.05 P = NS
One and 6 week old rats received a single i. p. injection of diluent or dexamethasone (7 mg/kg), stippled bars) (n = 3, ±SEM). mRNA was quantitated in arbitrary scanner units. NS = not significant.
Results The relative abundance of brain MR mRNA remained relatively constant throughout development (Fig. 1). The specificity of the probe was verified by the lack of significant hybridization to total liver RNA, an organ with abundant GR receptor mRNA (Kalinyak, Griffin, Hamilton, Bradshaw, Perlman and Hoffman 1989) (data not shown). The amount of actin m R N A decreased during development (data not shown). A different developmental pattern of MR gene expression was seen in the kidneys. High levels of kidney MR mRNA were found in the early neonatal period, but over the next 6 weeks, these levels decreased, reaching a nadir of 25 — 30 % of the neonatal concentrations (Fig. 1). The actin mRNA levels in the kidney did not change during development (data not shown).
To investigate the impact adrenal steroids had on the regulation of the MR mRNA, total brain and kidney RNA from pooled control or ADX 6 - 8 week old animals was obtained. Following adrenalectomy, the levels of MR mRNA increased by 35 % in the whole brain and by 97 % in the kidney (data not shown). Marked differences were found in the relative distribution of the MR and G R mRNA throughout the brain (Fig. 2). The MR mRNA was most abundant in the hippocampus and pituitary, while G R m R N A was more diffusely distributed. After adrenalectomy, the MR mRNA increased in the hippocampus (32 %) and cortex (35 %) but decreased in the caudate (40%), spetum (40%) and pituitary (50%), while the GR mRNA increased by 10 - < 200 % throughout all brain regions.
107
Downloaded by: University of British Columbia. Copyrighted material.
Horm. metab. Res. 24 (1992)
Mineralocorticoid Receptor mRNA
Horm. metab. Res. 24 (1992) To investigate whether glucocorticoids could modulate the MR gene expression in vivo, rats received a single intraperitoneal injection of dexamethasone (7 mg/kg) six hours prior to sacrifice. In 1 week old but not in six week old rats, high dose glucocorticoid treatment reduced the MR mRNA levels significantly in the kidney. In contrast to the kidney, high dose dexamethasone administration did not alter total brain MR mRNA levels (Table 1).
Judith E. Kalinyak, Joyce G. Bradshaw and A. J. Perlman In conclusion, we have demonstrated tissuespecific developmental patterns of MR mRNA accumulation in brain and kidney. Adrenalectomy caused an increase in MR mRNA in the hippocampus, in the cortex of the brain, and in the kidney, and dexamethasone decreased the relative abundance of renal MR mRNA in neonatal rats without influencing brain MR mRNA levels. Acknowledgements
In the brain, the expression of the MR gene differs from that of the G R gene in its ontogeny, regional distribution, and its regulation by adrenal steroids. While brain GR mRNA levels progressively increase during the first 3 weeks of life {Kalinyak et al. 1989), brain MR m R N A concentration did not vary substantially throughout the lifespan of the rat. The GR mRNA levels were widely distributed throughout the CNS, but the presence of MR mRNA was most abundant in the hippocampus (Arriza et al. 1987; Herman, Patel, Akil and Watson 1989; Chao, Choo and McEwen 1989). Using radioligand studies, Sarrieau, Sharma and Meany (1988) reported an increase in hippocampal GR but not MR number in the neonatal rat, and Rosenfeld et al. (1988) confirmed the divergent ontogenic regulation of the MR and GR in the hipocampus. They demonstrated an increase in GR receptors during the first 3 weeks of life, but did not detect any MR receptors until 8 days of age. We found that the brain M R mRNA is present as early as fetal day 15 and persists at this level after birth, a time when no MR was detected by binding assays. The difference between our findings and those of Rosenfeld et al. (1988) may reflect a greater sensitivity of our methods or ontogenic differences in the translation of the MR mRNA. In contrast to the brain, renal MR mRNA was under pronounced ontogenic control, with levels falling dramatically after 3 weeks of age. Adrenalectomy caused a 95 % increase in renal MR mRNA levels. The increase in brain MR mRNA was restricted to the hippocampus and cortical regions. Reul, van den Bosch and DeKloet (1987) showed that the MR receptor was localized almost exclusively to the hippocampus and that both the M R and G R increased after adrenalectomy. In contrast, Luttge, Rupp and Day da (1989) reported an increase in both type I and type II receptors soon after adrenalectomyovariectomy, but receptor levels, as measured by radioligand binding studies, returned to near baseline levels 16 days after surgery. Reul, Pearce, Funder and Krozowski (1989) demonstrated only a transient increase in hippocampal MR mRNA following adrenalectomy, while Herman et al. (1989) reported an increase in MR mRNA in a single subfield of the hippocampus 8 days after adrenalectomy. Chao, Choo and McEwen (1989) reported an increase in binding to the type II hippocampal receptor after adrenalectomy, but found no change in local MR mRNA abundance. Pharmacologic doses of dexamethasone caused a significant decrease in MR mRNA in the kidneys of 1 week old rats. The lack of down-regulation of the MR mRNA in the brain following dexamethasone treatment agrees with previous studies (Herman et al. 1989; Luttge, Rupp and Davda 1989).
This research was supported by grants from the NIH (HL 35351) and the American Heart Association (86N139A). JEK was a recipient of an American Heart Association (California Affiliate) Fellowship (87534A) and a Daland Fellowship from the American Philosophical Society.
References Arriza, J. L., C. Weinberger, G. Cerelli, T. M. Glaser, R. L. Handelin, D. E. Housman, R. M. Evans: Cloning of human mineralocorticoid receptor complementary DNAL. Structural and functional kinship with the glucocorticoid receptor. Science 237: 268—275 (1987) Chao, H. M., P. H. Choo, B. S. McEwen: Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain. Neuroendocrinology 50:365-371 (1989) Clayton, C. J., B. I. Grosser, W. Stevens: The ontogeny of corticosterone and dexamethasone receptors in rat brain. Brain Res. 134:445-453(1977) Funder, J. W., K. Shepard: Adrenocortical steroids and the brain. Ann. Rev. Physiol. 49:397-411(1987) Herman, J. P., P. D. Patel, H. Akil, S. J. Watson: Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat. Mol. Endocrinol. 3:1886-1894 (1989) Kalinyak, J. E., A. J. Perlman: Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone. J. Biol. Chem. 262:460-464(1987) Kalinyak, J. E., C. A. Griffin, R. W. Hamilton, J. G. Bradshaw, A. J. Perlman, A. R. Hoffman: Developmental and hormonal regulation of glucocorticoid receptor mRNA in the rat. J. Clin. Invest. 84: 1843-1848(1989) Kalinyak, J. E., R. I. Dorin, A. R. Hoffman, A. J. Perlman: Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone. J. Biol. Chem. 262:10441 -10444 (1987) Luttge, W. G, M. E. Rupp, M. M. Davda: Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors. Endocrinology 125:817-824(1989) Maniatis, R., E. F. Fritsch, J. K. Sambrook: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1982) Miesfleld, R., S. Okret, A.-C Wilkstrom, O. Wrange, J.-A. Gustafsson, K. R. Yamamoto: Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312: 779781 (1984) Patel, P. D., T. G. Sherman, D. J. Goldman, S. J. Watson: Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus. Mol. Endocrinol. 3: 1877—1885 (1989) Reul, J. M. H. M., F. R. van den Bosch, E. R. De Kloet: Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrin. 115:459-467 (1987) Reul, J. M. H. M., P. T. Pearce, J. W. Funder, Z. S. Krozowski: Type I and type II corticosteroid receptor gene expression in the rat: Effect of adrenalectomy and dexamethasone administration. Mol. Endo. 3:1674-1680(1989) Reul, J. M. H. M., E. R. De Kloet: Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradio-
Downloaded by: University of British Columbia. Copyrighted material.
Discussion
Mineralocorticoid Receptor mRNA
Horm. metab. Res. 24 (1992)
109
Downloaded by: University of British Columbia. Copyrighted material.
graphy and computerized image analysis. J. Steroid Biochem. 25: Requests for reprints should be addressed to: 269-272(1986) Rosenfeld, P., W. Sutanto, S. Levine, E. R. De Kloet: Ontogeny of typeJudith Kalinyak, M. D., Ph. D. I and type II corticosteroid receptors in the rat hippocampus. Dev. Division of Endocrinology Brainres.42:113-118(1988) Building 100, Room 286 Sarrieau, A., M. Vial, B. McEwen, Y. Broer, M. Dussaillant, D. Phil-San Francisco General Hospital ibert, M. Moguilewsky, W. Rostene: Corticosteroid receptors in rat San Francisco, CA 94110 (U. S. A.) hippocampal sections: Effect of adrenalectomy and corticosterone replacement. J. Steroid Biochem. 24:721 -724 (1986) Sarrieau, A., S. Sharma, M. J. Meany: Postnatal development and environmental regulation of hippocampal glucocorticoid and mineralocorticoid receptors. Dev. Brain Res.43:158-162(1988)
