Clinical and Experimental Pharmacology and Physiology (1992) 19,365-368
SHORT COMMUNICATION
THE HETEROGENEITY OF 11-P-HYDROXYSTEROID DEHYDROGENASE ACTIVITIES IN THE RAT HIPPOCAMPUS IMPLIES A COMPLEX REGULATION OF STEROID HORMONE ACTION R.E. Smith, W.R. Mercer, P. H. Provencher, V. Obeyesekere and Z. S. Krozowski Laboratory of Molecular Hypertension, Baker Institute, Prahran, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. The rat hippocampus 1 I-&hydroxysteroid dehydrogenase (1 I-HSD) displays a different substrate specificity to that of other tissues. S 1 nuclease analysis was used to determine whether the hippocampal messenger RNA is different from that found in other tissues. 2. S1 nuclease analysis using probes spanning the full length cDNA demonstrated that there were no differences in sequence between the hippocampal 1 1-HSD and the enzyme originally cloned from the liver. 3. These results suggest that there may be multiple 1 I-HSD isoforms in the hippocampus with different substrate specificities.
Key words: 11-/3-hydroxysteroiddehydrogenase, hippocampus, rat, steroid.
INTRODUCTION The syndrome of apparent mineralocorticoid excess (AME) is characterized by hypokalaemia and sodium retention in the presence of low plasma levels of renin and aldosterone (Ulick et al. 1977). In addition, AME patients exhibit marked increases in the urinary ratio of cortisol to cortisone metabolites. It has been proposed that AME is a result of a metabolic defect caused by deficient 11-p hydroxysteroid dehydrogenase (1 1-HSD) activity associated with unimpaired 11-/3 reductase. Thus, the molecular basis of this disease
would appear to be the inappropriate occupation of the mineralocorticoid receptor by glucocorticoids. It has been demonstrated that mineralocorticoids and glucocorticoids have equivalent affinities for the mineralocorticoid receptor (Krozowski 8z Funder 1983), and in vitro studies, using recombinant-derived mineralocorticoid receptor in the mouse mammary tumour virus - late terminal repeat (MMTV-LTR) luciferase reporter gene assay, clearly demonstrate that aldosterone and cortisol are equipotent activators
Correspondence: Dr Z. S. Krozowski, Laboratory of Molecular Hypertension, Baker Institute, Prahran, Vic. 3 181, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
R. E. Smith et al.
366
of gene transcription (Arriza et al. 1988). If mineralocorticoids are to occupy the mineralocorticoid receptor in the presence of much higher levels of glucocorticoids, then some specificity conferring mechanism must be operant. It has been previously demonstrated that in mineralocorticoid target tissues the presence of high levels of the 1I-HSD enzyme convert glucocorticoids to receptor inactive 11-keto metabolites (Funder et al. 1988). Conversely, inhibition of 11-HSD activity with carbenoxolone results in increased retention of labelled cortisol by the rat kidney and colon but not the hippocampus, where metabolism of cortisol has been shown to be low. Subsequent studies, however, demonstrated that corticosterone, the main rat glucocorticoid, was indeed extensively metabolized in the rat hippocampus (Moisan et al. 1990). In the hippocampus, but not in the kidney or colon, the enzyme clearly discriminates between cortisol and corticosterone as substrates. A possible explanation for these observations may be the presence of alternate isoforms of the 11-HSD enzyme in the hippocampus.
coding regions. Probe B also demonstrated a smaller band at 410 nucleotides in preparations of RNA from the hippocampus. However, this divergence in sequence maps to a position in the 5' untranslated region and is not expected to give rise to an altered protein (Krozowski et al. 1992).
DISCUSSION The ability of hippocampal 11-HSD to discriminate between cortisol and corticosterone suggests that a tissue-specific isoform may be present. The present study indicates that the isoform does not derive from the same gene that codes for the hepatic 11-HSD (Agarwal et al. 1989) and that there is at least one
METHODS Total RNA was isolated from tissues of male SpragueDawley rats by the guanidinium isothiocyanate method as previously described (Krozowski et al. 1990). Antisense RNA probes were used to perform solution hybridization analyses. Solution hybridization and S1 nuclease analyses were performed by a modification of a method previously described (Albiston et al. 1990). Briefly, 5 X 104 ct/min of riboprobe was added to 30pg of total tissue RNA or tRNA, denatured for 5 min at 85OC and hybridized at 60°C overnight. Each reaction mixture was then digested with 500 U of S1 nuclease for 50 min at 37OC.Reaction products were analysed on a 4% polyacrylamide, 8 moll L urea sequencing gel. Protected fragment sizes were calculated from a sequencing ladder run in parallel.
Pfdm B
Prob. A ' R o b e L
'
H
-
pr0b.L
H
1
'
RobrC Rube L
H
' -688
481
-488
-4t0
RESULTS Figure 1 shows the results of the S1 nuclease protection assays employing probes spanning the near full length liver 11-HSD cDNA (Agarwal et aZ. 19$$4). Three antisense RNA probes (A, B and C)were used to determine if any differences existed between the coding regions of the liver and the hippocampal 11HSD enzymes. All three probes were observed to be fully protected in both the liver and the hippocampus, demonstrating that there are no differences in the
ri.1. (a)
Schematic representation of antisense RNA probes used in the S1 nuclease protection analyses. The open box represents the liver 1 I-HSD coding region. 5'UT and 3'UT represent untranslated regions. (b) Autoradiograms showing the sizes of the protected RNA species in the liver ('L]'&nd hippocampus (H)using probes A, B and C. Numbers represent sizes (in nucleotides) of protected frag: ments. Differtnces in size between the probe and samples containing tissue RNA are due to polylinker sequences.
367
11-P-HSD in the rat hippocampus
other dehydrogenase present in the rat hippocampus; the second dehydrogenase gene would not appear to be closely related since recent studies have reported the existence of a single 11-HSD gene in the rat (Krozowski et al. 1992). Other studies have also identified I 1-HSD isoforms in the kidney and isolated collecting tubule cells. Other 1 I-HSD activities identified include an 11HSD2 enzyme with a different cofactor requirement and intrarenal distribution (Mercer & Krozowski 1992) and a higher affinity enzyme localized in distal tubule cells (Naray-Fejes-Toth et al. 1991). Previously, the cloned 11-HSD enzyme has also been shown to exist in several tissue-specific forms by Western blot studies (Monder & Lakshmi 1990) and S1 nuclease analysis (Krozowski et al. 1992). The apparent identity of the liver and hippocampal enzymes raises the question as to why previous studies have been unable to detect conversion of cortisol to cortisone in hippocampal slices. It is possible that this particular isoform is inactive or only activated under certain physiological conditions, or that net enzymatic activity is in the reductive direction in these cells (Murphy 1981). The presence of multiple 1 1-HSD activities in the hippocampus suggests a role for these enzymes in modulating the effects of adrenocorticosteroids. Early in viva studies in the rat demonstrated that the hippocampus retained high amounts of corticosterone (DeKloet et al. 1975). Since the level of binding is known to undulate with circadian rhythm without changes in mRNA levels for adrenocorticoid receptors, 11-HSD may modulate the total amount of glucocorticoid within glucocorticoid target cells in the hippocampus. These cells are thought to mediate the feedback action of corticosterone on stress-activated brain function (DeKloet & Reul 1987). In addition, a significant amount of aldosterone binding in the hippocampus cannot be displaced by corticosterone (Corini el al. 1983), which is consistent with the presence of high amounts of 11-HSD in hippocampal mineralocorticoid target cells. It is thought that these cells are involved in the co-ordination of circadian processes (McEwen & Brinton 1987) and the regulation of basal adrenocorticotrophic hormone (ACTH) secretion (Dallman et al. 1987). In summary, this study has provided evidence for the presence of multiple forms of 11-HSD in the rat hippocampus. The role of the 11-HSD isoforms may be that of cell-specific filters, allowing moderate but variable amounts of hormone to access glucocorticoid responsive cells, and protecting mineralocorticoid target cells from inappropriate occupation by glucocorticoid hormone.
REFERENCES Agarwal, A. K., Monder, C., Eckstein, B. & White, P. C. (1989) Cloning and expression of rat cDNA encoding corticosteroid 11-P-dehydrogenase. Journal of Biological Chemistry, 264, 18939-18946. Albiston, A. L., Lock, P., Burger, H. G . & Krozowski, Z. S. (1990) Cloning and characterization of the rat a-inhibin gene. Molecular and Cellular Endocrinology,68,121-128. Arr'iza, J., Simerly, R., Swanson, L. & Evans, R. M. (1988) The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron, 1, 887-900. Corini, H., Marusic, E. T., De Nicola, A. F., Rainbow, T. C. & McEwen, B. S. (1983) Identification of mineralocorticoid binding sites in rat brain by competition studies and density gradient centrifugation. Neuroendocrinology, 37,354-360. Dallman, M. F., Akana, S. F., Cascio, C. S., Darlington, D . N., Jacobson, L. & Levin, N. (1987) Regulation of ACTH secretion: Variations on a theme of B. Recent Progress in Hormonal Research, 43, 113-173. DeKloet, E. R. & Reul, J. M. H. M. (1987) Feedback action and tonic influence of corticosteroids on brain function: A concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology,12,83- 105. DeKloet, E. R., Wallach, G. & McEwen, B. S. (1975) Differences in corticosterone and dexamethasone binding in rat brain and pituitary. Endocrinology, 96,598-609. Funder, J. W., Pearce, P. T., Smith, R. & Smith, I. (1988) Mineralocorticoid action: Target tissue specificity is enzyme, not receptor, mediated. Science, 242,583-585. Krozowski, Z. S. & Funder, J. W. (1983) Renal mineralocorticoid receptors and hippocampal corticosterone binding species have identical intrinsic steroid specificity. Proceedings of the National Academy of Sciences, USA, 80,6056-6060. Krozowski, Z. S., Stuchbery, S., White, P., Monder, C. & Funder, J. W. (1990) Characterization of 1 I-P-hydroxysteroid dehydrogenase gene expression: Identification of multiple unique forms of messenger RNA in the rat kidney. Endocrinology, 127, 3009-3013. Krozowski, Z., Obeyesekere, V., Smith, R. & Mercer, W. (1992) Tissue-specific expression of an 1I-P-hydroxysteroid dehydrogenase with a truncated N-terminal domain. Journal of Biological Chemistry (in press). McEwen, B. S. & Brinton, R. E. (1987) Neuroendocrine aspects of adaptation. Progress in Brain Research, 72, 11-26. Mercer, W. R. & Krozowski, Z. S. (1992) Localization of an 1 1-P-hydroxysteroid dehydrogenase activity to the distal nephron of the rat kidney. Evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology, 130, 540-543. Moisan, M., Seckl, J. R. & Edwards, C. R. W. (1990) 11-phydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: Localization in hypothalamus, hippocampus and cortex. Endocrinology, 127, 1450- 1455.
368
R. E. Smith et al.
Monder, C. & Lakshmi, V. (1990) Corticosteroid 11-pdehydrogenase of rat tissues: Immunological studies. Endocrinology, 126,2435-2443. Murphy, B. E. P. (1981) Ontogeny of cortisol-cortisone interconversion in human tissues: A role for cortisone in human fetal development. Journal of Steroid Biochemistry, 14,8 11-8 17. Naray-Fejes-Toth, A., Watlington, C. 0. & Fejes-Toth, G.
(1991) 11-/3-hydroxysteroid dehydrogenase activity in the renal target cells of aldosterone. Endocrinology, 129, 17-21. Ulick, S., Ramirez, L. C. & New, M. (1977) An abnormality in steroid reductive metabolism in a hypertensive syndrome. Journal of Clinical Endocrinology and Metabolism, 44,799-802.
SHORT COMMUNICATION
THE HETEROGENEITY OF 11-P-HYDROXYSTEROID DEHYDROGENASE ACTIVITIES IN THE RAT HIPPOCAMPUS IMPLIES A COMPLEX REGULATION OF STEROID HORMONE ACTION R.E. Smith, W.R. Mercer, P. H. Provencher, V. Obeyesekere and Z. S. Krozowski Laboratory of Molecular Hypertension, Baker Institute, Prahran, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. The rat hippocampus 1 I-&hydroxysteroid dehydrogenase (1 I-HSD) displays a different substrate specificity to that of other tissues. S 1 nuclease analysis was used to determine whether the hippocampal messenger RNA is different from that found in other tissues. 2. S1 nuclease analysis using probes spanning the full length cDNA demonstrated that there were no differences in sequence between the hippocampal 1 1-HSD and the enzyme originally cloned from the liver. 3. These results suggest that there may be multiple 1 I-HSD isoforms in the hippocampus with different substrate specificities.
Key words: 11-/3-hydroxysteroiddehydrogenase, hippocampus, rat, steroid.
INTRODUCTION The syndrome of apparent mineralocorticoid excess (AME) is characterized by hypokalaemia and sodium retention in the presence of low plasma levels of renin and aldosterone (Ulick et al. 1977). In addition, AME patients exhibit marked increases in the urinary ratio of cortisol to cortisone metabolites. It has been proposed that AME is a result of a metabolic defect caused by deficient 11-p hydroxysteroid dehydrogenase (1 1-HSD) activity associated with unimpaired 11-/3 reductase. Thus, the molecular basis of this disease
would appear to be the inappropriate occupation of the mineralocorticoid receptor by glucocorticoids. It has been demonstrated that mineralocorticoids and glucocorticoids have equivalent affinities for the mineralocorticoid receptor (Krozowski 8z Funder 1983), and in vitro studies, using recombinant-derived mineralocorticoid receptor in the mouse mammary tumour virus - late terminal repeat (MMTV-LTR) luciferase reporter gene assay, clearly demonstrate that aldosterone and cortisol are equipotent activators
Correspondence: Dr Z. S. Krozowski, Laboratory of Molecular Hypertension, Baker Institute, Prahran, Vic. 3 181, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
R. E. Smith et al.
366
of gene transcription (Arriza et al. 1988). If mineralocorticoids are to occupy the mineralocorticoid receptor in the presence of much higher levels of glucocorticoids, then some specificity conferring mechanism must be operant. It has been previously demonstrated that in mineralocorticoid target tissues the presence of high levels of the 1I-HSD enzyme convert glucocorticoids to receptor inactive 11-keto metabolites (Funder et al. 1988). Conversely, inhibition of 11-HSD activity with carbenoxolone results in increased retention of labelled cortisol by the rat kidney and colon but not the hippocampus, where metabolism of cortisol has been shown to be low. Subsequent studies, however, demonstrated that corticosterone, the main rat glucocorticoid, was indeed extensively metabolized in the rat hippocampus (Moisan et al. 1990). In the hippocampus, but not in the kidney or colon, the enzyme clearly discriminates between cortisol and corticosterone as substrates. A possible explanation for these observations may be the presence of alternate isoforms of the 11-HSD enzyme in the hippocampus.
coding regions. Probe B also demonstrated a smaller band at 410 nucleotides in preparations of RNA from the hippocampus. However, this divergence in sequence maps to a position in the 5' untranslated region and is not expected to give rise to an altered protein (Krozowski et al. 1992).
DISCUSSION The ability of hippocampal 11-HSD to discriminate between cortisol and corticosterone suggests that a tissue-specific isoform may be present. The present study indicates that the isoform does not derive from the same gene that codes for the hepatic 11-HSD (Agarwal et al. 1989) and that there is at least one
METHODS Total RNA was isolated from tissues of male SpragueDawley rats by the guanidinium isothiocyanate method as previously described (Krozowski et al. 1990). Antisense RNA probes were used to perform solution hybridization analyses. Solution hybridization and S1 nuclease analyses were performed by a modification of a method previously described (Albiston et al. 1990). Briefly, 5 X 104 ct/min of riboprobe was added to 30pg of total tissue RNA or tRNA, denatured for 5 min at 85OC and hybridized at 60°C overnight. Each reaction mixture was then digested with 500 U of S1 nuclease for 50 min at 37OC.Reaction products were analysed on a 4% polyacrylamide, 8 moll L urea sequencing gel. Protected fragment sizes were calculated from a sequencing ladder run in parallel.
Pfdm B
Prob. A ' R o b e L
'
H
-
pr0b.L
H
1
'
RobrC Rube L
H
' -688
481
-488
-4t0
RESULTS Figure 1 shows the results of the S1 nuclease protection assays employing probes spanning the near full length liver 11-HSD cDNA (Agarwal et aZ. 19$$4). Three antisense RNA probes (A, B and C)were used to determine if any differences existed between the coding regions of the liver and the hippocampal 11HSD enzymes. All three probes were observed to be fully protected in both the liver and the hippocampus, demonstrating that there are no differences in the
ri.1. (a)
Schematic representation of antisense RNA probes used in the S1 nuclease protection analyses. The open box represents the liver 1 I-HSD coding region. 5'UT and 3'UT represent untranslated regions. (b) Autoradiograms showing the sizes of the protected RNA species in the liver ('L]'&nd hippocampus (H)using probes A, B and C. Numbers represent sizes (in nucleotides) of protected frag: ments. Differtnces in size between the probe and samples containing tissue RNA are due to polylinker sequences.
367
11-P-HSD in the rat hippocampus
other dehydrogenase present in the rat hippocampus; the second dehydrogenase gene would not appear to be closely related since recent studies have reported the existence of a single 11-HSD gene in the rat (Krozowski et al. 1992). Other studies have also identified I 1-HSD isoforms in the kidney and isolated collecting tubule cells. Other 1 I-HSD activities identified include an 11HSD2 enzyme with a different cofactor requirement and intrarenal distribution (Mercer & Krozowski 1992) and a higher affinity enzyme localized in distal tubule cells (Naray-Fejes-Toth et al. 1991). Previously, the cloned 11-HSD enzyme has also been shown to exist in several tissue-specific forms by Western blot studies (Monder & Lakshmi 1990) and S1 nuclease analysis (Krozowski et al. 1992). The apparent identity of the liver and hippocampal enzymes raises the question as to why previous studies have been unable to detect conversion of cortisol to cortisone in hippocampal slices. It is possible that this particular isoform is inactive or only activated under certain physiological conditions, or that net enzymatic activity is in the reductive direction in these cells (Murphy 1981). The presence of multiple 1 1-HSD activities in the hippocampus suggests a role for these enzymes in modulating the effects of adrenocorticosteroids. Early in viva studies in the rat demonstrated that the hippocampus retained high amounts of corticosterone (DeKloet et al. 1975). Since the level of binding is known to undulate with circadian rhythm without changes in mRNA levels for adrenocorticoid receptors, 11-HSD may modulate the total amount of glucocorticoid within glucocorticoid target cells in the hippocampus. These cells are thought to mediate the feedback action of corticosterone on stress-activated brain function (DeKloet & Reul 1987). In addition, a significant amount of aldosterone binding in the hippocampus cannot be displaced by corticosterone (Corini el al. 1983), which is consistent with the presence of high amounts of 11-HSD in hippocampal mineralocorticoid target cells. It is thought that these cells are involved in the co-ordination of circadian processes (McEwen & Brinton 1987) and the regulation of basal adrenocorticotrophic hormone (ACTH) secretion (Dallman et al. 1987). In summary, this study has provided evidence for the presence of multiple forms of 11-HSD in the rat hippocampus. The role of the 11-HSD isoforms may be that of cell-specific filters, allowing moderate but variable amounts of hormone to access glucocorticoid responsive cells, and protecting mineralocorticoid target cells from inappropriate occupation by glucocorticoid hormone.
REFERENCES Agarwal, A. K., Monder, C., Eckstein, B. & White, P. C. (1989) Cloning and expression of rat cDNA encoding corticosteroid 11-P-dehydrogenase. Journal of Biological Chemistry, 264, 18939-18946. Albiston, A. L., Lock, P., Burger, H. G . & Krozowski, Z. S. (1990) Cloning and characterization of the rat a-inhibin gene. Molecular and Cellular Endocrinology,68,121-128. Arr'iza, J., Simerly, R., Swanson, L. & Evans, R. M. (1988) The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron, 1, 887-900. Corini, H., Marusic, E. T., De Nicola, A. F., Rainbow, T. C. & McEwen, B. S. (1983) Identification of mineralocorticoid binding sites in rat brain by competition studies and density gradient centrifugation. Neuroendocrinology, 37,354-360. Dallman, M. F., Akana, S. F., Cascio, C. S., Darlington, D . N., Jacobson, L. & Levin, N. (1987) Regulation of ACTH secretion: Variations on a theme of B. Recent Progress in Hormonal Research, 43, 113-173. DeKloet, E. R. & Reul, J. M. H. M. (1987) Feedback action and tonic influence of corticosteroids on brain function: A concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology,12,83- 105. DeKloet, E. R., Wallach, G. & McEwen, B. S. (1975) Differences in corticosterone and dexamethasone binding in rat brain and pituitary. Endocrinology, 96,598-609. Funder, J. W., Pearce, P. T., Smith, R. & Smith, I. (1988) Mineralocorticoid action: Target tissue specificity is enzyme, not receptor, mediated. Science, 242,583-585. Krozowski, Z. S. & Funder, J. W. (1983) Renal mineralocorticoid receptors and hippocampal corticosterone binding species have identical intrinsic steroid specificity. Proceedings of the National Academy of Sciences, USA, 80,6056-6060. Krozowski, Z. S., Stuchbery, S., White, P., Monder, C. & Funder, J. W. (1990) Characterization of 1 I-P-hydroxysteroid dehydrogenase gene expression: Identification of multiple unique forms of messenger RNA in the rat kidney. Endocrinology, 127, 3009-3013. Krozowski, Z., Obeyesekere, V., Smith, R. & Mercer, W. (1992) Tissue-specific expression of an 1I-P-hydroxysteroid dehydrogenase with a truncated N-terminal domain. Journal of Biological Chemistry (in press). McEwen, B. S. & Brinton, R. E. (1987) Neuroendocrine aspects of adaptation. Progress in Brain Research, 72, 11-26. Mercer, W. R. & Krozowski, Z. S. (1992) Localization of an 1 1-P-hydroxysteroid dehydrogenase activity to the distal nephron of the rat kidney. Evidence for the existence of two species of dehydrogenase in the rat kidney. Endocrinology, 130, 540-543. Moisan, M., Seckl, J. R. & Edwards, C. R. W. (1990) 11-phydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: Localization in hypothalamus, hippocampus and cortex. Endocrinology, 127, 1450- 1455.
368
R. E. Smith et al.
Monder, C. & Lakshmi, V. (1990) Corticosteroid 11-pdehydrogenase of rat tissues: Immunological studies. Endocrinology, 126,2435-2443. Murphy, B. E. P. (1981) Ontogeny of cortisol-cortisone interconversion in human tissues: A role for cortisone in human fetal development. Journal of Steroid Biochemistry, 14,8 11-8 17. Naray-Fejes-Toth, A., Watlington, C. 0. & Fejes-Toth, G.
(1991) 11-/3-hydroxysteroid dehydrogenase activity in the renal target cells of aldosterone. Endocrinology, 129, 17-21. Ulick, S., Ramirez, L. C. & New, M. (1977) An abnormality in steroid reductive metabolism in a hypertensive syndrome. Journal of Clinical Endocrinology and Metabolism, 44,799-802.
