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Medical Research Council Blood Pressure Group (P.Y., S.M.M., R.F., J.M.C.C., E.D.), Division of Cardiovascular and Medical Sciences, Western Infirmary, Glasgow, Scotland G11 6NT, United Kingdom; and Molecular Endocrinology, Molecular Medicine Centre (C.J.K., J.R.S.), Western General Hospital, Edinburgh, Scotland EH4 2XU, United Kingdom
Address all correspondence and requests for reprints to: Dr. Eleanor Davies, Medical Research Council Blood Pressure Group, Division of Cardiovascular and Medical Sciences, Western Infirmary, Glasgow, Scotland G11 6NT, United Kingdom. E-mail: ed18g{at}clinmed.gla.ac.uk.
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Abstract |
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Introduction |
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Glucocorticoid receptors (GR) are expressed at high levels throughout the brain. Mineralocorticoid receptors (MR) are expressed at a high level only in the hippocampus, septum, and a few brainstem nuclei. The local synthesis of aldosterone and corticosterone at sites close to MR and GR may have profound pathophysiological consequences. For example, intracerebroventricular infusions of glucocorticoids and mineralocorticoids have major effects on systemic blood pressure, without significantly raising hormone concentrations in the systemic circulation (14). Mineralocorticoids in the brain also influence fluid homeostasis through effects on thirst and salt appetite (15, 16), and there is evidence that glucocorticoids can influence cognition and have neuroprotective or neurodegenerative properties under different circumstances via both MR and GR (17, 18).
In the adrenal cortex, the expression of the aldosterone synthase gene, CYP11B2, is mainly controlled by body sodium status via the renin-angiotensin system (RAS) (19). The transcription of adrenal CYP11B2 is enhanced by sodium deficiency, angiotensin (Ang)II, and small increases in dietary potassium, but it is suppressed by long-term administration of high doses of ACTH. In contrast, CYP11B1 expression is determined by physiological variations of ACTH (20). Whether extraadrenal steroid synthesis is similarly controlled and whether these steroids have the same actions on blood pressure and sodium acquisition as adrenally derived steroid is not known.
The elucidation of the control mechanisms controlling extraadrenal steroidogenesis is essential to assess the physiological and pathophysiological role of locally synthesized hormones. However, the low levels of CYP11B1 and CYP11B2 transcripts present in nonadrenal tissue render them extremely difficult to quantify. To study the transcriptional regulation of these genes, we developed a method for the quantitative detection of CYP11B1 and CYP11B2 transcripts using the Roche LightCycler system in combination with homologous RNA standards. We used this method to quantify CYP11B2 mRNA levels in the rat adrenal gland and various regions of the rat CNS and also to identify significant changes in gene expression caused by known modulators of adrenal CYP11B2 transcription, dietary sodium, and AngII. Simultaneous measurement of CYP11B1 mRNA expression was also performed.
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Materials and Methods |
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Effects of sodium depletion and high-sodium diet
Three groups of male Wistar Kyoto rats (n = 6) were fed normal (0.3%), high (3%), or low (0.03%) sodium diets (Special Diet Services Ltd., Witham, Essex, UK) for 12 d. The animals were then killed by decapitation at 12-h intervals, and trunk blood was collected for assay of plasma steroid levels and plasma renin activity (PRA). The adrenal glands, hippocampus, cerebellum, hypothalamus, brainstem, and cerebral cortex were removed, cleaned, and frozen at -70 C.
Effect of AngII
Two groups of animals (n = 6) were infused with normal saline vehicle or AngII (200 ng/kg·min) using Alzet osmotic minipump model 2002 (pump rate, 0.5 µl/h) (Alzet Osmotic Minipumps, Cupertino, CA) for 7 d. Blood and tissues were collected as described above.
Plasma steroid and renin measurement
Plasma aldosterone was determined by RIA (Diagnostic Products Corp., Los Angeles, CA). Plasma corticosterone was analyzed by direct RIA in diluted, heat-deproteinized plasma. A highly specific rabbit anticorticosterone antiserum and 3H-corticosterone were used. Bound radioactivity was measured with an antirabbit proximity scintillation reagent (Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK).
PRA was measured by a modified version of the method described by Morton and Wallace (21). Aliquots of plasma were incubated at 0 and 37 C for 30 min with AngI antiserum in Tris buffer (pH 7.4; 50 mM). The AngI generated and trapped was measured by RIA.
Total RNA isolation
Tissues were homogenized using a RiboLyzer Cell Disruptor (Hybaid, Middlesex, UK), and total tissue RNA was isolated using RNABee (200 mg/ml; Biogenesis, Poole, UK). RNA was treated with DNase (DNA-free, Ambion, Inc., Austin, TX), and the RNA quality was confirmed by electrophoresis on agarose gels. RNA concentration was measured using the RiboGreen RNA quantitation kit (Molecular Probes, Inc., Leiden, The Netherlands) and a Wallac Victor 1420 Multilabel Counter (PerkinElmer Life Sciences, Boston, MA).
Real-time quantitative RT-PCR (QRT-PCR)
The RB1 primers were designed to amplify a 324-bp fragment corresponding to positions 552–875 of the CYP11B1 gene, whereas the RB2 primers were designed to amplify a 324-bp segment corresponding to positions 558–881 of the CYP11B2 gene (Table 1
) (22). These primers are exon-spanning and therefore exclude contaminating genomic DNA. Primers were manufactured by MWG-Biotech (Ebersberg, Germany).
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Real time QRT-PCR
One-step real time QRT-PCR was performed using the LightCycler System (Roche Diagnostics, Mannheim, Germany) and the LightCycler RNA Master Hybridization Probes kit (Roche Diagnostics). Two pairs of gene sequence-specific hybridization probes homologous to exon 3 of rat CYP11B1 or CYP11B2 were designed and synthesized by TIB Molbiol (Berlin, Germany), each pair consisting of a fluorescein and LightCycler Red probe (Table 1
).
Tissue RNA (1 µg) was used as a template for LightCycler amplification. One-microliter aliquots of sequential 10-fold mRNA standard dilutions were used to construct a standard curve. The LightCycler protocol was as follows: reverse transcription (61 C for 20 min), denaturation (95 C for 30 sec), amplification (95 C for 1 sec, annealing temperature for 15 sec, fluorescence measurement, 72 C for 14 sec), for up to 85 cycles or until all of the positive samples reached the plateau phase; and cooling (40 C for 30 sec). Sample copy number was extrapolated from the standard curve. Each sample was measured three times and its crossing point was used to calculate CYP11B1 or CYP11B2 copy number (Fig. 1). A negative control was included in each run. No crossing point greater than 42 cycles resulted from tissue RNA in this study, and most samples yielded crossing points in the range of 20–35 cycles.
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The annealing temperatures were 52 C for CYP11B1 probes and 55 C for CYP11B2 probes.
The specificity of the CYP11B1 and CYP11B2 amplification was established by attempting to amplify the CYP11B1 standard with the RB2 primers and the CYP11B2 standard with the RB1 primers. No amplification was apparent in either case, demonstrating that the method is specific (data not shown).
Intra- and interassay variation
The LightCycler method uses fluorescent measurement to detect the point at which PCR enters the exponential phase of amplification. The fit point method was used to assess when samples entered this phase and to construct an optimized standard curve from the serial dilutions of homologous RNA standards (Fig. 1).
The intraassay precision of real-time QRT-PCR was assessed by performing 10 replicate CYP11B1 or CYP11B2 reactions in a single LightCycler run on adrenal or cerebral cortex total RNA. Interassay variation was assessed by CYP11B1 or CYP11B2 amplification of a single adrenal or cerebral cortex RNA sample over five separate LightCycler runs. The results of these are shown in Table 2. These show that interassay variation is greater than intraassay variation and that the relative variation (coefficient of variance) is greater in samples that contain lower levels of CYP11B1 or CYP11B2 transcript. Also, whereas crossing point is extremely consistent in all tests, the extrapolated copy number is more variable, presumably because of small sample changes in the amplification of standard. Again, these differences are more pronounced at lower copy number, as would be expected. The higher variability at lower levels of copy number will make very small changes in expression difficult to detect.
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Data analysis
mRNA expression rates and plasma hormone concentration data were analyzed by the Mann-Whitney U test. For all analyses, P < 0.05 was required for statistical significance. Data are expressed as the mean ± SE.
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Results |
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. PRA was significantly higher in the low-sodium diet group and significantly lower in the high-sodium diet group than in the normal diet group. This confirmed that low, normal, and high sodium status had been achieved in the animals. This distinction was not so clear in the plasma aldosterone levels due to the high variability in concentration between these small groups. This may have been due to inadequately controlled acute stress at death, particularly in the high-sodium group where plasma aldosterone was not significantly lower than in the normal group. Plasma aldosterone concentration in the low-sodium group was higher than that in the normal diet group, consistent with the PRA, although this did not quite reach statistical significance (P = 0.07). Nevertheless, these results strongly suggest that sodium diet manipulation, especially low-sodium diet, successfully altered aldosterone synthesis. There was no significant difference between the plasma corticosterone concentrations of the three groups, although the variability within the groups was high.
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Quantitative differences in CYP11B1 and B2 expression rates
In the adrenal glands of control animals (i.e. those in the normal sodium intake and vehicle infusion groups), CYP11B1 mRNA levels were 100- to 1000-fold higher than CYP11B2 mRNA. CYP11B1 expression was also consistently higher in all parts of the brain studied. Brain stem and hypothalamus had the highest CYP11B2 transcript levels of the brain regions studied. Cerebral cortex had the lowest CYP11B2 mRNA level, apart from hippocampus, but its CYP11B1 expression was up to 10-fold greater than any other brain region. (For example, control CYP11B1 levels were 1.9 x 107 ± 0.4 x 107 copies/µg RNA in cerebral cortex, 2.1 x 106 ± 0.3 x 106 copies/µg RNA in brainstem, and 6.3 x 105 ± 1.7 x 105 copies/µg RNA in hypothalamus.)
Effects of sodium intake on gene expression
Expression of both CYP11B1 and CYP11B2 was detected in adrenal glands, hippocampus, cerebellum, hypothalamus, brainstem, and cerebral cortex. Electrophoresis of the RT-PCR products on agarose gels demonstrated that no nonspecific products capable of causing erroneous results were generated in the course of the reaction (Fig. 2). Representative amplification curves are shown in Fig. 3.
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). Low-sodium diet had no significant effect on CYP11B2 expression in the brainstem or hypothalamus. High-sodium diet did not significantly decrease CYP11B2 expression in brain tissues. Sodium intake did not affect CYP11B1 expression in the adrenal gland or brain tissues (Fig. 5).
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). AngII infusion had no significant effect on CYP11B1 expression in any of these tissues (Fig. 7).
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Discussion |
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Expression and regulation of CYP11B1 and CYP11B2 in adrenal tissue
Using this method, the responses of adrenal CYP11B2 mRNA expression to changes in dietary sodium or AngII were as expected from previous published studies of their effects on steroid levels. Decreasing sodium intake caused the levels of adrenal CYP11B2 copy number to increase 57-fold, whereas increased sodium reduced it to a fifth of its basal level (the overall increase in expression from high-sodium diet to low-sodium diet was 300-fold). CYP11B1 mRNA levels did not respond significantly to these stimuli.
When comparing control animals from the two studies, it is apparent that adrenal CYP11B2 levels are approximately an order higher in vehicle animals (Fig. 6) than normal sodium diet animals (Fig. 4). However, both of these levels can be considered compatible with the normal range because plasma aldosterone concentration extends across one order of magnitude. Also, the composition of the chow fed to the two groups of control animals was not identical, and this may have contributed to the adrenal CYP11B2 differences.
Basal expression of CYP11B1 and CYP11B2 in brain tissue
As in previous studies, the levels of CYP11B1 and CYP11B2 transcripts detected in the brain were consistently much lower than those in the adrenal cortex (8, 23, 25). In animals on normal sodium intake, mRNA copy number was 10–1000 times lower in the brain than in the adrenal gland, depending on the brain region. Although this could be caused by consistently low levels of transcription across the whole brain region, we suggest that this reflects high expression in a very few cells within that region. Our previous immunohistochemical studies showed that aldosterone synthase and 11ß-hydroxylase were localized to particular cells within regions of the brain. For example, the Purkinje cells of the cerebellum express relatively high levels of aldosterone synthase and 11ß-hydroxylase, but account for a very small fraction of the total cerebellar mass (9). With this pattern of expression, extraadrenal hormones may act in a paracrine or an autocrine manner on the abundant GR and MR within the brain.
Our QRT-PCR results also demonstrate that expression and regulation of these genes across the brain are not uniform. The fact that copy number varies from region to region reflects the varying proportion of cells that express the CYP11B2 gene in each area, as well as the degree of expression within the individual cells. CYP11B2 transcript levels were particularly variable, covering a 60-fold range from 3.3 x 104copies/µg in brainstem to 5.4 x 102 copies/µg in hippocampus. CYP11B1 levels were higher, but they varied less between regions than those of CYP11B2.
As in previous studies, these results also suggest that CYP11B1 and CYP11B2 expression colocalize within the CNS, something that does not occur in the adrenal cortex due to its strict zonation. This colocalization raises some important questions. Because aldosterone synthase and 11ß-hydroxylase both use DOC as substrate, do they compete for substrate in tissues where they colocalize? What are their relative affinities for DOC? Can products of 11ß-hydroxylase be used by aldosterone synthase?
Another consideration involves receptor occupancy. Corticosterone binds not only to the GR but also to the MR. In aldosterone-selective tissues, such as the distal nephron, binding of corticosterone by MR is prevented by the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which inactivates corticosterone. However, the adult brain is, for the most part, nonselective, with high 11ß-HSD2 expression detectable in rat CNS only in the subcommissural organ and nucleus tractus solitarius (26), important sites of cardiovascular control. In the other CYP11B2-expressing CNS regions, aldosterone has no such priority and, because CYP11B1 expression is consistently higher in any given brain region, locally produced aldosterone may not achieve significant access to the MR. In such cases, there would be little point in regulating CYP11B2 expression unless 11ß-HSD2 was also expressed. It may be, therefore, that it is corticosterone and not aldosterone that is of importance in the majority of the adult rat brain. The 11ß-HSD1 enzyme, on the other hand, favors the production of corticosterone. Its presence is widespread throughout the brain and would, in effect, amplify the effects of local corticosterone production. Although the biological implications of this remain unexplored, knockouts of the local glucocorticoid amplifying enzyme, 11ß-HSD1, show the clear effects of local glucocorticoid regeneration in the CNS upon the hypothalamic-pituitary-adrenal axis (27) and hippocampal cognitive function (28), so local amplification by CYP11B1 may provide an effective extra boost to CNS glucocorticoid action when required.
Regulation of CYP11B1 and CYP11B2 in brain tissue
Brain CYP11B1 expression, like that in the adrenal cortex, did not respond to AngII or alterations in dietary sodium.
Restriction of dietary sodium did significantly increase CYP11B2 transcription within the cerebellum and the hippocampus, although the response was less pronounced than in the adrenal gland (a 57-fold increase in the adrenal gland, compared with a 14-fold increase in the hippocampus and a 5-fold increase in cerebellum). This difference between the tissues may partly be due to the activation of the RAS in the adrenal gland. Dietary sodium restriction is known to increase the adrenal concentration of AngII, and this correlates with raised plasma aldosterone levels (29). The increase in adrenal CYP11B2 mRNA during low-sodium treatment or AngII infusion is partly due to the hypertrophy and hyperplasia of the zona glomerulosa. It is highly unlikely that the brain could react in the same way, and therefore its increase in expression is limited to existing cells. No changes were observed in the hypothalamus or the brainstem, suggesting either that CYP11B2 expression is not subject to stimulation here, that any change was too small to detect, or that it is regulated differently to the cerebellum and the hippocampus.
The effects of AngII on CYP11B2 expression differ in two respects from those of a low-sodium diet. Firstly, the degree of adrenal stimulation with AngII is less than that with low-sodium diet, despite similar increments in plasma aldosterone. Secondly, there are no significant effects of AngII on patterns of CYP11B2 expression in any region of the brain. Differences in adrenal responsiveness might indicate that, in addition to activation of the RAS, other factors such as dopamine, serotonin, atrial natriuretic peptide, and plasma potassium are involved in adaptation to low-sodium diet. This is exemplified by a study comparing the antiproliferative effects of losartan, an angII type 1 (AT1) receptor antagonist; adrenal hyperplasia induced by AngII was prevented, but a similar response to low-sodium diet was unaffected (30).
Differences in responsiveness in the brain may reflect the fact that low-sodium diet activates the endogenous RAS, whereas AngII suppresses activity. Thus, increases in plasma AngII due to exogenous peptide infusion may be offset by decreased endogenous AngII synthesis. This argument is not dependent on whether AngII crosses the blood–brain barrier because there is clear evidence of an autonomous brain RAS. It is quite possible, for example, that peripheral infusions of pressor doses of AngII could inhibit the brain RAS via effects on blood pressure, whereas a low-sodium diet, which tends to lower blood pressure, might have the opposite effect. It is clearly important for future studies to investigate whether intracerebroventricular infusion of angiotensins or of inhibitors of the RAS can alter brain CYP11B2 mRNA expression independent of changes in the adrenal gland.
We have demonstrated that local expression of CYP11B2 in the CNS can be regulated by manipulation of sodium intake. The mechanism of this regulation is, as yet, unknown. To study this and to investigate whether differences in local expression might contribute to cardiovascular and other diseases will require methods of not only high precision and accuracy but also, because mRNA copy numbers are very low, great sensitivity. This method, we think, has such potential.
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Acknowledgments |
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Footnotes |
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Abbreviations: Ang, Angiotensin; CNS, central nervous system; DOC, 11-deoxycorticosterone; GR, glucocorticoid receptor(s); 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; MR, mineralocorticoid receptor(s); PRA, plasma renin activity; QRT-PCR, quantitative RT-PCR; RAS, renin-angiotensin system.
Received January 22, 2003.
Accepted for publication April 9, 2003.
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