Expression of cytochrome P45011B1 mRNA in the brain of normal and hypertensive transgenic rats

BRAIN RESEARCH ELSEVIER

Brain Research 733 (1996) 73-82

Research report

Expression of cytochrome P45011B 1 mRNA in the brain of normal and hypertensive transgenic rats B e t t i n a E r d m a n n a, *, H e l l m u t G e r s t a A n d r e a L i p p o l d t a H a n n e s B i l l o w a D e t l e v G a n t e n a K j e l l F u x e b, R i t a B e r n h a r d t c a Max-Delbr~ck-Centerfor Molecular Medicine (MDC), Robert-ROssle-Str. 10, D-13122 Berlin-Buch, Germany b Karolinska Institute, Department ofNeuroscience, Doctorsringen 12, S-17177 Stockholm, Sweden '~ Uni~,ersitiitdes Saarlandes, Department 12.4-Biochemistry, Postfach 15 11 50, D-66041 Saarbr~cken, Germany Accepted 30 April 1996

Abstract

Cytochrome P45011B1 (lll3-hydroxylase) was detected in the brain of male rats by in situ hybridization methods. Normal Sprague-Dawley rats were compared to the transgenic strain TGR(mRen2)27, characterized by the expression of the murine R e n - 2 ~ renin gene and the development of severe hypertension. Specific riboprobes were generated by in vitro transcription of a 152 base-pair long cDNA template. 35S-labeled riboprobes were hybridized to cryostat sections from adrenal glands and from two different levels of the brain using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared. Following autoradiography and counterstaining, cytochrome P45011B1 rnRNA was clearly localized in the zona fasciculata/reticularis of the adrenal cortex and in distinct layers of the cerebral cortex. High signal densities were obtained in the layers II-IV of the neocortex and in the layer II of the piriform cortex, although the concentrations of cytochrome P45011B1 mRNA were remarkably lower in the central nervous system as compared to adrenal glands. As revealed by the semi-quantitative analysis, there was a slight increase in adrenal 1l[3-hydroxylase mRNA in the transgenic rats, whereas the brain seems to express nearly the same amount of this enzyme in both strains. The cytochrome P45011B1 mRNA expression in distinct cells, probably nerve cells, and especially in regions with high densities of glucocorticoid receptors points to a possible function of brain derived corticosterone in receptor activation. Keywords: Cytochrome P45011B l; Cerebral cortex; In situ hybridization; Neurosteroid; Transgenic rat; Hypertension

1. Introduction There is increasing evidence for the synthesis and active role of a large number of cytochromes P450 in the brain (reviewed in [19,28,36,40]). Although the physiological role and toxicological significance of P450 enzymes in the central nervous system are still under discussion, they could have multiple functions e.g. in metabolism of drugs acting on the brain, in the synthesis and metabolism of steroids and in the generation of reactive oxygen radicals [1,36,39,41]. Among the different cytochrome P450 forms detected in the brain, some are induced following administration of drugs, nicotine or ethanol [1,2,12,18,39,41,42]. Other P450 forms were detected in untreated tissue following the

* Corresponding author. Fax: berdma @orion.rz.mdc-berlin.de

+49

(30)

949 4161;

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combined application of sensitive methods such as RTPCR, RNase protection assay and others [9,20,37]. Immunohistochemical approaches revealed the presence of several cytochrome P450 enzymes in brain tissue, like P4502B 1 [39], P4502E1 [10], aromatase [35,38], P45011A1 [14,17], P45011B1 [29], as well as of NADPH cytochrome P450 reductase [1,25] and glutathion S-transferase [19]. But, only in a few of these proteins, such as in aromatase [16] and NADPH cytochrome P450 reductase [25] could the corresponding mRNA distribution be detected using in situ hybridization methods. Cytochrome P450! 1B 1 (1 l[3-hydroxylase), catalyzing the terminal steps of the formation of the glucocorticoids cortisol and corticosterone, is expressed in the zona fasciculata/reticularis of adrenal glands in various species including rat [11,22,26,34,43] and man [7]. Cytochrome P45011B1 was first detected in rat brain by immunohistochemistry using a polyclonal antibody against bovine adrenal l ll3-hydroxylase which selectively localized the

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B. Erdmann et al. / Brain Research 733 (1996) 73-82

enzyme in tracts of myelinated fibres throughout the brain [29]. However, the corresponding mRNA showed a quite different distribution. Using RNase protection assays and RT-PCR, the mRNAs for P45011A1, P45011B1 as well as for the electron donor adrenodoxin were detected in cerebral cortex, amygdala, hippocampus and midbrain [20]. Interestingly, the 11 [3-hydroxysteroid dehydrogenase, converting corticosterone to inactive 11-dehydrocorticosterone, was found to occur in the same regions such as the hippocampus and cerebral cortex [15,18,23]. Because the immunohistochemical data do not seem to coincide with those for the corresponding mRNA, we applied in situ hybridization methods to clarify the sites for cytochrome P45011B1 mRNA synthesis in the rat central nervous system. Normal Sprague-Dawley rats were compared to the transgenic strain TGR(mRen2)27, expressing the murine Ren-2 d renin gene [24]. These transgenic animals develop severe hypertension (200-260 mmHg) and therefore represent a model in which the genetic basis for the disease is known. Although they have low amounts of renin in kidney and plasma, the adrenal glands show remarkable overexpression of mouse renin mRNA [3]. There is considerable evidence for a locally activated adrenal renin-angiotensin system and significant changes in the adrenal steroid metabolism in this transgenic strain. Corticosteroid concentrations and the basal expression of P45011B2 are significantly elevated, whereas the corticotropin-regulated expression of P45011A 1, P45011B 1 and P45011B2 mRNAs was only slightly influenced [33,34]. The aim of the present studies was, therefore, to localize cytochrome P45011B1 mRNA in normal and transgenic rats to find out whether the activated adrenal steroid metabolism may also affect the central nervous system via the hypothalamus-pituitary-adrenal axis.

2. Materials and methods 2.1. Animals

Experiments were performed with male heterozygous transgenic TGR(mRen2)27 rats [24] and Sprague-Dawley

Table 1 Characterization of the animals Strain

Number of rats used

Age (weeks)

Average body weight (g)

TGR SD TGR SD TGR

1 2 2 2 2

4 8 8 16 16

35 300 257 435 417

Transgenic rats carrying the mouse Ren-2 renin gene (TGR(mRen2)27) and control Sprague-Dawley (SD) rats of different ages and body weights were used in this study.

rats of different ages (Table 1). They were specific pathogen-free and were kept with free access to food pellets and tap water. The animals were deeply anaesthetized with sodium hexobarbital (300 mg/kg, i.p.) and decapitated. Brains and adrenals were removed immediately, rinsed quickly in ice-cold 0.9% saline and frozen in isopentane cooled to - 3 5 ° C with dry ice. The tissue was sectioned at a thickness of 14 ~m in a cryostat and mounted on "¥-aminopropyltriethoxysilaneactivated slides. Sections were taken from each brain at two different positions: at bregma - 0.3 mm and - 4.16 mm according to Paxinos and Watson [30]. The slides were stored at - 7 0 ° C until use. 2.2. RNA probe synthesis

The plasmids were obtained from Dr. S. Mellon (San Francisco, CA). They contain either a 152 or a 185-bp long cDNA template (representing positions 809-960 of the P45011B1 cDNA or positions 779-963 of the P45011B2 cDNA). Both are subcloned into the Hinc2 site of the vector pBluescript KS (Stratagene, La Jolla, CA), already containing the promoters for T3 and T7 polymerase, respectively. After linearization of the plasmid with BamHI or XhoI (Boehringer, Mannheim, Germany) the template DNA was extracted and purified from an agarose gel using the QIAEX gel extraction kit (Qiagen, Hilden, Germany). [35S]a-UTP labeled RNA probes were synthesized by in vitro transcription using 1 txg linearized plasmid DNA. The DNA was incubated in transcription buffer (40 mM Tris-HC1, pH 7.5, 6 mM MgC1 z, 2 mM spermidine) with 12.5 nmol ATP, CTP and GTP, 500 pmol UTP and 125 pmol [35S]~-UTP (1300 Ci/mmol, NEN, Du Pont de Nemours, Germany), 1 U/I~I RNase inhibitor (Boehringer Mannheim, Germany) and 1 U / ~ I T7 or T3 RNA polymerase (Boehringer Mannheim, Germany) at 37°C for 90 min. The DNA template was digested by addition of 20 ng/Ixl RNase free DNase I (Boehringer, Mannheim, Germany) for 15 rain at 37°C in the presence of lu/txl RNase inhibitor. The transcripts were then purified chromatographically on Nensorb cartridges (NEN, Du Pont de Nemours, Germany), checked by denaturing formaldehyde gel electrophoresis and used directly for in situ hybridization experiments. The specific activity of the probes was in the order of 5 × 108 dpm/Ixg RNA. 2.3. Northern blot and dot blot analysis

Poly(A) + RNA was prepared from brain tissue of Sprague-Dawley rats according to standard procedures [32]. 10 ~xg were separated on a denaturating agarose gel containing 2.2 M formaldehyde and blotted onto Hybond N membranes (Amersham Buchler, Braunschweig, Ger-

B. Erdmann et al. / Brain Research 733 (1996) 73-82

many). The membranes were hybridized at 61°C as recommended by the supplier with [32p]UTP (NEN, Du Pont de Nemours, Germany) labeled T7 antisense or T3 sense transcripts of the P45011B1 cDNA probe. Washing was carried out twice with 2 × SSPE (saline sodium phosphate-EDTA buffer, [32]), 0.1% SDS at 68°C, followed by a treatment with 50 Ixg/ml RNase in 2 X SSC (saline sodium citrate buffer, [32]) and a final washing step with 0.05 × SSPE, 0.1% SDS at 68°C. For dot blot analysis 50 pg each of P45011B1 or P45011B2 cold sense transcripts were spotted onto the membranes and treated as described above.

1

2

I

I

3.6kb ..~ 2.7kb -~

2.4. In situ hybridization The slides were brought to room temperature (RT) and fixed for 15 min in 4% paraformaldehyde in phosphate buffered saline (PBS) pH 7.0, followed by a rinse in PBS and two washes for 5 min in sterile water. After deproteination in 0.2 M HCI for 20 min and two short washing steps for 3 min in PBS the slides were acetylated for 20 min in 0.1 M triethanolamine, pH 8.0, and 0.25% acetic anhydride. The slides were washed two times for 3 min in PBS, dehydrated in graded ethanol and air dried. Subsequently, 150 ~1 prehybridization buffer (50% deionized formamide, 50 mM Tris-HC1, pH 7.6, 25 mM EDTA, pH 8.0, 20 mM NaC1, 0.25 m g / m l yeast tRNA, 2.5 × Denhardt's solution (0.05% Ficoll, 0.05% polyvinylpyrrolidone, 0.05% bovine serum albumin)) were applied onto each section and the slides were incubated in a humidified chamber at 37°C for 4 h. After draining the prehybridization buffer off the slides, 15 ~1 hybridization buffer (50% deionized formamide, 20 mM Tris-HC1, pH 7.6, 1 mM EDTA, pH 8.0, 0.3 M NaC1, 0.2 M dithiothreitol, 0.5 m g / m l yeast tRNA, 0.1 m g / m l

75

B P45011B1 .~ P45011B2 - . ~

Fig. 1. Northern blot (A) and dot blot (B) analysis. For Northern blot analysis 10 Ixg of poly(A) + RNA from rat brain were separated on a formaldehyde containing agarose gel and blotted onto Hybond N membranes. For dot blot analysis 50 pg each of cold sense transcripts of P4501lB1 and P45011B2 were spotted onto the membranes. In both cases membranes were hybridized with a labeled P45011BI antisense (lane 1) or sense (lane 2) transcript, respectively. The size of the transcripts is denoted on the left.

poly-A-RNA, 1 x Denhardt's solution, 10% dextransulphate) were used per section, containing either [35S]a-UTP labeled antisense or sense RNA for control experiments. The concentration of the probes was 10 pg/Ixl. The sections were covered with siliconized coverslips and incubated at 37°C for 17 h in a humidified chamber. Coverslips were removed by immersing the slides for 60 rain in 1 × SSC at 48°C, followed by two variants of post-hybridization washing conditions:

Table 2 Semi-quantitative densitometric evaluation of the film autoradiograms of different brain regions and adrenal glands of transgenic (TGR(mRen2)27) and Sprague-Dawley (SD) rats Strain

Age (weeks)

TGR

4

SD

8

TGR

8

SD

16

TGR

16

Exposure times of the film autoradiograms (d)

Tm T~d Tm T~o Tm T~d Tm T~d Tm T~d

Neocortex (Bregma - 0.3 mm)

Neocortex (Bregma - 4.16 mm)

Adrenal cortex

Measured area of adrenal cortex (mm 2)

0.713 0.136 0.819 0.112 0.721 0.146 0.835 0.073 0.834 0.071 21

0.722 0.168 0.771 0.195 0.744 0.143 0.860 0.073 0.834 0.088 21

0.121 0.194 0.274 0.245 0.170 0.220 0.404 0.308 0.228 0.270 8

2.19 3.74 5.65 8,31 9,27

Transmission mean values (Tm) reversely indicating the amount of cytochrome P45011B 1 mRNA in the tissue and their assigned standard deviations (T~d) are given for the cerebral cortex as mean values from all washing conditions used (formamide or RNase, 2 × 12 sections per variant, n = 24), and for the adrenal cortex (washing conditions formamide, 2 × 8 sections per variant, n = 16, see Section 2, Materials and methods).

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B. Erdmann et al. /Brain Research 733 (1996) 73-82

Fig. 2. Localization of cytochrome P45011B 1 mRNA in male rat adrenal glands. In situ hybridization with 35S-labeled riboprobes, washed at 56°C with formamide/SSC. A,C: control rat (Sprague-Dawley), B: transgenic rat (TGR(mRen2)27). A,B: antisense probe, labeling of cytochrome P45011B 1 mRNA in the zona fasciculata (f)/reticularis following autoradiography. Grains are shown as white dots following darkfield microscopy. C: sense probe (controls), no specific signals, g zona glomerulosa, m medulla. Counterstaining HE. Bar = 100 p~m.

1. 0.5 X SSC/50% formamide, 56°C, 4 X 60 rain; 1 x SSC, RT, 2 X 10 min; and 2. 1 X SSC, 48°C, 30 min; RNase A (Boehringer, Mannheim, Germany), 10 ~ g / m l in 10 mM Tris-HC1 pH 8.0, 0.5 M NaC1, 1 mM EDTA, 37°C, 30 min; 1 X SSC, 48°C, 2 X 10 rain; 0.5 X SSC, 60°C, 10 rain; 0.2 x SSC, RT, 10 min. In both cases, sections of the brains of different ages were hybridized in one and the same experiment to allow densitometric evaluation. Sections were dehydrated in graded ethanol, air dried and exposed to Hyperfilm-3H (Amersham Buchler, Braunschweig, Germany) for 8 days (adrenals) or 21 days (brains) at - 20°C. Slides were then dipped in Kodak NTB-2 autoradiography emulsion (Eastman Kodak, Rochester, USA) and exposed for 15 days (adrenals), 37 days (brains, RNasewashed) or 47 days (brains, formamid-washed) at 4°C. After developing the photographic signal, the tissue was either counterstained with haematoxylin/eosin (HE) (adrenals) or with cresyl violet (brain) and mounted with Entellan (Merck, Darmstadt, Germany). The specificity of the method used was determined by hybridization with [35S]oL-UTP labeled sense RNA at the

same specific activity, length and concentration as the antisense RNA. Photographs of the in situ autoradiograms were directly taken from the X-ray film (Fig. 3). Light microscopy was performed with an Axioplan Universal Microscope (Zeiss) equipped with Nomarski differential interference contrast.

2.5. Image analysis and densitometric eualuation Image acquisition and measurement of densitometric data on the cytochrome P45011B1 distribution was performed as described [7]; see also [45]. Briefly, the grey values (expressed as transmission mean values Tm) of the film autoradiograms were measured on a VIDAS 2.1 image analysis system (Kontron, Eching, Germany) with a PC486/33 host computer, 330MB hard disk, and a MTI CCD72 camera system running at 760 X 512 pixels, 8 bit resolution, mounted on an Axioplan Universal Microscope (Zeiss). All measured transmission mean values Tm, the assigned transmission standard deviations ~d , and the contributing areas A are stored in temporary files which conform to the VIDAS-system database standard. Every new measuring cycle comprises all sections (at least three per varian0 which carry the specific (labeled antisense

Fig. 3. Localization of cytochrome P45011B 1 mRNA in male rat brain. In situ hybridization with 35S-labeled riboprobes, washed with RNase. Micrographs taken directly from the X-ray film. A: bregma - 0 . 3 ram; B: bregma - 4 . 1 6 m m according to Paxinos and Watson [29]. Lines 1-6: animals of different ages ( 1 : 4 weeks, 2,3:8 weeks, 4 - 6 : 1 6 weeks) and different strains: * transgenic rats (TGR(mRen2)27), no label: control rats (Sprague-Dawley). Lines 1-5: antisense probe, labeling of cytochrome P45011B1 in the neocortex and in the piriform cortex in A. Line 6: sense probe (controls), no specific signals. Bar = 5 mm.

B. Erdmann et al. / Brain Research 733 (1996) 73-82

RNA) signals, and the corresponding sections which carry the unspecific (labeled sense RNA) signals. In the present study, each transmission mean value Tm results from the

+/

':~ i

77

measurement of 2 X 12 sections (12 with antisense, 12 with sense probes) cut from 2 animals per variant in the case of cerebral cortex, and from the measurement of

I

B. Erdmann et al. / Brain Research 733 (1996) 73-82

2 x 8 sections from one animal per variant in the case of the corresponding adrenals (see Table 2). The necessary normalization of data, the separation of hypothetically staged vertical layers as well as the partial integration of similar regions were performed by special post-processing programs.

3. Results

The riboprobes derived from the 152-bp long cDNA template specifically detected cytochrome P45011B1 mRNA. Northern blot analysis showed a specific signal of 3.6 kb if probed with the P45011B1 antisense transcript (Fig. 1A, lane 1). In addition, there is a second weaker signal of 2.7 kb. Hybridization of the blot with the corresponding sense transcript did not yield any signal (Fig. 1A, lane 2). P45011B1 and P45011B2 share sequence similarities of 68% in the region encompassed by the probes. In order to prove the specificity of the P45011BI probe we performed dot blot analysis with sense transcripts of the P45011B 1 and P45011B2 probes as targets. Hybridization with the labeled antisense and sense transcripts of P45011B 1 clearly demonstrated the specificity of the probe for P45011B1 (Fig. 1B). As shown in control experiments with rat adrenals, the antisense P45011B1 riboprobes hybridized to the zona fasciculata/reticularis of the adrenal cortex (Fig. 2A,B) with a few typical clusters of these cortical cells in the adrenal medulla (Fig. 2B), whereas the zona glomerulosa and the medulla itself were free of labeling. The control, sense probes did not yield any specific signal (Fig. 2C). Obviously, the adrenal cortex layers containing the 1113hydroxylase mRNA were larger in transgenic rats carrying the additional gene for mouse renin as compared to the normal Sprague-Dawley rats (Fig. 2B). Using these riboprobes, cytochrome P45011B1 mRNA could be clearly detected in rat brains, especially in the cerebral cortex including the piriform cortex (Fig. 3). Nearly independent of age and strain, P45011B1 mRNA was present in distinct cortical layers, as shown on X-ray film autoradiograms for the two regions examined (Fig. 3A,B). Transgenic animals (Fig. 3, lines with an asterisk) seem to express nearly the same amount of the enzyme as compared to the normal rats. The weak staining of the hippocampus (Fig. 3B) must be considered as unspecific, because it also appeared in control experiments with sense probes (line 6). Moreover, the staining in the hippocampus

79

was only present in sections washed with RNase, not in the variant washed with formamide/SSC (not shown). Autoradiography and further enlargement of the regions positive for P45011B1 mRNA revealed a distinct labeling of cells in the cerebral cortex including the piriform cortex (Fig. 4A,C,D). As seen as white patches in dark field microscopy, especially the layers II-IV of the neocortex were strongly labeled, whereas in layer V-VI a lower density of cells showed positive staining (Fig. 4A). In the piriform cortex the labeling is mainly confined to layer II with scattered positive cells in the deeper layers. Interestingly, the labeling is present in distinct types of large sized cells, perhaps representing nerve cells, as shown with bright field microscopy and cresyl violet counterstaining for layers II-III of the neocortex (Fig. 4D). Sense probes always caused a low and equally distributed number of silver grains in the autoradiograms (Fig. 4B,E). Initially, densitometric measurements of the film autoradiograms were separately made for the two different washing conditions used. Because nearly the same data were obtained for both variants in this way, the measurements were combined and therefore represent all the experiments. As far as the neocortex is concerned, the transmission mean values reversely indicate the amount of specific mRNA in the tissue section and are nearly constant throughout the different ages and strains examined (Table 2, see also Fig. 3). The adrenal glands contained remarkably higher amounts of the enzyme as compared to brain. It must be noted, however, that the exposure times on the X-ray films were quite different for adrenal and cerebral cortex (see Table 2 and Section 2, Materials and methods) and, therefore, they cannot be compared. In addition, the areas of the adrenal zones expressing P45011BI mRNA were clearly larger in transgenics than in normal animals.

4. Discussion

Northern blot analysis showed a major signal at 3.6 kb and a minor signal at 2.7 kb (Fig. 1A). The latter has been reported to be the only transcript in rat adrenals [13], but the same report demonstrated a P45011B2 transcript of a similar length in this tissue. In order to exclude cross-reaction of our probe with P45011B2 we performed dot blot analysis which clearly demonstrated the specificity for P45011BI (Fig. 1B). Thus, possibly an alternative polyadenylation signal is used in the brain resulting in a transcript size of 3.6 kb.

Fig. 4. Localization of cytochrome P45011B1 mRNA in male rat brain. In situ hybridization with 35S-labeled riboprobes, washed at 56°C with formamide/SSC. Transgenic rat (TGR(mRen2)27), 8 weeks old, bregma - 0 . 3 mm according to Paxinos and Watson [29]. A,C,D: antisense probe, labeling of cytochrome P45011B 1 in the cerebral cortex (A: layers I1-VI, D: details of layer II-III) and in the piriform cortex (C: layers II-III). The labeling is found over large, probably nerve cells. B: sense probe, control to C. E: sense probe, control to D. A-C: darkfield; D,E: bright field with Nomarski differential interference contrast. Counterstaining cresyl violet. Bar = 100 ~m.

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B. Erdmann et a l . / Brain Research 733 (1996) 73-82

Using these probes, the present study firstly demonstrates the expression of cytochrome P45011B1 mRNA in distinct areas of the rat brain, mainly in the neocortex and the piriform cortex and its probable presence in nerve cells. It also gives further evidence that the enzyme can be synthesized de novo in the brain. The amount of mRNA detectable by in situ hybridization was, however, remarkably lower in the brain as compared to adrenal glands. Similar results were obtained for other P450 forms such as aromatase [16]. In the adrenal gland, the riboprobes specifically hybridized to the zona fasciculata/reticularis with a few typical clusters of these cell types in the adrenal medulla. This points again to a functional interaction of steroid producing and chromaffin cells [5]. Although the zonal distribution of P45011B 1 remained unchanged in normal and transgenic rats, the labeled areas of the zona fasciculata/reticularis were larger in the hypertensive transgenic variant. Previous experiments revealed an activated adrenal steroid metabolism in this transgenic strain with an increase in adrenal weight and number of glomerulosa cells, an enhanced urinary excretion of 11-deoxycorticosterone (DOC), corticosterone, 18-hydroxycorticosterone and aldosterone, as well as an overexpression of cytochrome P45011B2 producing aldosterone [33,34]. Contrary to these studies which could not detect differences in basal cytochrome P45011B1 mRNA expression between normal and hypertensive transgenic rats, in our case the semiquantitative evaluation of the in situ hybridization also revealed, besides the larger area, a clear increase in the concentration of adrenal cytochrome P45011B 1 mRNA in the transgenic strain as compared to the normotensive controls. In the brain, cytochrome P45011B 1 mRNA was, for the first time, localized by in situ hybridization methods. Specific signals were obtained in distinct neocortical layers and in layer II of the piriform cortex throughout the different ages, strains and washing conditions used (Figs. 3 and 4). As shown in bright field microscopy in combination with cresyl violet staining, the signals were probably localized in nerve cells. Previous results on RNase protection, RT-PCR and Southern blotting in normal rats pointed to the occurrence of cytochrome P45011B1 mRNA in these regions [20]. Polyclonal antibodies against purified bovine adrenocortical P45011B1, however, seem to react with other neuronal structures such as myelinated fibres in the white matter [29]. As far as the neocortex is concerned, mRNA expression, enzyme activity or immunoreactivity are described for ll[3-hydroxysteroid dehydrogenase [15,23], cytochrome P4502E1 [10], NADPH cytochrome P450 reductase [25] and other (possibly multiple) forms of cytochrome P450 [1]. The occurrence of 1 l[3-hydroxylase as well as 1 l[3-hydroxysteroid dehydrogenase in this region fits well with earlier findings on the existence of high densities of neuronal nuclear glucocorticoid receptors all

over the neocortex concentrated mainly in layers II-III and VI [6,8] controlling gene expression. This pattern largely mimics the patterns of the 1 l[3-hydroxylase distribution and suggests a certain morphological link as well as the participation of this enzyme in control of both sensory and motor functions of the neocortex, possibly involving glucocorticoid receptors. Although it was proven that the rat brain has the potential to make steroids de novo from cholesterol because it contains P45011A1 activity as well as the respective protein and mRNA [20], the functions of neurosteroids are still under discussion [4]. Corticosterone may modulate behavioral responses [27] and rapidly inhibit reproductive behavior, apparently through corticosteroid binding sites in neuronal membranes that are independent of GABA A receptors [28]. On the other hand, the l l[3-hydroxysteroid dehydrogenase may regulate the access of corticosterone to classical central nervous mineralo- a n d / o r glucocorticoid receptors and thus modulate corticosteroid effects on brain function through genomic actions [23]. The cytochrome P45011B1 mRNA signals in the neocortex and the piriform cortex were confined to apparent nerve cells with no signals in the white matter (Fig. 4D). In agreement, mixed primary glial cultures and a C6 glioma cell line do not express 1l[3-hydroxylase, although they synthesize cytochrome P45011A1 [20]. These findings also point to a preferred neuronal expression of cytochrome P45011B 1, which has already been shown for NADPH cytochrome P450 reductase [25] and microsomal P450 forms [1]. In general, glia and neurons tend to be highly specific in the forms of P450 they express [19]. The present study was confined to two brain rostrocaudal levels and does, therefore, not exclude the expression of cytochrome P45011B 1 in other brain regions. However, the hippocampus being very rich in mineralo- and glucocorticoid receptors [8,20] came out negative in the present study. It should be noted that sex-specific differences in cytochrome P45011B 1 mRNA expression were obtained in the hippocampus [20] where a clear expression of this enzyme occurred only in female rats. Hence, there is a large need for a more comprehensive study on the distribution of cytochrome P45011B1 mRNA in the brain. The immunoreactivity for P45011B1 in the myelinated tracts [29] remains unclear in view of the lack of a mRNA signal in the oligodendrocytes for this cytochrome enzyme. In TGR(mRen2)27 transgenic rats the additional mouse R e n - 2 d gene is mainly expressed in the adrenal glands. Despite the slightly enhanced cytochrome P45011B1 mRNA signals in the adrenals of the transgenic rats, the brain seems to contain nearly the same levels of this enzyme as the normal Sprague-Dawley rats (Fig. 3). High 1 l[3-hydroxylase expression in the brain was detected in young transgenic rats at the ages of 4 and 8 weeks (Table 2, Tm values around 0.7), at a time when the fulminant hypertension develops [24]. But this tendency is not nearly as obvious as in the adrenal, where a markedly changed

B. Erdmann et al. / Brain Research 733 (1996) 73-82

steroid m e t a b o l i s m has been p r o v e n [31,33,34,44] with a significant increase in the expression o f c y t o c h r o m e P 4 5 0 1 1 B 2 (aldosterone synthase). This points again to the existence o f a true local r e n i n - a n g i o t e n s i n system acting i n d e p e n d e n t l y in different organs. It remains to be determ i n e d in w h i c h w a y the closely related aldosterone synthase and n e w l y detected c y t o c h r o m e P45011 forms of this f a m i l y [21] contribute to the different forms of hypertension m e d i a t e d by steroid h y d r o x y l a t i n g e n z y m e s . In conclusion, the relatively high expression o f c y t o c h r o m e P45011B1 in the n e o c o r t e x and the p i r i f o r m cortex o f the male rat indicate the existence o f b r a i n - d e r i v e d corticosterone in these areas, representing a possible local source for the activation o f g l u c o c o r t i c o i d receptors in these cortical regions with c o n s e q u e n c e s for regulation o f gene expression and m e m b r a n e function.

Acknowledgements W e w o u l d like to thank Dr. U. G a n t e n and Dr. H.-J. H e r r m a n n for their help in obtaining and preparing the animals. W e also thank Mrs. R. V o g e l for technical assistance. This project was supported in part by a f e l l o w s h i p f r o m the D e u t s c h e Akademie der Naturforscher ' L e o p o l d i n a ' and the B u n d e s m i n i s t e r i u m fiir F o r s c h u n g und T e c h n o l o g i e to B.E. and in part by grants f r o m the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t to R.B. (Be 1 3 4 3 / 2 - 4 and Be 1 3 4 3 / 6 - 1 ) .

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