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Abteilung Nephrologie und Rheumatologie (E.B., G.A.M., J.S.), Abteilung Pathologie (P.M.), Abteilung Vegetative Physiologie und Pathophysiologie (A.B., Y.H., G.B., J.S.) Universität Göttingen, 37073 Göttingen; Abteilung Endokrinologie (H.S.W., S.R.B.), Universität Düsseldorf, 40225 Düsseldorf; and Anatomisches Institut, Lehrstuhl I (H.K.), Universität Würzburg, 97070 Würzburg, Germany
Address all correspondence and requests for reprints to: Dr. Jürgen Steffgen, Abteilung Vegetative Physiologie und Pathophysiologie, Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany. E-mail: jsteffgen{at}gmx.de.
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Introduction |
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In the proximal tubule of rat kidney, probenecid-inhibitable transport of cortisol via the p-aminohippurate (PAH)-transport has been described (5), mediated most likely through the organic anion transporter (OAT) 1 (6, 7). Probenecid, a classical inhibitor of OAT (8, 9), reduced both [3H]PAH uptake into, and cortisol release from, bovine adrenocortical cells to comparable degrees (10, 11). Unlabeled PAH as well as cortisol in the incubation medium inhibited [3H]PAH uptake, indicating that PAH and cortisol share a common transporter. Moreover, cortisol release from adrenocortical cells was trans-stimulated by extracellular glutarate and PAH. These cis-inhibition and trans-stimulation experiments supported the concept of an anion exchange mechanism being involved in cortisol release from, and organic anion uptake into, adrenocortical cells. Indeed, a probenecid-inhibitable PAH transport was expressed after injection of mRNA from adrenocortical cells in Xenopus laevis oocytes (10), suggesting a PAH transporter in the adrenocortical cells. However, a direct demonstration of such a transporter at the molecular level was missing.
In other tissues, transport of conjugated steroid hormones has been reported by members of the oatp family (12, 13). Moreover, corticosterone inhibited organic cation transport by rat organic cation transporters (rOCT)1 and rOCT2, showing an 38-fold higher affinity for rOCT2 (14). Thus, these transport proteins might also be involved in steroid release. Therefore, rat adrenal cortex was analyzed for the existence of such transport proteins by a homology cloning procedure, in situ hybridization, and immunohistochemistry. Furthermore, expression of these transporters was studied in rats that were either pretreated with ACTH or hypophysectomized. Part of the results has been published in abstract form (15).
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Materials and Methods |
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PCR
To amplify OAT1 and oatp1–3 from rat adrenal cDNA, primers were designed based on the nucleotide sequence aligments of previously cloned OAT1 from flounder (f), rat (r), and human (h), and oatp1–3 from rat. The PCR for OAT1 was performed in a 50-µl reaction volume with 3 mM MgCl2, 20 pmol of each primer, 10 mM deoxynucleotide triphosphate (dNTP) mixture, 200 ng cDNA, 4% dimethylsulfoxide, and 5 U Taq polymerase (Pfu Turbo DNA polymerase, Stratagene, Cambridge, UK). Melting temperature was 94 C for 45 sec, extension temperature was 72 C for a time of 1 min/kb dependent on the length of the product, and annealing temperature was 55 C for 30 sec. For optimal amplification of oatps, 3.5 mM MgCl2 was used and annealing performed for 15 sec at 55 C. PCR products were visualized on a 1–2% agarose gel with ethidium bromide, excised, and cloned into pPCR-Script Amp SK(+) (Stratagene) for sequencing and in vitro transcription.
The following forward (F) and reverse (R) primers were used:
pPCR-Script Amp cloning and analysis of positive clones with PCR
The blunt-ended PCR products generated by Pfu polymerase were ligated into pPCR-Script vector, using the pPCR-Script Amp cloning kit (Stratagene). A PCR cocktail consisting of PCR buffer, dNTPs, primers (M13 F and M 13 R), and Taq polymerase was prepared. White colonies were picked and analyzed individually with a 25 cycles PCR (94 C for 45 sec, 54 C for 30 sec, and 72 C for 1 min with a final incubation for 10 min at 72 C). The PCR products were visualized on a 2% agarose gel with ethidium bromide. The positive colonies were cultured overnight in Luria-Bertani medium containing 50 µg/ml ampicillin. Plasmid DNA was isolated from the overnight cultures with QIAprep Spin miniprep kit (QIAGEN) according to the manufacturer’s instructions.
Sequencing
Using T3-T7 vector-specific primers and sequence-specific primers, the cloned PCR products were sequenced by dye terminator cycle sequencing (Applied Biosystems, Weiterstadt, Germany) on an automatic sequencer (ABI 377, Applied Biosystems). Sequence homology searches were performed online using Fasta3 database searches at the European Bioinformation Institute (http://www.ebi.ac.uk/fasta3/).
Nonradioactive in situ hybridization on paraffin-embedded material
For the generation of riboprobes, RNA probes of the different transporters were produced by a modified protocol using PCR-generated templates for in vitro transcription. Briefly, PCR-amplified products of the different transporters cloned into pPCR-Script vector were used as a template for a PCR, using oligonucleotide primers specific for the T3-(5'-CAATTAACCCT-CACTAAAGGG-3') and T7-promoter (5'-GTAATACGACTCACTATAGGGC-3') regions of the vector. PCR products were generated in an OmniGene thermocycler (Hybaid, Middlessex, UK). Fifty microliters reaction mixture contained 200 ng plasmid DNA, 20 pmol of each primer, 1 µl dNTP (10 mM each), 5 µl Taq buffer (10x), and 1 U Taq polymerase (Pharmacia). After an initial 95 C denaturation step (2 min), 30 cycles were carried out at 94 C (45 sec), 55 C (30 sec), and 72 C (1 min), followed by a final extension step of 72 C for 10 min. PCR products were visualized on a 1% agarose gel and stained with ethidium bromide. The PCR products were excised and purified using QIAquick gel extraction kit. Digoxigenin (DIG)-11-uridine 5-triphosphate-labeled sense and antisense probes were generated by in vitro transcription by using purified T3 or T7 PCR products. Purification of the probes was performed with 4 M LiCl and ethanol precipitation.
Adrenals were taken from Wistar rats, fixed in 4% paraformaldehyde, and embedded in paraffin. In situ hybridization was performed according to the method described by Breitschopf et al. (16). Briefly, tissue sections were deparaffinized with Roticlear (Carl Roth GmbH, Karlsruhe, Germany), rehydrated in serial dilutions of ethanol (100%, 90%, and 70%), and postfixed in 4% paraformaldehyde. Samples were permeabilized using 10 µg/ml proteinase K (Roche Molecular Biochemicals GmbH, Mannheim, Germany) for 30 min at 37 C. Digestion was stopped by washing the samples in PBS (pH 7.4). To block the endogenous alkaline phosphatase, slides were incubated with 0.25% acetic anhydride and dehydrated in serial dilutions of ethanol (70, 90, and 100%).
DIG-labeled riboprobes were diluted 1:100 in hybridization buffer (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). After application of sense and antisense probes, the slides were covered with sterile coverslips and placed on a hot plate for 5 min at 85 C to denature the ribonucleotides. Hybridization was performed overnight at 55–58 C in a sealed humidified chamber containing 50% formamide. Nonspecific binding or unbound probes were removed by the following posthybridization washes: 1x saline sodium citrate (Fluka Biochemika, Taufkirchen, Germany)/0.1% SDS at room temperature (2 x 5 min) and 0.2x saline sodium citrate/0.1% SDS at hybridization temperature (2 x 10 min). Finally, the sections were washed in Tris-buffered saline containing 0.1% Tween 20 (Roche Molecular Biochemicals). DIG-labeled RNA probes were detected, after hybridization to target nucleic acid, by enzyme linked immunoassay using an antibody-conjugate (antidigoxigenin alkaline phosphatase). A subsequent enzyme-catalyzed color reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt produced an insoluble blue precipitate, which visualized hybrid molecules. In the case of the oatp probes, the slides were counterstained with hemalaun.
To analyze the influence of ACTH on expression of the transporters, male Wistar rats were either daily pretreated for 5 d with 2.5 µg ACTH (synacten) or hypophysectomized. The animal experiments were commercially performed by Biotech Pipelines (Trige, Denmark), and studies were approved by the Danish Animal Ethical Committee. For hypophysectomy, rats were placed in a stereotactic apparatus and the pituitary was removed transauricularly by suction through a syringe when a vacuum was activated. Correct hypophysectomy was controlled twice in the following week by weighing of the rats. Animals were kept on a 12-h day and night rhythm. To correct for the influence of the stress caused by the injection, all groups were always treated in parallel. Hypophysectomized rats were injected with 0.9% saline at the same time points when one group of rats was treated with synacten. Twelve hours after the last injection, animals were killed in the morning and adrenal glands prepared for in situ hybridization.
RIA
The concentrations of corticosterone in the blood serum of the treated rats were determined by a RIA. The antiserum employed was raised against corticosterone but showed 100% cross-reactivity with cortisol. The cross-reactivity with other steroids like aldosterone, progesterone, androstendione, or testosterone was less than 0.1%. The antibody was diluted 1:2000 with PBS buffer containing 0.1% gelatin. The [3H]corticosterone tracer was purchased from NEN Life Science Products (Köln, Germany), and unlabeled corticosterone used as a standard was supplied by Sigma (Deisenhofen, Germany). The sample volume was either 10 or 25 µl serum. Bound and free tracers were separated by dextran-coated charcoal. The limit of detection of this RIA was 1 ng/ml. Inter- and intraassay coefficient of variation, as determined by multiple duplicate determination of a pool of sera, were 15.6 and 7.5%, respectively.
Oocytes, injection, and uptake experiments
Oocytes were prepared from Xenopus laevis (Nasco, Fort Atkinson, WI) ovaries by treatment with collagenase (type CLSII, Biochrom) and subsequent washing in Ca2+-free Ringer’s solution (see below) as described earlier (10). Oocytes were injected with 25 ng cRNA-encoding rat OAT1 using a micropump (Drummond, Broomall, PA) and maintained at 18 C in Barth’s solution [in mM: 90 NaCl, 2.4 NaHCO3, 1 K2SO4, 0.8 MgSO4, 0.3 Ca(NO3), 0.4 CaCl2, and 5 HEPES (pH 7.5)] containing 12 mg/liter gentamicin (Refobacin, Merck, Darmstadt, Germany). The medium was changed daily, and damaged oocytes were discarded. After 3 d, oocytes were incubated for 10 min at room temperature in Ringer’s solution [in mM: 90 NaCl, 3 KCl, 2 CaCl2, and 5 HEPES (pH 7.5)] containing 10 µM of the radiolabeled substrate [3H]PAH (1 mCi/mmol; NEN Life Science Products) in the presence and absence of 500 µM corticosterone.
Immunohistology
Adrenal glands were taken from Wistar rats, fixed in 4% paraformaldehyde, and embedded in paraffin. The tissues were sectioned, deparaffinized, and rehydrated. Antigens were retrieved employing 1% Triton X-100 in Tris-buffered saline (pH 7.6) before endogenous peroxidase was blocked with 0.3% H2O2 and 10% methanol for 15 min. For detection of OAT1 immunoreactive protein, we employed the catalyzed signal amplification method with the reagents provided by DakoCytomation (Hamburg, Germany). Sections were preincubated with 2% normal swine serum and exposed to a 1:200 dilution of the polyclonal rabbit antirat OAT1 affinity-purified antiserum (Alpha Diagnostic International, San Antonio, TX) supplemented with 2% rat normal serum for 60 min. For negative control, the antiserum was diluted and eventually replaced by rabbit IgG (DakoCytomation). No nonspecific staining was observed. Bound antibodies were detected by sequential incubations with a biotinylated pig antirabbit antibody, streptavidin-biotin-peroxidase complex, biotinyl tyramide, hydrogen peroxidase, and streptavidin-peroxidase, each step lasting about 15 min. Between the incubation steps specimens were washed thoroughly in Tris-HCl (pH 7.6), containing 0.3 M NaCl and 0.1% Tween 20. Visualization was achieved through 3-amino-9-ethyl-carbazole (ImmunoTech, Hamburg, Germany) for 10 min. Slides were counterstained with hematoxylin for 15 sec, rinsed in tap water, and mounted with glycerin gelatin.
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Results |
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Cattle and rat differ with respect to the main synthesized glucocorticoid, which is cortisol in cattle but corticosterone in rat. So far, experimental evidence for interactions of glucocorticoids with the cloned organic anion transporter OAT1 was based only on studies with cortisol. Therefore, we tested whether corticosterone interacts with the cloned rat renal OAT1. Rat renal OAT1 was cloned by us, and the sequence of this cloned rat OAT1 was identical with that reported by Sekine et al. (6). The mRNA encoding for rat OAT1 was injected into Xenopus laevis oocytes, and 3 d after injection the uptake of [3H]PAH, the model substrate of OAT1, was measured in the absence and presence of 500 µM corticosterone. Corticosterone almost abolished [3H]PAH uptake in oocytes expressing OAT1, whereas there was no effect on uptake into water-injected control oocytes (Fig. 1
). Thus, it is possible that OAT1 is also involved in corticosterone release in rat adrenal cells.
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, lane A). The product was sequenced and revealed 98% identity to the rat renal OAT1.
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, lane B). The 2% difference in sequence most likely was due to PCR and/or sequencing errors.
Because members of the oatp family interacted with steroid hormone derivates (12), cDNA from adrenal cortex was analyzed for the existence of these transporters using PCR primers based on regions in which all three oatps are highly homologous. The oatp_679Fb F and oatp_958R reverse primers and the oatp_368Fa F and the oatpR reverse primers were expected to yield PCR products of 279 bp and 590 bp, respectively. As a positive control, these primers were tested with rat liver cDNA, and the products were confirmed by sequencing. With cDNA from rat adrenal glands, the two products of the predicted size were amplified (Fig. 2, lanes F and I). The 279-bp product had 98% identity to oatp3, and the 590-bp product showed 96% identity to oatp2.
To determine whether oatp1–3 are present in the adrenal gland, specific primers were designed to each individual oatp (oatp1_-2 F, oatp2_-12F, oatp3_1F). The primers fitted to the ends of the open reading frame of the respective transporter and the PCR parameters were optimized to provide high-stringency conditions, especially by an annealing temperature of 60 C. Each of these individual oatp primers was combined with a common reverse primer (oatp123_2013R).
The whole open reading frames of oatp2 (Fig. 2, lane D) and oatp3 (Fig. 2, lane C) were successfully amplified. For oatp1 reamplification of the purified end product was needed to yield acceptable amounts of oatp1 for further analysis (Fig. 2, lane E). The open reading frames of all these three oatps, oatp1, oatp2, and oatp3, were confirmed by sequence analysis, indicating that the three members of the oatp family are present in the rat adrenal gland.
This finding raised the question of the specific localization of these transporters. Sequence regions with low identity between the different OATs were selected as templates for nonradioactive in situ hybridization. The sequence amplified with f,r-OAT1 F and R primers (cf. Fig. 2) was chosen as template for OAT1 (position 1043–1283). Because the open reading frames of oatp1, oatp2, and oatp3 share about 86% identities, probes for oatp1 and oatp2 were selected from their 3'-untranslated regions (position 2074–2682 for oatp1 and position 2096–2764 for oatp2). Using these riboprobes the respective products for oatp1 (Fig. 2, lane H) and oatp2 (Fig. 2, lane G) were amplified. Because the 3'-untranslated region of oatp3 was unknown, the fragment amplified with oatp_679Fb and oatp_958R primers (position 679–958) at high-stringency conditions was chosen for in situ hybridization of oatp3. Sequencing confirmed the identity of the probes for the three transporters.
By in situ hybridization (Fig. 3), signals for OAT1 were localized in the outer zona fasciculata and also in some cells of the zona glomerulosa (Fig. 3, A and B). Signals for oatp1 (Fig. 3, D and E) as well as those for oatp2 (Fig. 4, A and B) were localized in the inner zona fasciculata. Oatp3 mRNA (Fig. 4, D and E) was localized in the zona glomerulosa. Control testing with sense probes of each transporter did not show any staining (Figs. 3 and 4, C and F each). These results illustrate a specific, zonal expression of OATs in the rat adrenal cortex.
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), whereas no signal was obtained with the sense probe (Fig. 5B). An OCT1-specific probe did not reveal any signal (data not shown).
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Serum corticosterone levels increased more than 2-fold on ACTH treatment and dropped to less than 5% in the hypophysectomized rats. In rats pretreated with ACTH, an intense expression of OAT1 was detected in the whole (inner and outer) zona fasciculata as well as in the zona glomerulosa and zona reticularis (Fig. 6A). In hypophysectomized rats, the signal for OAT1 was abolished (Fig. 6B).
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). However, in contrast to the expression of OAT1, the signal for oatp3 did not spread to the inner zona fasciculata or the zona reticularis after ACTH treatment. In hypophysectomized rats (Fig. 6B), the message for oatp3 in the zona glomerulosa was weaker but, in contrast to the signal for OAT1, still detectable. The effect of ACTH on the expression pattern of OCT2 was similar to its effect on oatp3. In ACTH-treated rats (Fig. 6A), the intensity of the signal for OCT2 mRNA was higher than in hypophysectomized rats and was restricted to the zona glomerulosa and the outer zona fasciculata.
To exclude a possible dissociation between transcript and protein and provide evidence for this transporter at the protein level, rat adrenal tissue sections were incubated with polyclonal rabbit antirat OAT1 antibodies. These antibodies bound specifically against cells in the outer zona fasciculata and to some cells in the zona reticularis (Fig. 7, A and C). Nonspecific staining was excluded by the negative control (Fig. 7, B and D). Therefore, immunohistochemistry confirmed the localization of OAT1 in the adrenal cortex.
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Discussion |
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Because steroid hormones interfere with oatp transporters, we tested by a PCR-based screening assay, whether oatp1, oatp2, and oatp3 are also present in rat adrenal gland. All three oatp transporters were detected. Thus, these studies provide for the first time molecular evidence for the existence of OAT1 as well as of oatp1, oatp2, and oatp3 in the rat adrenal gland.
To localize these transporters within the different zones of the adrenal gland, nonradioactive in situ hybridization was performed. Specific riboprobes recognized all four tested anion transporters in the adrenal cortex. OAT1 was identified mainly in the outer zona fasciculata, and oatp3 in the zona glomerulosa. Some cells of the inner zona fasciculata and zona reticularis were positive for oatp1 and oatp2. Because glucocorticoids are synthesized mainly in the outer zona fasciculata, the distribution of OAT1 perfectly matches the localization of glucocorticoid synthesis. Because dehydroepiandrosterone is synthesized in the zona reticularis, localization of oatp2 in this zone suggests a role of this transporter in hormone release.
Former stop flow measurements in rat renal tubules indicated interactions of steroids not only with the PAH transporter but also with OCT (22). Uptake of tetraethylammonium into oocytes expressing the cloned rOCT was inhibited by corticosterone (14). So far, adrenal glands were not analyzed for the presence of OCTs. In this study, OCT2 was localized in the adrenal cortex, mainly in the zona glomerulosa, but also in the outer zona fasciculata, whereas OCT1 was not detected. Based on its localization in the outer zona fasciculata, OCT2 could also be involved in glucocorticoid release from adrenocortical cells.
It is well known that the adrenal cortex increases the synthesis of cortisol or corticosterone in response to ACTH, resulting in higher levels of these steroids in the blood. Engeland et al. (23) demonstrated that injection of a high dose of ACTH increased the mRNA of the key enzyme for cortisol synthesis, cytochrome P450-11ß hydroxylase, within 24 h. They observed an expansion of the area of hybridization to the inner zones of the adrenal cortex, suggesting that ACTH increased the number of P450-11ß hydroxylase mRNA-expressing cells. After chronic ACTH treatment, an extension of fasciculata-like cells toward the adrenal capsule was observed (24). Ho and Vinson (25) demonstrated that P450-11ß hydroxylase was also expressed in the zona glomerulosa following an ACTH treatment. A change in the mitochondrial christae from the tubular to the vesicular form suggested an ACTH induced transformation of zona glomerulosa cells to zona fasciculata cells (26). Recruitment of steroidogenic cells was discussed as a novel mechanism for amplifying the steroid response to adrenal activation.
If a transporter plays a role in hormone release from adrenocortical cells, the expression could also be regulated. Therefore, distribution of OAT1 and OCT2, the two transport proteins localized in the outer zona fasciculata in the adrenal cortex, and oatp3 in the zona glomerulosa were analyzed in rats treated with ACTH and, as a negative control, in hypophysectomized rats. Because stress has been shown to increase corticosterone synthesis (23), hypophysectomized rats were injected with saline at the same time that injections were performed in ACTH-treated rats.
Activation of the adrenal cortex by ACTH increased the amount of OAT1 mRNA, now being present in all zones of the adrenal cortex. In hypophysectomized rats the signal for OAT1 was no longer detectable. The results unravel a regulatory effect of ACTH on OAT1 expression and suggest a recruitment of OAT1-positive cells most likely involved in cortisol release. Such an effect of ACTH has so far not been described for any organ-expressing OAT1 (kidneys and brain). In ACTH-treated rats, mRNAs for oatp3 and OCT2 were more abundant than in hypophysectomized rats. Unlike OAT1, oatp3 and OCT2 mRNAs did not spread to the whole zona fasciculata.
The pattern of OAT1 expression showed the most prominent dependence on ACTH. These findings are in accordance with our previous studies showing an ACTH-induced stimulation of cortisol release and [3H]PAH uptake into bovine adrenal cells (11). Moreover, the regulation and distribution of OAT1 in the adrenal cortex in dependence of ACTH parallels the reported expression of P450-11ß hydroxylase (23, 25) and the amount of corticosterone release in intact animals.
Immunohistochemistry with specific polyclonal antibodies confirmed the localization of OAT1 in the outer zona fasciculata and demonstrated that the presence of the OAT1 transcript reflects the presence of the corresponding protein. Thus, there is now functional as well as molecular evidence for a close relationship between OAT1 and glucocorticoid release. Thereby, OAT1 may be directly involved in cortisol release or/and needed to support the cell with substrates necessary for their endocrine function. Similarly, the other transporters, OCT2 and oatp1, 2, and 3, detected in rat adrenal gland may be of functional importance, a conclusion supported by their strict zonal distribution. Further studies should reveal the function of all transporters identified in the adrenal cortex and address the question whether inhibitors of these transporters are of therapeutic use in case of hormone overproduction.
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Acknowledgments |
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Footnotes |
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Abbreviations: dNTP, Deoxynucleotide triphosphate; f, flounder; F, forward primer; h, human; OAT, organic anion transporter; oatp, organic anion transporter polypeptide; OCT, organic cation transporter; PAH, p-aminohippurate; r, rat; R, reverse primer; rOCT, rat organic cation transporter.
Received September 25, 2002.
Accepted for publication June 16, 2003.
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