RAPID COMMUNICATION |
Correspondence to: Barbara Peters, Dept. of Pharmacology, Univ. of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany..
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Summary |
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Steroid acute regulatory protein (StAR) plays an essential role in steroidogenesis because it is responsible for the transfer of cholesterol from cellular stores to the inner mitochondrial membrane. We investigated the distribution and regulation of StAR expression in association with aldosterone production in the rat adrenal gland in vivo. Using nonradioactive in situ hybridization, we demonstrate that the outermost five to seven parenchymal cell layers express the StAR gene only weakly and inhomogeneously. The strongest expression is found in the zona fasciculata and zona reticularis. In addition, some cells in the adrenal medulla also stained positively. To differentiate between functionally active glomerulosa and inactive intermediate cells, we compared the expression pattern of StAR with that of aldosterone synthase. The expression of the latter is localized to two or three cell layers only, located immediately below the capsule. However, the cells of the intermedia are capable of expressing both genes prominently, as shown after stimulation with bilateral nephrectomy for 2 days. All zones of the adrenal cortex by then expressed StAR gene to the same extent. This was accompanied by a 50-fold elevated plasma aldosterone concentration. Our data demonstrate that the width of the aldosterone-producing zone can increase within a short period of time by recruiting hormonally inactive cells to steroidogenesis. (J Histochem Cytochem 46:12151221, 1998)
Key Words: rat adrenal, glomerulosa, intermediate zone, nephrectomy, angiotensin II, StAR, aldosterone synthase, in situ hybridization
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Introduction |
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Acute stimulation of steroidogenesis involves, in addition to enhanced activity of steroidogenic enzymes, increased substrate delivery for the reaction. This is accomplished by the recently described steroid acute regulatory protein (StAR), which transports cholesterol to the inner mitochondrial membrane (
Recently, an intermediate zone in the rat adrenal cortex was defined by immunohistological criteria, being located between the zona glomerulosa and zona fasciculata (
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Materials and Methods |
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Animals, Nephrectomy, Hemodialysis, and Drug Treatment
Adult male SpragueDawley rats weighing 400520 g were housed under alternating 12-hr light and dark cycles at a constant temperature between 20C and 22C. They were fed the standard laboratory diet of Ssniff (R/M-H; Soest, Germany) and had free access to tapwater. All animal experiments were conducted in accordance with federal and local laws as well as institutional regulations.
All animals received chronic indwelling catheters into the femoral artery and vein under Ketamine/Xylazine anesthesia (75 mg/kg and 6 mg/kg, respectively) and were further allocated to one of three groups of six rats each. Each animal received an osmotic minipump (Alzet pumps, model 2ML1; Charles River, Sulzfeld, Germany) which supplied 10 µl per hour at a constant rate. The pumps were implanted SC for application of physiological saline in Groups 1 and 3 or ANGII (Sigma; Deisenhofen, Germany) in Group 2. The calculated infusion rate of ANGII was 200 ng/kg/min. Furthermore, the animals of Group 3 underwent bilateral nephrectomy (NX), whereas the animals of Groups 1 and 2 were sham-operated. All groups received hemodialysis on each day after surgery as described previously (
Determination of Plasma Aldosterone and ANGII
Plasma samples were obtained from conscious rats via a catheter at the end of the experiment, 55 hr after operation. Plasma aldosterone and ANGII were measured by specific radioimmunoassays after extraction, as described previously (
Statistical Analysis
Data on plasma aldosterone concentrations were given as means ± SEM. Differences between groups were evaluated by analysis of variance, followed by the post hoc test of Bonferroni. Only p values less than 0.05 were accepted to indicate significant differences.
Tissue Preparation
Rats were anesthetized with Ketamine/Xylazine (75 mg/kg and 6 mg/kg, respectively). Fixation of the adrenal glands was performed by retrograde perfusion from the abdominal aorta with 2% freshly prepared paraformaldehyde in PBS, pH 7.4, for 90 sec at a pressure of 220 mmHg and for 90 sec at a pressure of 170 mmHg, followed by perfusion with 18% sucrose in PBS, adjusted to 800 mOsm/kg for another 3 min at the same pressure level.
For in situ hybridization, the adrenal glands were removed, mounted quickly onto small pieces of styrofoam and then snap-frozen in liquid nitrogen-cooled isopentane. All tissues were stored at -80C.
In Situ Hybridization
A full-length cDNA of StAR protein was obtained from rat adrenal cDNA. Because the published cDNA sequences of rat StAR differ in their 5'-region (
DIG1-11-UTP-labeled sense and anti-sense riboprobes were synthesized from the linearized plasmids using T3 and T7 RNA polymerases, respectively. In situ hybridization was carried out as described previously (
The specificity of the obtained in situ hybridization signal was verified by parallel incubation with anti-sense and sense riboprobes on alternate sections. Throughout all experiments, sense probes did not produce any detectable signal.
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Results |
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Plasma ANGII and Aldosterone Concentrations
After a period of 55 hr, plasma ANGII levels were elevated about tenfold by ANGII infusion and decreased to less than 30% of controls by bilateral nephrectomy (sham 59 ± 13 pg/ml; ANGII infusion: 607 ± 110 pg/ml; NX 18 ± 2 pg/ml). At 55 hr after chronic infusion of ANGII, aldosterone levels were increased (27 ± 12 pg/ml) compared with levels in sham-operated rats (11 ± 3 pg/ml). Owing to the slight differences and small number of rats, the effect did not reach significance level. In contrast, bilateral nephrectomy increased plasma aldosterone concentrations prominently and significantly (465 ± 90 ng%; p < 0.001). Corticosterone levels were not different between the groups (sham 29 ± 8 µg%; ANGII 41 ± 13 µg%; NX 48 ± 10 µg%).
Distribution of CyP11B2 and StAR mRNA in the Adrenal Glands of Control Animals
By in situ hybridization with CyP11B2 anti-sense probe, staining was observed in parenchymal cells of the zona glomerulosa only, clustered within the first two to three subcapsular cell layers (Figure 1a). No staining was observed in the capsule, the inner parts of the cortex, or in the medulla. This demonstrates the specificity of the probe used, because no crossreaction with CYP11B1 mRNA occurred.
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In contrast, StAR anti-sense probe revealed only a faint and inhomogeneous staining in the parenchymal cells of the zona glomerulosa and intermedia (the outermost five to seven cell layers) (Figure 1b), with a slight preference for the subcapsular cell layer. An intense homogeneous signal was observed in parenchymal cells of the zona fasciculata and zona reticularis. In the adrenal medulla, some single cells or groups of two to three cells stained positive for StAR mRNA (Figure 1c), whereas CYP11B2 always was negative in this area.
Effect of ANGII on Expression of StAR and CYP11B2
Under stimulation with ANGII, no significant change in StAR mRNA was observed in the outer adrenal cortex (Figure 2a and Figure 2b), while the signal per cell for CYP11B2 mRNA became more intense and the number of positive cells increased (Figure 2c and Figure 2d). There was no increase in width of the aldosterone-producing zone.
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Effect of Nephrectomy on Expression of StAR and CYP11B2
The prominent increase of plasma aldosterone concentrations by nephrectomy was accompanied by an intense staining for StAR mRNA in the zona glomerulosa and intermedia (control, Figure 3a*; nephrectomy, Figure 3b*). The entire outer cortex became positive to a degree similar to that of the zona fasciculata and zona reticularis (Figure 3b). Furthermore, after nephrectomy we observed that an area of five to seven cell layers now stained intensely positive for CYP11B2 (Figure 3c*), whereas the zona fasciculata/reticularis and medulla still remained without staining (Figure 3c).
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Discussion |
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StAR protein is an important factor in steroidogenesis, because there is evidence that the delivery of cholesterol to the inner mitochondrial membrane is the rate-limiting step of steroidogenesis (
In control rats, the StAR gene was expressed with high intensity in the zona fasciculata and zona reticularis. The zona glomerulosa and the intermediate zone exhibited only faint staining, if at all. This is in contrast to the human adrenal, in which no considerable difference between zona glomerulosa and zona fasciculata was observed by immunohistological means, whereas the zona reticularis stained to a lower extent than the outer regions (-hydroxylase has been observed in single cells or groups of cells in the medulla of pig and human, some being in connection to the adrenal cortex. The detection of StAR mRNA in the rat adrenal medulla supports the view that steroids are produced at that site and suggests interactions with chromaffin cells.
The pattern of StAR expression throughout the cortex and its upregulation by bilateral nephrectomy provides us with important information regarding the role of the intermediate zone. Adaptation processes in the adrenal cortex were believed to occur slowly and to involve proliferation of cells. Recently,
Associated with a 50-fold elevation of plasma aldosterone concentration, together with the rise of StAR mRNA, the CYP11B2-positive region increased from one or two to five to seven cell layers. This demonstrates that an increase in width of the aldosterone-producing zone can happen within a short period of time and that all cells in the intermediate zone can become steroidogenically active. It also suggests that the increase in glomerulosa width in our experiment is solely the result of activation of previously quiescent cells and not of proliferation.
Surprisingly, ANGII administration did not induce a notable increase of StAR mRNA, whereas it stimulated CYP11B2 expression considerably. A stimulation of StAR protein by ANGII in vitro has been reported (
In summary, we have demonstrated changes in the distribution pattern of StAR mRNA in the rat adrenal gland after acute stimulation in vivo and concomitant changes in aldosterone production. The abundance of StAR mRNA in all layers of the outer adrenal cortex after stimulation confirms the function of the intermediate zone as a reservoir of steroidogenic cells.
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Acknowledgments |
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Supported by grant Pe 366/3-2 from the Deutsche Forschungsgemeinschaft and by the Forschungsfond der Fakultät für Klinische Medizin Mannheim of the University of Heidelberg.
We thank Hiltraud Hosser for technical support and I. Ertel for photographic work.
Received for publication July 30, 1998; accepted August 4, 1998.
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