Localisation of 11β-Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular Nuclei

Abstract

An accumulating body of evidence suggests that the activity of the mineralocorticoid, aldosterone, in the brain via the mineralocorticoid receptor (MR) plays an important role in the regulation of blood pressure. MR was recently found in vasopressin and oxytocin synthesising magnocellular neurosecretory cells (MNCs) in both the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus. Considering the physiological effects of these hormones, MR in these neurones may be an important site mediating the action of aldosterone in blood pressure regulation within the brain. However, aldosterone activation of MR in the hypothalamus remains controversial as a result of the high binding affinity of glucocorticoids to MR at substantially higher concentrations compared to aldosterone. In aldosterone-sensitive epithelia, the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) prevents glucocorticoids from binding to MR by converting glucocorticoids into inactive metabolites. The present study aimed to determine whether 11β-HSD2, which increases aldosterone selectivity, is expressed in MNCs. Specific 11β-HSD2 immunoreactivity was found in the cytoplasm of the MNCs in both the SON and PVN. In addition, double-fluorescence confocal microscopy demonstrated that MR-immunoreactivity and 11β-HSD2-in situ hybridised products are colocalised in MNCs. Lastly, single-cell reverse transcriptase-polymerase chain reaction detected MR and 11β-HSD2 mRNAs from cDNA libraries derived from single identified MNCs. These findings strongly suggest that MNCs in the SON and PVN are aldosterone-sensitive neurones.

Keywords: oxytocin; vasopressin.

Figure 1

Immunocytochemical localisation of mineralocorticoid receptor (MR) in coronal section of the hypothalamus from Wistar–Kyoto (WKY) and Wistar rats. (a, b) Prominent MR‐immunoreactive neurones were observed only in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) within the hypothalamic region in both WKY rats (a) and Wistar rats (b). (c, d) MR‐immunoreactivity in the SON from a WKY rat (c) and a Wistar rat (d). (e) The intensity of the MR‐labelling in the SON and PVN was measured as the optical density (OD) and normalised to OD values of nonspecific staining areas outside the SON or PVN. The mean normalised OD value of the SON from WKY rats was significantly higher than that from Wistar rats (n = 6 for both strains; *P = 0.0021). (f) Relative expression of MR‐mRNA in the SON and PVN was significantly higher in WKY rats than in Wistar rats (n = 6 for both strains; *P = 0.0271). (g) Immunocytochemical localisation of MR was also performed in the kidney. An intense MR immunoreactivity was present in the tubules where the cellular morphology and location resembles that of the distal tubules of the kidney cortex (arrows). In the kidney medulla, a strong 11β‐hydroxysteroid dehydrogenase type 2 immunoreactivity was present in collecting ducts (arrowheads). OC, optic chiasm. Scale bar = 250 μm in (b), 50 μm in (d) and 1 mm in (g).

Figure 2

Colocalisation of mineralocorticoid receptor (MR) immunoreactivity with both vasopressin (VP)‐neurophysin (NP) and oxytocin (OT)‐NP immunoreactivity in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) from a Wistar–Kyoto (WKY) rat. All confocal photomicrographs are in a coronal 3‐μm optical section. MR‐immunoreactivity was labelled with DyLight 488‐conjugated secondary antibody (pseudo‐coloured in green). VP‐NP and OT‐NP immunoreactivities were labelled with DyLight 649‐conjugated secondary antibody (pseudo‐coloured in red). (a, d) MR‐immunoreactivity in the SON. Most magnocellular neurosecretory cells (MNCs) in the SON appeared to possess some degree of MR‐immunoreactivity in their cell bodies. (b) VP‐NP immunoreactivity in the same section and optical plane as in (a). (c) A merged image of (a) and (b) demonstrated that all the VP‐NP immunoreactive MNCs retain MR‐immunoreactivity. (e) OT‐NP immunoreactivity in the same section and image plane as in (d). (f) A merged image of (d) and (e) demonstrated that all OT‐NP immunoreactive MNCs possess MR immunoreactivity. (g, j) MR‐immunoreactivity in the PVN. Although the MR‐immunoreactivity was not robust as in the SON, a prominent MR immunoreacitivty was observed in MNC within the PVN. Most of these MR‐immunoreactive MNCs are located in a cluster of cells in the posterior magnocellular region. (h) VP‐NP immunoreactivity in same section and image plane as in (g). Note that the VP‐NP immunoreactive cells form a cluster in the lateral portion of the PVN. (i) A merged image of (g) and (h) showed that all VP‐NP immunoreactive MNCs expressed MR‐immunoreactivity. (k) OT‐NP immunoreactivity in same section and image plane as in (j). (l) Merged images of (j) and (k) revealed that MR‐immunoreactivity is colocalised with OT‐NP immunoreactivity within MNCs in the PVN. Scale bar = 50 μm in (a) and 100 μm in (g).

Figure 3

Immunocytochemical localisation of 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) in sections of the hypothalamus and kidney from a Wistar–Kyoto (WKY) rat. (a) 11β‐HSD2 immunoreactivity was observed in essentially all neuronal nuclei in the brain section. This ubiquitous nuclear labelling was most likely nonspecific labelling; however, the immunoreactivity was dense in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). (b) In the SON, magnocellular neurosecretory cells (MNCs) containing cytoplasmic immunoreactivity were observed. Note that cells outside of the SON are devoid of cytoplasmic immunoreactivity. (c) In the PVN, the cells possessing cytoplasmic immunoreactivity were mostly located in the posterior magnocellular cluster. (d) A higher power view demonstrates cytoplasmic immunoreactivity in MNCs in the PVN. (e) Immunocytochemical localisation of 11β‐HSD2 was also performed in the kidney. In the kidney cortex, an intense 11β‐HSD2 immunoreactivity was present in the tubules for which the cellular morphology and location resemble that of the distal tubules (arrows). In the kidney medulla, a strong 11β‐HSD2 immunoreactivity was present in collecting ducts (arrowheads). OC, optic chiasm. Scale bar = 500 μm in (a) and (e), 50 μm in (b–d).

Figure 4

In situ hybridisation detection of 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) mRNA in the hypothalamus of a Wistar–Kyoto (WKY) rat. (a) Weak ubiquitous labelling of cell nuclei was observed elsewhere in the brain section; however stronger hybridisation labelling was seen in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) in which cytoplasmic labelling were present in neurones. (b) Within the PVN, more prominent cytoplasmic labelling was observed in the posterior magnocellular region; however, noticeable cytoplasmic labelling was also observed in the parvocellular regions as well. (c) Discrete cytoplasmic labelling was found in magnocellular neurosecretory cells (MNCs) in the SON. (d) The sense probe that was processed in parallel with the experimental bran sections did not produce detectable labelling in the SON. (e) In situ hybridisation of 11β‐HSD2 was also performed in the kidney. The expression pattern of the in situ labelling in the kidney was resembled to that of immunoreactivity (Fig. 3 e). An intense hybridisation labelling was present in the distal tubules in the cortex (arrows) and in the collecting ducts in the medulla region (arrowheads). OC, optic chiasm. Scale bar = 500 μm in (a), 100 μm in (b), 50 μm in (c) and (d), and 1 mm in (e).

Figure 5

Double fluorescent labelling of 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) in situ hybridisation and mineralocorticoid receptor (MR) immunocytochemistry. MR immunoreactivity was labelled with DyLight 488‐conjugated secondary antibody (pseudo‐coloured in green). (a, d) Intense immunoreactivity to MR was observed in the MNCs in the paraventricular nucleus (PVN) (a) and supraoptic nucleus (SON) (d). (b, e) 11β‐HSD2 hybridisation materials were labelled with DyLight 649‐conjugated secondary antibody (pseudo‐coloured in blue) in same section and image plane as in (a) and (d). The 11β‐HSD2 hybridisation materials were observed in the magnocellular neurosecretory cells (MNCs) in the PVN (b) and SON (e). (c, f) The merged images revealed that all MR immunoreactivity was co‐localised with vasopressin (VP) 11β‐HSD2 hybridisation materials within the MNCs in the PVN (c) and SON (f). (g, h) Confocal images at 1 μm optical section acquired with a × 100 objective lens were obtained to observe the subcellular distribution of MR immunoreactivity (g) and 11β‐HSD2 hybridisation labelling (h) in the SON MNCs. Intense MR immunoreactivity was clumped in the perinuclear zone of the cytoplasm with a distinct granular appearance along with more diffuse immunoreactivity within the entire cytoplasm (g). Strong 11β‐HSD2 hybridisation labelling was observed in the peripheral region of the cytoplasm (h). (i) A merged image demonstrated that MR immunoreactivity was co‐localised with 11β‐HSD2 hybridisation product in MNCs, although their subcellular distributions were different from each other. Scale bars = 50 μm.

Figure 6

Single cell reverse transcriptase‐polymerase chain reaction detection of transcripts in individual dissociated magnocellular neurosecretory cells (MNCs) from a Wistar–Kyoto (WKY) rat. Libraries of cDNA were derived from twelve cells dissociated from punched supraoptic nucleus (SON) tissue from a WKY rat. All cells had mRNA for vasopressin (VP) and oxytocin (OT), confirming they were MNCs. Of these MNCs, mineralocorticoid receptor (MR) mRNA was found in MNCs except #3 and 7; 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) mRNA was found in cells #1, 4, 7, 8 and 10. Thus, mRNAs for MR and 11β‐HSD2 were colocalised in four MNCs (#1, 4, 8 and 10) out of 12 MNCs isolated. Both MR and 11β‐HSD2 mRNAs were found in cDNA library derived from punched SON tissues. M, Marker; N, Negative control.