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Department of Pharmacology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA
1 Department of Pathology,
2 Division of Biocomputing, Department of Biochemistry and Molecular Biology and
3 Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA
(Requests for offprints should be addressed to E Brailoiu; Email: ebrailou{at}temple.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß (ER and ERß ), which function as transcription factors (Jensen & DeSombre 1973, Mosselman et al. 1996). However, a number of cell responses are induced in seconds to minutes following estrogen stimulation, for example, nitric oxide generation, calcium mobilization, and activation of tyrosine kinases (Morley et al. 1992, Brubaker & Gay 1999, Karpuzoglu et al. 2006). For this reason, the existence of non-genomic ERs mediating rapid cell signaling has been proposed in a number of studies (Lagrange et al. 1997, Pietras & Szego 1977, Singh et al. 2000, Toran-Allerand et al. 2002, Morales et al. 2003, Qiu et al. 2003). Two groups independently proposed that the G protein-coupled receptor (GPR 30), is the non-genomic ER (Revankar et al. 2005, Thomas et al. 2005). A high-affinity, displaceable, single binding site specific for estrogen was detected respectively in plasma membranes of SKBR3 breast cancer cells that express GPR30 but lack nuclear ERs, and in COS7 cells transfected with GPR30 (Revankar et al. 2005, Thomas et al. 2005). As GPR30 is expressed in cell lines in these studies (Revankar et al. 2005, Thomas et al. 2005), the distribution of GPR30 in the brain has not been investigated. An earlier study demonstrates that COS7 cells transfected with GPR30 responded to estrogen with a large influx of Ca2+ (Revankar et al. 2005). The present study was undertaken to determine the distribution of GPR30 in the central nervous system of adult rats of either sex, and to characterize the calcium response of native GPR30 in dissociated and cultured rat hypothalamic neurons in response to the GPR30-specific ligand G-1 (Bologa et al. 2006).
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Materials and Methods |
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Adult male and female Sprague–Dawley rats, weighing 225–250 g (Ace Animals Inc., Boyertown, PA, USA), were used in this study. Animal protocols were reviewed and approved by the Institution Animal Care and Use Committee.
Animals were anesthetized with urethane (1.2 g/kg, i.p.) and intracardially perfused with 0.1 M PBS followed by 4% paraformaldehyde/0.2% picric acid in PBS. Brains and pituitary glands were removed, post-fixed for 2 h, and stored in 30% sucrose/PBS solution overnight.
In single-staining, tissues were processed for GPR30-immunoreactivity (irGPR30) by the avidin–biotin complex procedure (Brailoiu et al. 2005). Tissues were first treated with 3% H2O2 to quench endogenous peroxidase, washed several times, blocked with 10% normal goat serum, and incubated in GPR30 antiserum (1:1000 dilution). GPR30 antiserum was a rabbit polyclonal antibody directed against the human C-terminus GPR30 (CAVIPDSTEQSDVRFSSAV; Revankar et al. 2005). The specificity of GPR30 antiserum has been previously confirmed (Revankar et al. 2005). After thorough rinsing, sections were incubated in biotinylated anti-rabbit IgG (1:150 dilution, Vector Laboratories, Burlingame, CA, USA) for 2 h, rinsed with PBS, and incubated in avidin–biotin complex solution for 1 h (1:100 dilution, Vector Laboratories). Following several washes in Tris-buffered saline, sections were incubated in 0.05% diaminobenzidine/0.001% H2O2 solution and washed for at least 2 h with Tris-buffered saline. Sections were mounted on slides with 0.25% gel alcohol, air-dried, dehydrated with absolute alcohol followed by xylene, and coverslipped with Permount. For control experiments, sections were processed with pre-immune serum in the place of GPR30 antiserum.
In the case of double-labeling experiments, hypothalamic and medullary sections were first blocked with normal goat serum (1:10 in PBS, 0.5% BSA, 0.4% Triton X- 100) and then incubated with GPR30 antiserum (1:500 dilution). Following several washes with PBS, sections were incubated with biotinylated anti-rabbit IgG (1:50, Vector Laboratories), washed with PBS, and incubated with fluorescein isothiocyanate (FITC). After rinsing with PBS, the hypothalamic sections were incubated with either oxytocin (OT) antibody (1:1000, a monoclonal antibody from Chemicon, Tamecula, CA, USA) or vasopressin (VP) antiserum (1:1500, a guinea pig polyclonal antibody from Chemicon); medullary sections were incubated with choline acetyltransferase (ChAT) antiserum (1:1000; a guinea pig polyclonal antibody from Bachem Bio Sciences Inc., King of Prussia, PA, USA) for 48 h in a cold room. In the case of double labeling with the neuronal marker neuron-specific enolase, cultured hypothalamic neurons were fixed in 4% paraformaldehyde/0.2% picric acid, blocked with 10% normal goat serum, and incubated with GPR30 antiserum (1:500 dilution). They were washed with PBS and incubated with biotinylated anti-rabbit IgG, followed by avidin FITC. After rinsing with PBS, the cells were blocked with normal donkey serum and incubated with neuron-specific enolase antiserum (1:150, a mouse monoclonal antibody from Chemicon). Tissues and cells were incubated in Avidin Texas Red conjugated to appropriate secondary antibodies, washed with PBS, and mounted in Citifluor and coverslipped. Sections and cells were examined under a confocal scanning laser microscope (Leica Microsystems Inc., Exton, PA, USA) with excitation/emission wavelengths set to 488/520 nm for FITC and 543/620 nm for Texas Red in the sequential mode.
Neuronal cell culture
Cells were isolated from the hypothalamus of postnatal 1- to 3-day-old rats by enzymatic digestion with 0.5 mg papain/100 mg tissue (Dun et al. 2006). Cells were plated at a density of 103/mm2 in a Neurobasal-A medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µ g/ml streptomycin (Invitrogen), and maintained at 37 ° C in a humidified atmosphere with 5% CO2. Glial cell growth was inhibited by the mitotic inhibitor cytosine ß-arabino furanoside (1 µ M; Sigma). Neurons were cultured for 5 days. Cells were transferred to a medium without fetal serum 12 h prior to Ca2+ measurements.
Cytosolic Ca2+ concentrations
Cytosolic Ca2+ measurements were performed as described previously (Brailoiu et al. 2006). Cells were incubated with 5 µ M Fura-2 AM (Molecular Probes, Eugene, OR, USA) in Hanks’ balanced salt solution (HBSS) at room temperature for 45 min in the dark, washed thrice with dye-free buffer, and then incubated for another 45 min to allow for complete de-esterification of the dye. Coverslips were subsequently mounted in a custom-designed bath on the stage of an Eclipse TE 2000-U Nikon inverted microscope equipped with a Roper Scientific CCD camera (Optical Apparatus Co., Ardmore, PA, USA). Cells were routinely superfused with HBSS at a flow rate of 1 ml/min. Fura-2 fluorescence (emission = 510 nm), following alternate excitation at 340 and 380 nm, was acquired at a frequency of 0.33 Hz. Images were acquired and analyzed using Metafluor software. For Ca2+ -free experiments, HBSS without Ca2+ supplemented with 2.5 mM EGTA was used.
Chemicals
G-1 was a gift from Chemical Diversity Inc. (San Diego, CA, USA).
Statistical analysis
In calcium measurement experiments, statistical significance between groups was tested using one-way ANOVA followed by Bonferroni test, P < 0.05 being considered significantly different.
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Results |
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Examination of sections prepared from four female and five male rats revealed a similar pattern of distribution of irGPR30 in the brain and pituitary. Results described below are derived from male rats unless stated otherwise. The nomenclature follows that used in Paxinos & Watson (1998).
Forebrain
A high density of irGPR30 was detected in the Islands of Calleja (ICj), major island (ICjM), ventral pallidum (VP), and olfactory tubercle (Tu); a lesser density of irGPR30 was present in the nucleus of vertical limb diagonal band (VDB; Fig. 1A–D
). A high density of irGPR30 was detected in the rostrocaudal extent of lateral globus pallidus (Fig. 1E and F). In the thalamus, irGPR30 was detected in the reticular thalamic nucleus (Fig. 1E) and medial habenular nucleus (not shown).
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). Within the PVN, irGPR30 was identified mostly in the lateral magnocellular part (PaLM), medial parvicellular part (PaMP), ventral part (PaV), dorsal cap (PaDC), and less in the anterior and posterior parvicellular parts (Fig. 2B). Few cells in the suprachiasmatic nucleus, retrochiasmatic nucleus, and periventricular hypothalamic nucleus were immunoreactive (Fig. 2A). The arcuate nucleus showed a moderate number of irGPR30 cells (not shown).
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. Pituitary gland
With respect to the pituitary gland, the anterior pituitary contained a large number of immunoreactive cells (Fig. 2E); numerous terminal-like endings were irGPR30 in the posterior pituitary (Fig. 2F).
Hippocampus
Cells in the hippocampal formation including CA1, CA2, CA3 regions and dentate gyrus (DG) were irGPR30 (Fig. 3A and B).
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At the level of pons, a high concentration of irGPR30 was seen in the substantia nigra reticular and compact parts (SNR, SNC; Fig. 3C and D). In the medulla, positively labeled cells were noted in the area postrema (AP), dorsal motor nucleus of the vagus (10), nucleus of the solitary tract (Sol), and hypoglossal nucleus (12, Fig. 4A and B). A cluster of irGPR30 cells was conspicuously present in the nucleus ambiguus (Amb; Fig. 4C–D). In addition, irGPR30 cells were distributed sparsely throughout the reticular formation (Fig. 4A).
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Double-labeling of the hypothalamic sections with GPR30-antiserum and OT or VP antiserum revealed that ~40 and 32% irGPR30 cells were OT-positive, and about 60 and 70% irGPR30 cells were VP-positive, in the hypothalamic paraventricular and SOs respectively (Fig. 5A–D). In the medulla oblongata, nearly all irGPR30 cells in the Amb, dorsal motor nucleus of the vagus, and hypoglossal nucleus were ChAT-positive (Fig. 5E and F). With respect to cultured hypothalamic neurons, irGPR30 was co-localized with neuron-specific enolase (Fig. 6A and B), affirming that irGPR30 was expressed in cultured neurons.
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Administration of G-1 (100 nM), the ligand that selectively activates GPR30 but not the nuclear ERs ER and ERß (Bologa et al. 2006), induced an increase in [Ca2+ ]i by 964 ± 8.6 nM in 26 of 109 cells tested; an example is shown in Fig. 7A. The rise of [Ca2+ ]i was rapid and sustained, reaching a plateau in about 3–6 min. In hypothalamic neurons perfused with a Ca2+ -free medium, G-1 (100 nM) elevated [Ca2+]i by 58 ± 2.2 nM (n = 6); a representative experiment is shown in Fig. 7B.
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Discussion |
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and ERß . Using an antiserum directed against the human C-terminus GPR30, irGPR30 was detected in the Tu, hypothalamus, hippocampal formation, substantia nigra, medulla oblongata, and pituitary. As tissues processed with pre-immune serum, instead of GPR30 antiserum, resulted in no positive labeling, the irGPR30 appears to be specific. Further, the expression pattern of GPR30 (or CMKRL2 or CEPR) mRNA in human brain tissues (Owman et al. 1996, Feng & Gregor 1997, O’Dowd et al. 1998) is similar to the receptor distribution profile observed here in the rat brain. For example, GPR30 mRNA is highly expressed in the Tu, hippocampus, supraoptic, paraventricular, arcuate, and suprachiasmatic nuclei; the signal is less abundant in the cortex and caudate nucleus (O’Dowd et al. 1998). CEPR mRNA is highly expressed in the caudate nucleus, hippocampus, substantia nigra, subthalamic nucleus, thalamus, hypothalamus, medulla oblongata, and spinal cord (Feng & Gregor 1997).
Our results show that the pattern of irGPR30 expression is similar in the adult male and female rat brain. This is in agreement with the report in which a gender difference relative to the distribution of ER and ERß is not observed in the rat brain (Laflamme et al. 1998). Several areas, such as the ICj and Tu, express ER and ERß (Laflamme et al. 1998) as well as irGPR30. Other areas, for example, the substantia nigra and AP express irGPR30 and ER mRNA, whereas the paraventricular hypothalamic and supraoptic nuclei express irGPR30 and ERß mRNA. Several regions including the amygdala, ventromedial hypothalamic nucleus, and ventral premammillary nucleus, where a strong hybridization signal to ER and ERß mRNA is detected (Laflamme et al. 1998), contain little or no irGP30. In spite of partial overlapping with one or the other ER mRNA in different brain regions, our results indicate that the pattern of irGPR30 expression is distinct from that of ER and ERß in the rat brain.
The co-localization study provides information relative to the phenotype of cells expressing irGPR30 in the hypothalamus and brainstem. Even if GPR30 and ERß are present in the paraventricular hypothalamic nucleus, they seem to be localized in different cell populations: ERß is reported to be highly (60–80%) expressed in corticotropin releasing factor-positive cells, whereas, the percentage of hypothalamic neurons expressing both ERß and VP varies with different studies, ranging from 5% (Laflamme et al. 1998) to 88–99% (Hrabovszky et al. 2004). In our study, approximately 60% of irGPR30 neurons expressed VP in the paraventricular hypothalamic nucleus. Co-expression of OTand ERß is reported to be about 40% (Laflamme et al. 1998), which is similar to the percentage of co-localization of irGPR30 and OT in the paraventricular hypothalamic nucleus. Estrogens regulate the activity of OT (Akaishi & Sakuma 1985) and VP neurons (Hartley et al. 2004), and activation of GPR30 could be one of the signaling pathways. With respect to the nucleus Amb, irGPR30 neurons are ChAT-positive, thus are cholinergic.
There is evidence that GPR30 is localized to the endoplasmic reticulum of COS7 cells transfected with the receptor (Revankar et al. 2005). In another study, GPR30 is shown to be localized to the plasma membrane of human breast cancer cells (Thomas et al. 2005). A more recent study shows that GPR30 is localized to the plasma membrane of rat hippocampal CA2 neurons, and in GPR30-transfected HeLa cells the receptor is translocated to the cytoplasm upon agonist stimulation (Funakoshi et al. 2006). In the case of our immunohistochemical study, irGPR30 seems to be distributed throughout the cytoplasm in some of the neurons; e.g. dorsal motor nucleus of the vagus (DMNV; Fig. 4B) and Amb (Figs 4D and 5E). On the other hand, irGPR30 may be affiliated with plasma membranes of hypothalamic and hippocampal neurons (Figs 2B, C and 3B). Immunofluorescent staining clearly shows GPR30 is localized intracellularly; i.e. perinuclear in cells shown in Figs 5A and 6A, and dispersed throughout the cytoplasm in cells illustrated in Fig. 5E. Electron microscopic examination will be needed to resolve the issue of receptor localization. The possibility of a cell type-specific or state-dependent localization of GPR30 is intriguing.
The physiological consequence of activation of GPR30 in a specific neural axis is largely unknown. In addition to its well-documented hormonal action, estradiol is being considered as a neurotransmitter in the brain (Balthazart & Ball 2006). Estradiol by intrahippocampal injection enhanced spatial memory in male rats (Packard et al. 1996). Since the expression of ER and ERß mRNA in the hippocampus is limited (Laflamme et al. 1998), GPR30 may prove to be the major receptor subtype through which estrogen produces its enhancing effect.
In the midbrain, distribution of irGPR30 appears to be restricted to the substantia nigra, compact and reticular parts. ER is detected in low levels in the substantia nigra compact part of the rat (Laflamme et al. 1998). Estrogen is reported to have a protective role on dopaminergic neurons in a mouse model (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced) of Parkinson’s disease. However, both the ER and ERß appear to be absent in neurons of the substantia nigra of the mouse (Shughrue 2004). Viewed in this context, the neuroprotective effect of estrogen on dopaminergic neurons may be related to activation of GPR30 and independent of nuclear receptors.
With respect to the brainstem, irGPR30 is detected in the AP, nucleus of the solitary tract, nucleus Amb, and dorsal motor nucleus of the vagus. These nuclei are directly or indirectly associated with autonomic regulation of cardiovascular/gastrointestinal activity. Moreover, estradiol administered directly to the nucleus Amb increased the phenylephrine-induced baroreflex within 5 min of injection (Saleh et al. 2000). A participation of GPR30 in this response may be anticipated.
Similar to the COS7 cells transfected with GPR30 (Revankar et al. 2005), G-1 elicited a large increase of [Ca2+]i in dissociated and cultured hypothalamic neurons. Under physiological conditions, circulating estrogens are bound to the carrier proteins, sex hormone-binding globulin, and albumin (Anderson 1974); however, only free estrogen is thought to enter the cell (through diffusion). Unlike the nuclear ERs that activate transcription, protein-bound estrogens mediate non-nuclear receptor signaling (Ramirez et al. 1996, Zheng et al. 1996, Kahn et al. 2002). Estradiol-BSA (E2-BSA) has been shown to elevate [Ca2+]i through non-genomic ERs in several cell models; for example, E2-BSA elevates [Ca2+ ]i in gonadotropin-releasing hormone-1 neurons (Temple & Wray 2005), human granulocytes (Stefano et al. 2000a), and cultured human pre-osteoclastic cells (Fiorelli et al. 1996). Fast calcium mobilization by E2-BSA has also been reported in rat aorta endothelial cells (Rubio-Gayosso et al. 2000), as well as in thoracic and human arterial endothelia (Stefano et al. 2000b). The observation that G-1, the GPR30 selective agonist (Bologa et al. 2006), rapidlyelevates [Ca2+]i in hypothalamic cells is consistent with a non-nuclear effect of estrogen. Another novel observation is that the Ca2+ response elicited by a second application of G-1 was significantly smaller as compared with that evoked by the first application, indicating that the non-genomic ER undergoes rapid desensitization. Calcium mobilization and receptor desensitization are two common features of GPRs. Viewed in this context, our observations support the thesis that GPR30 is a GPR.
In a Ca2+ -free medium, G-1 causes an elevation of [Ca2+ ]i with a magnitude much smaller than that elicited in a Ca2+ -containing medium, suggesting that activation of GPR30 mobilizes extracellular as well as intracellular Ca2+ stores. Increase of [Ca2+]i in neurons may lead to neurotransmitter release (Brailoiu et al. 2003), modulation of neurite outgrowth (Jacques-Fricke et al. 2006), and hormone secretion (Stojilkovic et al. 2005). Activation of GPR30 by estrogen may influence cell physiology according to the type of cells in question.
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Acknowledgements |
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References |
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Received in final form 21 February 2007
Accepted 28 February 2007
Made available online as an Accepted Preprint 6 March 2007
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