ARTICLE |
Correspondence to: Isabel Siegle, Auerbachstr. 112, 70376 Stuttgart, Germany. E-mail: isabel.siegle@ikp-stuttgart.de
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Summary |
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Prostacyclin (PGI2) is a labile, lipid-derived metabolite of arachidonic acid synthesized through the sequential action of cyclo-oxygenase (COX) and prostacyclin synthase (PGIS). In addition to its well-characterized vasodilatory and thrombolytic effects, an increasing number of studies report an important role of PGI2 in nociception in various animal species. In this study we investigated the regional distribution of PGIS in human brain by immunohistochemistry and in situ hybridization. PGIS-immunoreactive (ir) protein was localized to blood vessels throughout the brain. Neuronal cells and glial cells, such as microglia and oligodendrocytes, also showed intense labeling. The strongest expression of PGIS was seen in large principal neurons, such as pyramidal cells of the cortex, pyramidal cells of the hippocampus, and Purkinje cells of the cerebellum. Abundance of PGIS mRNA was observed in blood vessels and large neurons and correlated well with the immunohistochemical findings. The expression of PGIS in human brain was further demonstrated by immunoblotting and detection of 6-keto-PGF 1, the stable degradation product of prostacyclin in human brain homogenate. These results demonstrate a widespread expression of PGIS in the central nervous system and suggest a potentially important role of prostacylin in modulating neuronal activity in human brain. (J Histochem Cytochem 48:631641, 2000)
Key Words: prostacyclin synthase, central nervous system, nociception, in situ hybridization, immunohistochemistry
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Introduction |
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Prostacyclin (PGI2) belongs to the family of prostanoids, which are labile bioactive lipids acting as local hormones (
Lately, there is new interest in the role of prostanoids in the CNS owing the discovery of the inducible isoenzyme of cyclo-oxygenase (COX-2). Although cyclo-oxygenase-1 (COX-1) is present under normal conditions in most cells, COX-2 is the predominant isoform in brain. In contrast to most other tissues, COX-2 is expressed under physiological conditions in forebrain neurons, particularly in neocortex, amygdala, hippocampus, and limbic cortices (
In this study we have investigated by immunohistochemistry and in situ hybridization the in vivo regional and cellular distribution of PGIS in various areas of the human brain, including frontal cortex, hippocampus, cerebellum, medulla oblongata, substantia nigra, and thalamus.
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Materials and Methods |
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Postmortem Specimens
Tissue blocks from brains of three donors with no history of neurological illness were obtained from the Department of Pathology of the Robert Bosch Hospital (Stuttgart, Germany). The underlying illnesses were chronic respiratory insufficiency (female, 42 years) and illicit drug consumption (males, 33 years and 53 years). Postmortem delay time ranged from 20 to 26 hr. Tissue samples were fixed in buffered 4% formalin for 24 hr, dehydrated, and embedded in paraffin (Paraffin Plus; Shandon, Pittsburgh, PA). Sections were cut (3 µm) and mounted on glass slides. Fresh brain samples were also obtained from cortical and hippocampal surgical waste collected perioperatively from three patients with epilepsy. The removed specimens were immediately frozen in liquid nitrogen and stored at -80C before use as positive control for activity assays and Western blotting analysis.
Activity Assay of PGIS
PGIS activity in human brain tissues was determined by two independent methods. The conversion of 100 µM [14C]-PGH2 to the various [14C]-prostanoids in brain homogenates was measured by thin-layer chromatography (TLC) as described previously (
Second, PGIS activity in brain preparations was measured by enzyme-linked immunoassay (EIA) using 30 µM PGH2 as substrate, according to instructions of the supplier (SPI Bio). As above, the amount of spontaneously formed 6-keto-PGF1 in buffer was subtracted from the individual samples.
Anti-PGIS Antibody
Anti-human PGIS polyclonal antibodies were used for immunohistochemical detection of PGIS (
Immunoprecipitation of PGIS
Extracts of 3 mg solubilized brain homogenates were precleared by addition of 40 µl of protein A/Sepharose CL-4B and the supernatant was incubated (18 hr, 4C) with 5 µg of the polyclonal antibody against PGIS (
Western Blotting
Precipitated proteins were separated by 7.5% SDS-PAGE (30 mA, 1 hr) and blotted for 1 hr with a constant current of 200 mA onto a nitrocellulose membrane in a semidry blotting procedure (48 mM Tris/39 mM glycine/20% methanol/0.037% SDS). Proteins were visualized with a 0.1% Ponceau 5 solution in 5% acetic acid to check transfer efficiency. After destaining, the membrane was blocked with 5% milk powder in PBS/0.1% Tween-20 for 2 hr at room temperature (RT) and incubated with a monoclonal antibody against PGIS (Oxford Biochemical Research; 5 µg/ml). After washing three times with PBS/0.1% Tween-20, the membrane was further incubated with a goat anti-mouse antibody at a dilution of 1:7500 for 45 min. Antibody binding was visualized by the ECL technique (Amersham; Braun-schweig, Germany).
Immunohistochemical Staining for PGIS
Immunostaining was performed with a modification of the avidinbiotinperoxidase complex (ABC) technique (
To test the specificity of immunostaining, a 10-fold molar excess of the PGIS peptide was added to the anti-PGIS antibody solution 60 min before staining. No staining was observed. False-positive results originating from endogenous biotin (
Morphology
To characterize general cellular patterns, sections were stained with hematoxylin/eosin. In addition, double staining experiments for GFAP (glial fibrillary acidic protein, dilution 1:250; Progen, Heidelberg, Germany) and von Willebrandt factor (vW factor, dilution 1:400; Dako, Hamburg, Germany) and PGIS were performed. GFAP is a astroglia-associated antigen and vW factor is an endothelial cell marker. The double immunostaining was carried out with a sequential method. First the sections were stained with the polyclonal antibody against PGIS by the ABC technique. Color development was achieved by DAB/H2O2 with addition of 6.8 mg imidazole, 2 ml 1% nickel acetate, and 2.5 ml 1% CoCl2 (to each 100 ml of incubation solution), which produced a black color instead of brown. Thereafter, the sections were stained for GFAP or vW factor using an APAAP method with one repetition. The APAAP complex was developed with New Fuchsin and naphthol-AS-BI (red color) as described previously (
Generation of 35S-labeled Riboprobes and In Situ Hybridization
Antisense and sense probes for the human prostacyclin mRNA were prepared as follows. RT-PCR with the depicted primer pair was performed using total human kidney RNA. A PCR fragment (420 bp) was cloned into pCR 2.1 plasmid (Invitrogen; Groningen, The Netherlands). Cloned cDNA fragments were sequenced according to the dideoxy method to confirm identity and orientation of the inserts.
In situ hybridization was performed as described previously (
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Results |
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Western Blot Findings
Western blotting analysis was performed to demonstrate the presence of PGIS immunoprecipited with polyclonal anti-PGIS antibodies from human brain homogenates. In brain samples, monoclonal antibodies against PGIS recognized a single protein band of 52 kD (Fig 1 , Lanes 24), consistent with the molecular weight of partially purified PGIS from bovine aortic endothelium (Fig 1, Lane 1). In general, the amount of immunoprecipited PGIS protein was unchanged in cortex of postmortem tissues (Fig 1, Lane 4) compared with the same areas of freshly obtained surgical material (Fig 1, Lanes 2 and 3, respectively).
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Incubation of solubilized brain extracts with PGH2, the immediate substrate of PGIS, caused significant production of 6-keto-PGF 1, the stable degradation product of prostacyclin, as shown in Fig 1. Compared to freshly obtained material, the activity of PGIS in postmortem tissues was decreased to 31% and 67% in cortex and hippocampus, respectively. This decline in enzymatic activity was most likely related to inactivation of PGIS due to an ischemic period before death, as proposed by
from [14C]-PGH2 in freshly obtained cortical tissues. Considerably larger amounts of PGE2 (40.3 ± 5.5%) and PGD2 (19.3 ± 2.1%) were formed as the result of spontaneous decay of PGH2 in aqueous solutions. In good agreement with the Western blotting analysis, these results clearly indicated that PGIS is expressed in notable amounts in different regions of human brain.
Immunohistochemical Findings
Incubation of brain sections with an antibody raised against a peptide homologous to human PGIS caused staining of large and small blood vessels throughout the brain. In addition, neuronal cells, glial cells of presumed microglial identity, and oligodendrocytes were found to contain PGIS immunoreactivity in most areas of the brain. No immunostaining was detected in tissue sections when any of the crucial steps of the immunostaining procedure was omitted or competitive inhibition experiments with an excess of PGIS peptide were performed. Fig 2A shows PGIS-ir Purkinje cells in cerebellum. This staining of PGIS-positive cells was completely eliminated when pre-absorbed antibody was used as primary antibody (Fig 2B). The immunoreactivity in cerebral blood vessels appeared to be mainly located in endothelial cells (Fig 3A). Fig 3B shows double immunostaining for PGIS-like immunoreactivity and vW factor, an endothelial cell marker. All of the PGIS-positive structures overlapped in staining with vW factor-positive structures. In most vessels, however, vW factor-ir cells were further surrounded by PGIS-positive cells. This staining pattern was consistent with the well known expression of PGIS in endothelial cells as well as in underlying vascular smooth muscle cells (
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Immunolocalization of PGIS in frontal cortex showed widespread staining in all cortical laminae but occurred predominantly in the cortical Layers III/V and VI, in which large neurons are located (Fig 4A). In most PGIS-positive samples, intense immunostaining was detected in neurons, either pyramidal or fusiform. These cells displayed a characteristic PGIS-ir in the cytosol, sometimes extending towards dendritic processes but without nuclear staining ( Fig 4B). In some cases, immunoreactivity was also localized to the neuropil. Moderate staining was abundant in the majority of small cells, which morphologically appeared to be glial cells (Fig 4C). Hence, judging from the double staining experiment with PGIS and GFAP, a cell marker for astrocytes, it can be assumed that these cells are most likely microglial cells or oligodendrocytes rather than astrocytes (Fig 4D ). These findings are in contrast to those of
The hippocampus exhibited a distinct pattern of immunostaining for PGIS. Similar to the staining of cortical tissues, the immunoreactivity for PGIS was quite extensive. A dense accumulation of PGIS-positive neurons was found in granule cells of the dentate gyrus and in the pyramidal cell layer of Ammon's horn, starting with PGIS-positive CA3 neurons in the hilus of dentate gyrus and extending into the subiculum as a broad band of slightly to moderately stained immunoreactive CA1 neurons (Fig 5A and Fig 5B). Shorter and longer dendritic processes of these cells contained immunoreactivity ( Fig 5C). Moderate staining of glial cells was detected in the molecular layer of the dentate gyrus. In general, the staining intensity among the various cells was similar, but some neurons of the gyrus dentatus appeared to express more PGIS, as shown in Fig 5D.
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Immunostaining for PGIS in cerebellum was primarily located in Purkinje cell bodies and processes and in discrete cells of the molecular and granular layers. As in other regions of the brain, PGIS was expressed in neurons, whereas astrocytes in the Purkinje cell layer lacked detectable PGIS immunoreactivity (Fig 6A and Fig 6B). The small cells positive for PGIS in the Purkinje layer were closely associated with Purkinje cells, suggesting that these cells might be Bergmann glia. In the granular layer adjacent to the Purkinje cell layer, there were also occasional PGIS-positive cells (Fig 6C). In the molecular layer, staining for PGIS was present, most likely in stellate and basket cells (Fig 6D).
Strong immunostaining was also observed in motor neurons of the medulla oblongata (Fig 7A) and in the ependymal lining of the ventricular walls (Fig 7B). Furthermore, the melanin-containing cells in the substantia nigra showed PGIS-positive staining (not shown), as did the large neurons in the thalamus (Fig 7C).
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In Situ Hybridization Findings
To confirm the immunohistochemical data, we determined mRNA expression of PGIS using [35S]-cRNA. The following control experiments were performed to validate the specificity of in situ hybridization. (a) Hybridization with sense probe resulted in very low labeling with nonspecific distribution. (b) Pretreatment of sections with RNase A before hybridization completely prevented the appearance of positively stained cells (not shown). In general, the results of in situ hybridization corresponded well to the immunohistochemical findings. Expression of the PGIS gene was relatively strong in large neurons, with a distinct pattern throughout the cytoplasm, as shown in Fig 8A and Fig 8B for neurons in the frontal cortex. In contrast, only diffuse labeling was apparent in glial cells. However, significant mRNA staining was observed in the adventitia and media of cerebrovascular blood vessels, being equally intense in both layers (Fig 8C and Fig 8D), whereas in hippocampus the hybridization signal was most prominent over the dentate gyrus. However, the high cellularity of this structure and the scattering of the silver grains precluded a unequivocal identification of PGIS mRNA-positive cells (not shown).
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Discussion |
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In this study, expression of authentic PGIS in human brain is evidenced by four independent methods. (a) 6-Keto-PGF1, the stable breakdown product of PGIS activity, was detected after incubation of brain extract with the unlabeled and 14C-labeled precursor PGH2 by EIA and TLC methods. (b) Western blotting analysis using monoclonal anti-PGIS antibodies showed a band co-migrating with purified PGIS. (c) Pre-absorption of the polyclonal anti-PGIS antisera discriminated specific from nonspecific staining in brain tissues sections. Finally (d), the overall distribution the PGIS immunoreactivity matched the PGIS mRNA expression. It should be noted that PGIS mRNA was detected mainly in neurons, whereas only diffuse staining appeared in glial cells. Brain tissue samples were obtained 2026 hr postmortem. Therefore, it is possible that the prolonged time before fixation caused degradation of mRNA and hence a decrease in in situ hybridization signals. Our major finding is that most of the brain tissue samples analyzed exhibited PGIS-ir protein and mRNA expression in neuronal cell bodies and fibers and, to a lesser extent, in glial cells. Prominent immunostaining for PGIS was observed in a number of different neuronal cell types, including pyramidal cells in neocortex and CA3 pyramidal cells in hippocampus, acetylcholine-containing motor neurons in medulla oblongata, and dopaminergic neurons in substantia nigra.
Substantial evidence indicates that prostanoids such as PGE2, PGD2, and PGF2 play an important role in modulating neuronal functions, including those involving sleep, body temperature changes, and neuronal signaling (
Until now, only one IP receptor has been cloned (
Considerable evidence confirms that prostacyclin contributes to the control of cerebral blood flow (CBF) and circulation. For example, intracarotid infusion of PGI2 in baboons increased CBF (
The results obtained in this study show a widespread expression of prostacyclin synthase in human brain tissues. Although the specific function of PGIS still remains to be established, these findings suggest that prostacyclin might play an important role in neuronal activity.
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Footnotes |
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1 Present address: Biochemistry Department, Byk Gulden, Konstanz, Germany.
2 Present address: Vanderbilt University School of Medicine, Nashville, Tennessee.
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Acknowledgments |
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Supported by the Robert Bosch Foundation.
We thank Dr M. Breyer for use of equipment and advice on performing in situ hybridization.
Received for publication September 7, 1999; accepted December 27, 1999.
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