54
Brain Research, 538 (1991) 54-53 Elsevier
BRES 16206
A vasoactive peptide, endothelin-3, is produced by and specifically binds to primary astrocytes Hannelore Ehrenreich 1, John H. Kehrl 1, Robert W. Anderson 2, Peter Rieckmann 1, Ljubisa Vitkovic 1, John E. Coligan 2 and Anthony S. Fauci ~ 1Laboratory of Immunoregulation, and 2Biological Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Accepted 31 July 1990) Key words: Astrocyte; Glial cell; Brain; Endothelin-3; Vasoactive peptide; Endothelin-receptor
Primary rat astrocytes were found by immunohistochemistry to display positive staining for endothelin-3, located predominantly in the perinuclear area. The ability of these cells to produce and release endothelin-3 was confirmed by a combination of reverse-phase HPLC and radioimmunoassay, specific for endothelin-3, which demonstrated immunoreactive peptide in cellular extracts and astrocyte-conditioned medium. In addition, astrocytes were shown to possess a single class of binding sites for endothelin with comparable high affinity for endothelin-1, -2 and -3. These results suggest that astrocytes, by virtue of their ability to produce and secrete endothelin-3, serve as a potential extravascular source of intracerebral vasoregulation capable of influencing regional cerebral blood flow.
INTRODUCTION A new family of peptides, the endothelins, are among the most p o t e n t recognized vasoconstrictors of m a m m a lian vessels 11'36. Endothelin-1 was originally isolated and sequenced from the supernatant of cultured porcine aortic endothelial cells36. This peptide elicits extremely p o t e n t and long-lasting vasoconstricting responses in brain arteries in vitro and in vivo 2'9'29, and requires either d a m a g e d e n d o t h e l i u m in o r d e r to be active from the luminal side of the blood vessel or application from the adventitiai side to p r o v o k e vasoconstriction 25'28. Cross-hybridization studies with endothelin-1 genomic p r o b e s have been used to isolate related genes and led to the prediction of two o t h e r potential endothelin-like peptides. The d e d u c e d amino acid sequences for endothelin-2 and endothelin-3 differ from endothelin-1 by 2 and 6 amino acids, respectively 11. Like endothelin-1, these two peptides have been shown to exert potent vasoconstrictor/pressor effects, although they vary somewhat in their pharmacological properties H. Cloning and sequencing of an endothelin-related gene in the rat revealed that the predicted rat endothelin was identical to h u m a n endothelin-335. I m m u n o r e a c t i v e endothelin-3 has been found in extracts from rat brain and from porcine spinal cord 21'31. While endothelin-1 was d e m o n s t r a t e d to
be present in h u m a n and porcine spinal cord neurons 6'31 by in situ hybridization and immunohistochemistry, as well as in the porcine and rat p o s t e r i o r pituitary system 37, and, on the basis of northern blot analysis, has been assumed to be synthesized by glial cells2°, the precise cellular localization of endothelin-3 in the nervous system has r e m a i n e d obscure. W e have now identified endothelin-3 in primary rat astrocytes. MATERIALS AND METHODS Cell culture. Primary astrocytes were obtained from the brains of 1-day-old Sprague-Dawiey rats and cultured in neuron-free conditions, as described previously3°. Cells were grown to confluence on plastic flasks (for extraction procedures) or 24 well-plates with (immunohistochemistry) or without (radioreceptor assay) glass coverslips in glutamine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The medium was exchanged every other day. Cells were used for experiments after 2-3 weeks in culture. At least 12 h before each experiment, cells were brought into serum- and phenol red-free medium. The purity of astrocyte cultures was routinely determined by immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) (rabbit anti-bovine GFAP, from R. Lipsky, NINDS, NIH, Bethesda, MD 20892), an astrocyte-specific marker. Rhodamine-conjugated goat anti-rabbit antiserum (Boehringer Mannheim, Indianapolis, IN) was used as the second antibody. Confluent monolayers of astrocytes showed 98-99% positive staining for GFAP. Contamination of these cultures with microglia as assessed by using microglia-specific markers was consistently less than 2.3% 4. Checking of our cultures for the presence of endothelial
Correspondence: H. Ehrenreich, National Institute of Allergy and Infectious Diseases, Building 10, Room 11B-13, Bethesda, MD 20892, U.S.A.
55 cells and fibroblasts by anti-yon Willebrand factor (Dakopatts, Glostrup, Denmark) and anti-fibronectin (Kirkegaard & Perry, Gaithersburg, MD) staining, respectively, gave negative results. lmmunohistochemistry. Astrocyte monolayers grown on glass coverslips in 24-well-plates to approximately 80% confluence were fixed in methanol (-20 °C, 10 min) and treated with 10% normal goat serum (room temperature [RT], 20 min) to reduce non-specific background staining. Rabbit anti-endothelin-3 and, alternatively, anti-endothelin-1 antibody (Peninsula, Belmont, CA, 1:100, RT, 180 min) was used as the first antiserum and rhodamine-labeled goat-anti-rabbit IgG (Boehringer Mannheim, 1:1000, RT, 30 min) as the second antiserum. To test the specificity of the immunohistochemical reaction, normal rabbit serum or antiserum, preabsorbed with synthetic endothelin-3 (Peninsula) was substituted for the primary antiserum. For double-staining for GFAP and endothelin-3, incubation with a mouse monoclonal anti-GFAP antibody (Boehringer Mannheim, 1:40, RT, 30 min) followed the procedure described above. FITC-labeled goat anti-mouse antiserum (Kirkegaard & Perry, 1:200, RT, 30 min) was used as second antiserum. Extraction and chromatographic procedures. The serum-free astrocyte-conditioned medium was centrifuged (2000 g, 10 min, 2 °C) and concentrated by extraction through a Sep-Pak C18 cartridge (Waters Associates, Milford, MA), pretreated with ace-
tonitrile containing 5 mM trifluoroacetic acid (TFA). The retained material was washed with 5 mM TFA, eluted with methanol and speed-vac-dried. For HCl-extraction, cells were washed 2 times in ice-cold PBS, mechanically detached and centrifuged (2000 g, 10 min, 2 °C). Hot 0.1 N HCI (approximately 3 ml/100 x 106 cells) was added to the cell pellet. The suspension was well mixed, boiled in a waterbath for 4 rain and immediately put on ice. The cells were then minced with a polytron and the suspension centrifuged (3000 g, 15 min, 2 °C). The supernatant was collected and lyophilized. The extracts were analyzed by reverse-phase liquid chromatography using a 3.9 mm x30 cm, 300 A pore, 15-/~m particle, C18 Delta-Pak column (Waters Associates). The peptide was eluted with a step gradient of 5-50% at 30 min and 100% at 45 rain with acetonitrile (Burdick-Jackson, Muskegon, MI) containing 0.1% TFA (Pierce, Rockford, IL) at a flow rate of 1 ml/minute. Data were collected and analyzed with a Waters 990 photodiode array detector and chromatographic work station. Fractions (1 ml) were collected, speedvac-dried, redissolved in radioimmunoassay (RIA)-buffer and subjected to RIA. For RIA, an endothelin-3 RIA-kit (Peninsula) was used, that showed less than 2% cross-reactivity with endothelin-1. The detection limit of this assay was 1 pg/tube. Recoveries of standard endothelin-3 (Peninsula) from extracted cells or media were 75% and 68%, respectively. Binding studies. Confluent primary astrocytes in 24-well-plates
Fig. 1. Immunofluorescent staining of primary rat astrocytes with anti-endothelin-3 antiserum [original magnification x 133 (A) and x 210 (B)] as well as double-staining for endothelin-3 (C: red) and GFAP (D: green).
56
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Brain Research, 538 (1991) 54-53 Elsevier
BRES 16206
A vasoactive peptide, endothelin-3, is produced by and specifically binds to primary astrocytes Hannelore Ehrenreich 1, John H. Kehrl 1, Robert W. Anderson 2, Peter Rieckmann 1, Ljubisa Vitkovic 1, John E. Coligan 2 and Anthony S. Fauci ~ 1Laboratory of Immunoregulation, and 2Biological Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Accepted 31 July 1990) Key words: Astrocyte; Glial cell; Brain; Endothelin-3; Vasoactive peptide; Endothelin-receptor
Primary rat astrocytes were found by immunohistochemistry to display positive staining for endothelin-3, located predominantly in the perinuclear area. The ability of these cells to produce and release endothelin-3 was confirmed by a combination of reverse-phase HPLC and radioimmunoassay, specific for endothelin-3, which demonstrated immunoreactive peptide in cellular extracts and astrocyte-conditioned medium. In addition, astrocytes were shown to possess a single class of binding sites for endothelin with comparable high affinity for endothelin-1, -2 and -3. These results suggest that astrocytes, by virtue of their ability to produce and secrete endothelin-3, serve as a potential extravascular source of intracerebral vasoregulation capable of influencing regional cerebral blood flow.
INTRODUCTION A new family of peptides, the endothelins, are among the most p o t e n t recognized vasoconstrictors of m a m m a lian vessels 11'36. Endothelin-1 was originally isolated and sequenced from the supernatant of cultured porcine aortic endothelial cells36. This peptide elicits extremely p o t e n t and long-lasting vasoconstricting responses in brain arteries in vitro and in vivo 2'9'29, and requires either d a m a g e d e n d o t h e l i u m in o r d e r to be active from the luminal side of the blood vessel or application from the adventitiai side to p r o v o k e vasoconstriction 25'28. Cross-hybridization studies with endothelin-1 genomic p r o b e s have been used to isolate related genes and led to the prediction of two o t h e r potential endothelin-like peptides. The d e d u c e d amino acid sequences for endothelin-2 and endothelin-3 differ from endothelin-1 by 2 and 6 amino acids, respectively 11. Like endothelin-1, these two peptides have been shown to exert potent vasoconstrictor/pressor effects, although they vary somewhat in their pharmacological properties H. Cloning and sequencing of an endothelin-related gene in the rat revealed that the predicted rat endothelin was identical to h u m a n endothelin-335. I m m u n o r e a c t i v e endothelin-3 has been found in extracts from rat brain and from porcine spinal cord 21'31. While endothelin-1 was d e m o n s t r a t e d to
be present in h u m a n and porcine spinal cord neurons 6'31 by in situ hybridization and immunohistochemistry, as well as in the porcine and rat p o s t e r i o r pituitary system 37, and, on the basis of northern blot analysis, has been assumed to be synthesized by glial cells2°, the precise cellular localization of endothelin-3 in the nervous system has r e m a i n e d obscure. W e have now identified endothelin-3 in primary rat astrocytes. MATERIALS AND METHODS Cell culture. Primary astrocytes were obtained from the brains of 1-day-old Sprague-Dawiey rats and cultured in neuron-free conditions, as described previously3°. Cells were grown to confluence on plastic flasks (for extraction procedures) or 24 well-plates with (immunohistochemistry) or without (radioreceptor assay) glass coverslips in glutamine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The medium was exchanged every other day. Cells were used for experiments after 2-3 weeks in culture. At least 12 h before each experiment, cells were brought into serum- and phenol red-free medium. The purity of astrocyte cultures was routinely determined by immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) (rabbit anti-bovine GFAP, from R. Lipsky, NINDS, NIH, Bethesda, MD 20892), an astrocyte-specific marker. Rhodamine-conjugated goat anti-rabbit antiserum (Boehringer Mannheim, Indianapolis, IN) was used as the second antibody. Confluent monolayers of astrocytes showed 98-99% positive staining for GFAP. Contamination of these cultures with microglia as assessed by using microglia-specific markers was consistently less than 2.3% 4. Checking of our cultures for the presence of endothelial
Correspondence: H. Ehrenreich, National Institute of Allergy and Infectious Diseases, Building 10, Room 11B-13, Bethesda, MD 20892, U.S.A.
55 cells and fibroblasts by anti-yon Willebrand factor (Dakopatts, Glostrup, Denmark) and anti-fibronectin (Kirkegaard & Perry, Gaithersburg, MD) staining, respectively, gave negative results. lmmunohistochemistry. Astrocyte monolayers grown on glass coverslips in 24-well-plates to approximately 80% confluence were fixed in methanol (-20 °C, 10 min) and treated with 10% normal goat serum (room temperature [RT], 20 min) to reduce non-specific background staining. Rabbit anti-endothelin-3 and, alternatively, anti-endothelin-1 antibody (Peninsula, Belmont, CA, 1:100, RT, 180 min) was used as the first antiserum and rhodamine-labeled goat-anti-rabbit IgG (Boehringer Mannheim, 1:1000, RT, 30 min) as the second antiserum. To test the specificity of the immunohistochemical reaction, normal rabbit serum or antiserum, preabsorbed with synthetic endothelin-3 (Peninsula) was substituted for the primary antiserum. For double-staining for GFAP and endothelin-3, incubation with a mouse monoclonal anti-GFAP antibody (Boehringer Mannheim, 1:40, RT, 30 min) followed the procedure described above. FITC-labeled goat anti-mouse antiserum (Kirkegaard & Perry, 1:200, RT, 30 min) was used as second antiserum. Extraction and chromatographic procedures. The serum-free astrocyte-conditioned medium was centrifuged (2000 g, 10 min, 2 °C) and concentrated by extraction through a Sep-Pak C18 cartridge (Waters Associates, Milford, MA), pretreated with ace-
tonitrile containing 5 mM trifluoroacetic acid (TFA). The retained material was washed with 5 mM TFA, eluted with methanol and speed-vac-dried. For HCl-extraction, cells were washed 2 times in ice-cold PBS, mechanically detached and centrifuged (2000 g, 10 min, 2 °C). Hot 0.1 N HCI (approximately 3 ml/100 x 106 cells) was added to the cell pellet. The suspension was well mixed, boiled in a waterbath for 4 rain and immediately put on ice. The cells were then minced with a polytron and the suspension centrifuged (3000 g, 15 min, 2 °C). The supernatant was collected and lyophilized. The extracts were analyzed by reverse-phase liquid chromatography using a 3.9 mm x30 cm, 300 A pore, 15-/~m particle, C18 Delta-Pak column (Waters Associates). The peptide was eluted with a step gradient of 5-50% at 30 min and 100% at 45 rain with acetonitrile (Burdick-Jackson, Muskegon, MI) containing 0.1% TFA (Pierce, Rockford, IL) at a flow rate of 1 ml/minute. Data were collected and analyzed with a Waters 990 photodiode array detector and chromatographic work station. Fractions (1 ml) were collected, speedvac-dried, redissolved in radioimmunoassay (RIA)-buffer and subjected to RIA. For RIA, an endothelin-3 RIA-kit (Peninsula) was used, that showed less than 2% cross-reactivity with endothelin-1. The detection limit of this assay was 1 pg/tube. Recoveries of standard endothelin-3 (Peninsula) from extracted cells or media were 75% and 68%, respectively. Binding studies. Confluent primary astrocytes in 24-well-plates
Fig. 1. Immunofluorescent staining of primary rat astrocytes with anti-endothelin-3 antiserum [original magnification x 133 (A) and x 210 (B)] as well as double-staining for endothelin-3 (C: red) and GFAP (D: green).
56
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