Acta Neuropathol (1992) 84:457 -460
kta Neuropathologtca @ Springer-Verlag 1992
Immunohistochemical evidence of endothelin-1 in human choroid plexus* M. H. Jiang, L. Eriksson, and Y. Olsson Laboratory of Neuropathology, University Hospital, S-75185 Uppsala, Sweden Received January 28, 1992/Revised, accepted March 19, 1992
Summary. An immunohistochemical investigation was carried out on 17 specimens of human choroid plexus obtained post mortem, 1 biopsy of normal choroid plexus including part of the lateral ventricle and 1 papilloma of the choroid plexus removed surgically The material was fixed in formalin. Paraffin and cryostat sections were used. A polyclonal antiserum to endothelin-1 served as a primary antibody The avidin-biotinperoxidase method was applied to demonstrate the immunoreaction. The epithelial cells of the choroid plexuses, the choroid papilloma and most ependymal cells of the lateral ventricle showed a distinct brown reaction product in their cytoplasm indicating antigenic sites to endothelin-1. The reaction was of lesser intensity in the ependymal cells. The connective tissue in choroid plexus was unstained. A positive immunoreaction was present in the walls of some vessels in the choroid plexus in cryostat sections. This is the first report on the presence of antigenic sites to endothelin-1 in the epithelial cells of the human choroid plexus. The role of endothelin in these cells should be investigated to ascertain if the cells synthesize this biologically active peptide or if it is merely bound to receptors in them. Key words: Choroid plexus - Choroid plexus papilloma - Endothelin-1 - H u m a n brain - Immunohistochemistry
Endothelin, a newly discovered peptide, was isolated from the supernatant of a culture of porcine aortic endothelial cells [25]. It is present in the vascular endothelial cells and other cells of the brain, lung,
intestine, kidney and adrenal gland [12]. After binding to receptors in the smooth muscle cells of blood vessels, endothelins can induce marked vasoconstriction and can act as a growth factor [1, 14, 21, 25] and probably have many additional biological effects. It has been suggested that endothelins may act as neuropeptides in the central nervous system and modulate neuronal activity [4, 24]. Based on experiments with cloned endothelinrelated genes, human endothelin was found to exist in three distinct isoforms, endothelin-1, endothelin-2 and endothelin-3 [6]. Endothetins are produced in cells from the precursor, pre-proendothelin which is converted to "big endothelin" [25] and then to the various endothelins by enzymes [6]. Biochemical investigations on the central nervous system have demonstrated the presence of endothelin-1 in the spinal cord [26], the hypothalamus, and brain stem [23] and that endothelin-3 is the major form in the pituitary gland [23]. Using 125I-labeled endothelin, the presence of receptors for endothelin-1 and endothelin-3 was demonstrated in the basal ganglia [13], brain stem [3, 16], hippocampus [8, 16], hypothalamus [16, 23], cerebral cortex [16, 23] and cerebellum [16]. During an immunohistochemical investigation, we observed that very strong immunoreactivity to endothelin-1 occurred in the human choroid plexus. Receptors for endothelin are present in the choroid plexus of the rat [8, 13] and endothelin occurs in human cerebrospinal fluid [24]. As we have not found any publication on the presence of endothelin in the human choroid plexus we would like to report our observations.
Material and methods * Supported by grants from Swedish Medical Research Council, project 03020, 1987 Ars stiftelse f6r strokeforskning, Selanders stiftelse, Ahlen-stiftelsen and Stiftelsen GamlaTj~inarinnor,Stockholm, Sweden Correspondence to: M.-H. Jiang (address see above)
The material included portions of choroid plexus obtained 2-4 days post mortem from 13 males and 4 females aged 39 to 84 years, part of the choroid plexus, temporal lobe and ventricle removed for the treatment of severe epilepsy, and a choroid papilloma from a boy of 1 year removed surgically.
458
Paraffin embedding Cubes measuring about I cm3 which included the medial temporal lobe, its wall and choroid plexus from the autopsy cases were fixed for 48 h by immersion in 4 % purified para-formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The surgical specimen of the choroid plexus and medial part of the temporal lobe and the wall of the temporal ventricle and the choroid plexus papilloma were fixed in the same solution for about 24 h. The tissues were dehydrated, infiltrated with paraffin at 54 ~ overnight, then embedded in paraplast. Sections, 5 ~m thick, were placed on slides coated with poly-L-lysine and de-paraffinized.
Cryostat sections Seven autopsy cases were used for cryostat sectioning. Samples of the choroid plexus were fixed in 4 % paraformaldehyde in 0.1 M solium borate buffer (pH 9.6) for 4-8 h, washed in phosphatebuffered saline (PBS) containing 15 % w/v sucrose and 0.01% sodium azide for 2-4 h and dried at 20 ~ for 2-4 h as described by Giaid et al. [4]. Sections, 10 ~m thick, were placed on slides coated with poly-L-lysine. Immunohistochemistry was then carried out as follows. Unspecific binding of the primary antiserum to the tissue was blocked with methanol and hydrogen peroxide. After rinsing the sections in PBS, various dilutions of a primary antibody (1 : 6.000, 1:8.000, 1:10.000, 1:12.000 and 1:16.000 in diluted goat serum) were applied to the sections in the form of a polyclonal rabbitanti-human endothelin-1 antiserum obtained from Cambridge Research Biochemicals, UK. The antiserum was allowed to react with the antigens in the sections overnight at + 4 ~ Thereafter the sections were rinsed repeatedly in PBS.
Antibodies bound to antigens in the sections were located by the avidin-biotin peroxidase method [5] using 3,3'-diaminobenzidine tetrahydrochloride as chromogen (Vectastain Elite Kit,Vector Laboratories, Burlingame, Calif.). Mayers hematoxylin was used to stain the nuclei. Negative controls were run by omitting the primary antibody from the immunohistochemical procedure.
Results T h e r e was a distinct b r o w n reaction p r o d u c t in the c y t o p l a s m o f almost all epithelial ceils o f the choroid plexus, indicating antigenic sites to h u m a n endothelin-1 in b o t h f r o z e n and p a r a f f i n - e m b e d d e d sections (Fig. 1). T h e walls of s o m e b l o o d vessel were stained in the cryostat sections but n o t in the paraffin material. T h e connective tissue c o m p o n e n t s in the plexus did n o t show any reaction p r o d u c t . This p a t t e r n of staining was seen in all the investigated cases. Most e p e n d y m a l cells lining the ventricles s h o w e d i m m u n o r e a c t i v i t y in their c y t o p l a s m b u t usually this reaction had a lower intensity t h a n that in the epithelial cells of the c h o r o i d plexus. M a n y nerve cell bodies o f the t e m p o r a l tobe specim e n s were i m m u n o - s t a i n e d . This o c c u r r e d m o s t frequently in the C A 4 sector o f t h e h i p p o c a m p u s . Glial cells and the walls of b l o o d vessels of the brain p a r e n c h y m a were usually unstained. T h e t u m o r cells o f the plexus papilloma s h o w e d a m a r k e d i m m u n o r e a c tion.
Fig. 1. Color photograph from the choroid plexus of one autopsy case, fixed by immersion in formalin and embedded in paraffin. Note the strong positive (brown)immunostaining of the epithelial cells of the plexus and its absence in the stroma and the blood vessels
459
Discussion This is the first investigation which d e m o n s t r a t e s the presence of antigenic sites to endothelin-1 in the h u m a n choroid plexus. R e c e p t o r s for endothelins are k n o w n to be present in the choroid plexus of the rat [8, 13]. Big endothelin-1, endothelin-1 and endothelin-3 are k n o w n to be present in h u m a n cerebrospinal fluid [24] but f r o m where they arise is unknown. In view of our findings, the epithelial cells of the choroid plexus m a y participate in controlling the levels of endothelin in the cerebrospinal fluid. As our investigation indicates endothelin-1 is present in the epithelial cells of the choroid plexus, but w h e t h e r this is due to binding of endothein-1 to surface receptors in these cells or to its synthesis in t h e m is not known. If it is synthesized in the epithelial cells then it m a y be secreted and contribute to the content of this peptide in the cerebrospinal fluid. In addition to being present in the choroid plexus, receptors to endothelins occur in m a n y regions of the central nervous system [8, 16, 23] but details of their function are not yet known. T h e y m a y cause constriction of blood vessels b o t h in vivo and in vitro and, therefore, m a y be implicated in the control of vascular tone [7, 10, 11, 20, 22]. Intracerebral injection of endothelin causes elevation of the arterial blood pressure in anesthetized and conscious rats but this response is m u c h less m a r k e d after intravenous administration [17, 18]. It would a p p e a r that endothelin-1 acts on the adventitial aspect rather than the luminal aspect of cerebral arteries [15, 22]. Since endothelin circulating in blood does not cross the blood-brain barrier [13], it has b e e n speculated that endothelin m a y have o t h e r actions in the central nervous system that are at present unknown. Recent investigations have shown that endothelin circulating in doses having no effect on intracerebral blood vessels causes vasoconstriction and reduced blood flow blood in the vessels of the choroid plexus [9]. The endothelial cells lining the blood vessels of the choroid plexus being m u c h m o r e p e r m e a b l e than the cells lining vessels of the brain p a r e n c h y m a [2, 19] permit endothelin to pass m o r e easily into the choroid plexus than into the brain p a r e n c h y m a . T h e experiments of Kadel et al. [9] indicate that circulating or locally produced endothelin m a y be involved in the regulation of production of cerebrospinal fluid.
Acknowledgement. We are grateful to Ms. Gunilla Tibbling for kindly providing chemistry.
technical
advice regarding immunohisto-
References 1. Bobik A, Grooms A, Millar JA, Mitchell A, Grinpukel S (1990) Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol 258:C408-C415 2. Dohrmann G (1970) The choroid plexus: a historical review. Brain Res 18:197-218
3. Fuxe K, Angg~rd E, Lundgren K, Cintra A, Agnati LE Galton S,Vane J (1989) Localization of [125I]endothelin-1 and [125I]endothelin-3 binding sites in the rat brain. Acta Physiol Scand 137:563 -564 4. Giaid A, Gibson SJ, Ibrahim NBN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM (1989) Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root gangia. Proc Natl Acad Sci USA 86:7634-7638 5. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577-580 6. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T (1989) The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 86:2863-2867 7. Jansen I, Fallgren B, Edvinsson L (1989) Mechanisms of action of endothelin on isolated feline cerebral arteries: in vitro pharmacology and electrophysiology. J Cereb Blood Flow Metab 9:743-747 8. Jones CR, Hiley CR, Pelton JY, Mohr M (1989) Autoradiogrpahic visualization of the binding sites for [1;SI]endothelin in rat and human brain. Neurosci Lett 97:276-279 9. Kadel KA, Heistad DD, Faraci FM (1990) Effects of endothelin on blood vessels of the brain and choroid plexus. Brain Res 518:78-82 10. Kauser K, Rubanyi GM, Harder DR (1989) Endotheliumdependent modulation of endothelin-induced vasoconstriction and membrane depolarization in cat cerebral arteries. J Pharmacol Exp Ther 252:93-97 11. Kobayashi H, Hayashi M, Kobayashi S, Kabuto M, HandaY, Kawano H (1990) Effect of endothelin on the canine basilar artery. Neurosurgery 27:357-361 12. Koseki C, Imai M, Hirata Y, Yanagisawa Y, Masaki T (1989) Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol 256:R858-866 13. Koseki C, Imai M, HirataY, Yanagisawa M, Masaki T (1989) Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain. Neurosci Res 6:581-585 14. Lin HY, Kaji EH, Winkel GK, Ives HE, Lodish HF (1991) Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor. Proc Natl Acad Sci USA 88:3185-3189 15. MimaT, Yanagisawa M, ShigenoT, Saito A, Goto K,Takakura K, Masaki T (1989) Endothelin acts in faline and canine cerebral arteries from the adventitial side. Stroke 20:1553-1556 16. Nambi E Pullen M, Feuerstein G (1990) Identification of endothelin receptors in various regions of rat brain. Neuropeptides 16:195-199 17. Nishimura M, Takahashi H, Matsusawa M, Ikegaki I, Yoshimura M (1990) Intracerebroventricular injections of endothelin increase arterial pressure in conscious rats. Jpn Circ J 54:662-670 18. Nishimura M, Takahashi H, Matsusawa M, Ikegaki I, Sakamoto M, Nakanishi T, Hirabayashi M, Yoshimura M (1991) Chronic intracerebroventricular infusions of endothelin elevate arterial pressure in rats. J Hypertens 9:71-76 19. Rapoport SI (1976) Blood-brain barrier in physiology and medicine. Raven Press, New York 20. Saito A, Shiba R, Kimura S,Yanagisawa M, Goto K, Masaki T (1989) Vasoconstrictor response of large cerebral arteries of cats to endothelin, an endothelium-derived vasoactive peptide. Eur J Pharmacol 162:353-358 21. Schiffrin EL,Turgeon A,Tremblay J, Deslongchamps M (1991) Effects of ANR angiotensin, vasopressin, and endothelin on ANP receptors in cultured rat vascular smooth muscle cells. Am J Physiol 260:H58-H65
460 22. ShigenoT, MimaT, Takakura K,Yanagisawa M, Saito A, Goto K, Masaki T (1989) Endothelin-1 acts in cerebral arteries from the adventitial but not from the luminal side. J Cardiovasc Pharmacol 13:S174-176 23. Takahashi K, Ghatei MA, Jones PM, Murphy JK, Lam H-C, O'Halloran D J, Bloom SR (1991) Endothelin in human brain and pituitary gland: pressence of immunoreactive endothelin, endothelin messenger ribonucleic acid, and endothelin receptors. J Clin Endocrin Metab 72:693-699 24. Yam@ T, Johshita H, Ishibashi M, Takaku F, Ohno H, Suzuki N, Matsumoto H, Fujino M (1990) Endothelin family in
human plasma and cerebrospinal fluid. J Clin Endocrin Metab 71:1611-1615 25. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y,Yazaki Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415 26. Yoshizawa T, Kimura S, Kanazawa I, UchiyamaY, Yanagisawa M, Masaki T (1989) Endothelin localizes in the dorsal horn and acts on the spinal neurones: possible involvement of dihydropyridine-sensitive calcium channels and substance P release. Neurosci Lett 102:179-184
kta Neuropathologtca @ Springer-Verlag 1992
Immunohistochemical evidence of endothelin-1 in human choroid plexus* M. H. Jiang, L. Eriksson, and Y. Olsson Laboratory of Neuropathology, University Hospital, S-75185 Uppsala, Sweden Received January 28, 1992/Revised, accepted March 19, 1992
Summary. An immunohistochemical investigation was carried out on 17 specimens of human choroid plexus obtained post mortem, 1 biopsy of normal choroid plexus including part of the lateral ventricle and 1 papilloma of the choroid plexus removed surgically The material was fixed in formalin. Paraffin and cryostat sections were used. A polyclonal antiserum to endothelin-1 served as a primary antibody The avidin-biotinperoxidase method was applied to demonstrate the immunoreaction. The epithelial cells of the choroid plexuses, the choroid papilloma and most ependymal cells of the lateral ventricle showed a distinct brown reaction product in their cytoplasm indicating antigenic sites to endothelin-1. The reaction was of lesser intensity in the ependymal cells. The connective tissue in choroid plexus was unstained. A positive immunoreaction was present in the walls of some vessels in the choroid plexus in cryostat sections. This is the first report on the presence of antigenic sites to endothelin-1 in the epithelial cells of the human choroid plexus. The role of endothelin in these cells should be investigated to ascertain if the cells synthesize this biologically active peptide or if it is merely bound to receptors in them. Key words: Choroid plexus - Choroid plexus papilloma - Endothelin-1 - H u m a n brain - Immunohistochemistry
Endothelin, a newly discovered peptide, was isolated from the supernatant of a culture of porcine aortic endothelial cells [25]. It is present in the vascular endothelial cells and other cells of the brain, lung,
intestine, kidney and adrenal gland [12]. After binding to receptors in the smooth muscle cells of blood vessels, endothelins can induce marked vasoconstriction and can act as a growth factor [1, 14, 21, 25] and probably have many additional biological effects. It has been suggested that endothelins may act as neuropeptides in the central nervous system and modulate neuronal activity [4, 24]. Based on experiments with cloned endothelinrelated genes, human endothelin was found to exist in three distinct isoforms, endothelin-1, endothelin-2 and endothelin-3 [6]. Endothetins are produced in cells from the precursor, pre-proendothelin which is converted to "big endothelin" [25] and then to the various endothelins by enzymes [6]. Biochemical investigations on the central nervous system have demonstrated the presence of endothelin-1 in the spinal cord [26], the hypothalamus, and brain stem [23] and that endothelin-3 is the major form in the pituitary gland [23]. Using 125I-labeled endothelin, the presence of receptors for endothelin-1 and endothelin-3 was demonstrated in the basal ganglia [13], brain stem [3, 16], hippocampus [8, 16], hypothalamus [16, 23], cerebral cortex [16, 23] and cerebellum [16]. During an immunohistochemical investigation, we observed that very strong immunoreactivity to endothelin-1 occurred in the human choroid plexus. Receptors for endothelin are present in the choroid plexus of the rat [8, 13] and endothelin occurs in human cerebrospinal fluid [24]. As we have not found any publication on the presence of endothelin in the human choroid plexus we would like to report our observations.
Material and methods * Supported by grants from Swedish Medical Research Council, project 03020, 1987 Ars stiftelse f6r strokeforskning, Selanders stiftelse, Ahlen-stiftelsen and Stiftelsen GamlaTj~inarinnor,Stockholm, Sweden Correspondence to: M.-H. Jiang (address see above)
The material included portions of choroid plexus obtained 2-4 days post mortem from 13 males and 4 females aged 39 to 84 years, part of the choroid plexus, temporal lobe and ventricle removed for the treatment of severe epilepsy, and a choroid papilloma from a boy of 1 year removed surgically.
458
Paraffin embedding Cubes measuring about I cm3 which included the medial temporal lobe, its wall and choroid plexus from the autopsy cases were fixed for 48 h by immersion in 4 % purified para-formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The surgical specimen of the choroid plexus and medial part of the temporal lobe and the wall of the temporal ventricle and the choroid plexus papilloma were fixed in the same solution for about 24 h. The tissues were dehydrated, infiltrated with paraffin at 54 ~ overnight, then embedded in paraplast. Sections, 5 ~m thick, were placed on slides coated with poly-L-lysine and de-paraffinized.
Cryostat sections Seven autopsy cases were used for cryostat sectioning. Samples of the choroid plexus were fixed in 4 % paraformaldehyde in 0.1 M solium borate buffer (pH 9.6) for 4-8 h, washed in phosphatebuffered saline (PBS) containing 15 % w/v sucrose and 0.01% sodium azide for 2-4 h and dried at 20 ~ for 2-4 h as described by Giaid et al. [4]. Sections, 10 ~m thick, were placed on slides coated with poly-L-lysine. Immunohistochemistry was then carried out as follows. Unspecific binding of the primary antiserum to the tissue was blocked with methanol and hydrogen peroxide. After rinsing the sections in PBS, various dilutions of a primary antibody (1 : 6.000, 1:8.000, 1:10.000, 1:12.000 and 1:16.000 in diluted goat serum) were applied to the sections in the form of a polyclonal rabbitanti-human endothelin-1 antiserum obtained from Cambridge Research Biochemicals, UK. The antiserum was allowed to react with the antigens in the sections overnight at + 4 ~ Thereafter the sections were rinsed repeatedly in PBS.
Antibodies bound to antigens in the sections were located by the avidin-biotin peroxidase method [5] using 3,3'-diaminobenzidine tetrahydrochloride as chromogen (Vectastain Elite Kit,Vector Laboratories, Burlingame, Calif.). Mayers hematoxylin was used to stain the nuclei. Negative controls were run by omitting the primary antibody from the immunohistochemical procedure.
Results T h e r e was a distinct b r o w n reaction p r o d u c t in the c y t o p l a s m o f almost all epithelial ceils o f the choroid plexus, indicating antigenic sites to h u m a n endothelin-1 in b o t h f r o z e n and p a r a f f i n - e m b e d d e d sections (Fig. 1). T h e walls of s o m e b l o o d vessel were stained in the cryostat sections but n o t in the paraffin material. T h e connective tissue c o m p o n e n t s in the plexus did n o t show any reaction p r o d u c t . This p a t t e r n of staining was seen in all the investigated cases. Most e p e n d y m a l cells lining the ventricles s h o w e d i m m u n o r e a c t i v i t y in their c y t o p l a s m b u t usually this reaction had a lower intensity t h a n that in the epithelial cells of the c h o r o i d plexus. M a n y nerve cell bodies o f the t e m p o r a l tobe specim e n s were i m m u n o - s t a i n e d . This o c c u r r e d m o s t frequently in the C A 4 sector o f t h e h i p p o c a m p u s . Glial cells and the walls of b l o o d vessels of the brain p a r e n c h y m a were usually unstained. T h e t u m o r cells o f the plexus papilloma s h o w e d a m a r k e d i m m u n o r e a c tion.
Fig. 1. Color photograph from the choroid plexus of one autopsy case, fixed by immersion in formalin and embedded in paraffin. Note the strong positive (brown)immunostaining of the epithelial cells of the plexus and its absence in the stroma and the blood vessels
459
Discussion This is the first investigation which d e m o n s t r a t e s the presence of antigenic sites to endothelin-1 in the h u m a n choroid plexus. R e c e p t o r s for endothelins are k n o w n to be present in the choroid plexus of the rat [8, 13]. Big endothelin-1, endothelin-1 and endothelin-3 are k n o w n to be present in h u m a n cerebrospinal fluid [24] but f r o m where they arise is unknown. In view of our findings, the epithelial cells of the choroid plexus m a y participate in controlling the levels of endothelin in the cerebrospinal fluid. As our investigation indicates endothelin-1 is present in the epithelial cells of the choroid plexus, but w h e t h e r this is due to binding of endothein-1 to surface receptors in these cells or to its synthesis in t h e m is not known. If it is synthesized in the epithelial cells then it m a y be secreted and contribute to the content of this peptide in the cerebrospinal fluid. In addition to being present in the choroid plexus, receptors to endothelins occur in m a n y regions of the central nervous system [8, 16, 23] but details of their function are not yet known. T h e y m a y cause constriction of blood vessels b o t h in vivo and in vitro and, therefore, m a y be implicated in the control of vascular tone [7, 10, 11, 20, 22]. Intracerebral injection of endothelin causes elevation of the arterial blood pressure in anesthetized and conscious rats but this response is m u c h less m a r k e d after intravenous administration [17, 18]. It would a p p e a r that endothelin-1 acts on the adventitial aspect rather than the luminal aspect of cerebral arteries [15, 22]. Since endothelin circulating in blood does not cross the blood-brain barrier [13], it has b e e n speculated that endothelin m a y have o t h e r actions in the central nervous system that are at present unknown. Recent investigations have shown that endothelin circulating in doses having no effect on intracerebral blood vessels causes vasoconstriction and reduced blood flow blood in the vessels of the choroid plexus [9]. The endothelial cells lining the blood vessels of the choroid plexus being m u c h m o r e p e r m e a b l e than the cells lining vessels of the brain p a r e n c h y m a [2, 19] permit endothelin to pass m o r e easily into the choroid plexus than into the brain p a r e n c h y m a . T h e experiments of Kadel et al. [9] indicate that circulating or locally produced endothelin m a y be involved in the regulation of production of cerebrospinal fluid.
Acknowledgement. We are grateful to Ms. Gunilla Tibbling for kindly providing chemistry.
technical
advice regarding immunohisto-
References 1. Bobik A, Grooms A, Millar JA, Mitchell A, Grinpukel S (1990) Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol 258:C408-C415 2. Dohrmann G (1970) The choroid plexus: a historical review. Brain Res 18:197-218
3. Fuxe K, Angg~rd E, Lundgren K, Cintra A, Agnati LE Galton S,Vane J (1989) Localization of [125I]endothelin-1 and [125I]endothelin-3 binding sites in the rat brain. Acta Physiol Scand 137:563 -564 4. Giaid A, Gibson SJ, Ibrahim NBN, Legon S, Bloom SR, Yanagisawa M, Masaki T, Varndell IM, Polak JM (1989) Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root gangia. Proc Natl Acad Sci USA 86:7634-7638 5. Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29:577-580 6. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki T (1989) The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 86:2863-2867 7. Jansen I, Fallgren B, Edvinsson L (1989) Mechanisms of action of endothelin on isolated feline cerebral arteries: in vitro pharmacology and electrophysiology. J Cereb Blood Flow Metab 9:743-747 8. Jones CR, Hiley CR, Pelton JY, Mohr M (1989) Autoradiogrpahic visualization of the binding sites for [1;SI]endothelin in rat and human brain. Neurosci Lett 97:276-279 9. Kadel KA, Heistad DD, Faraci FM (1990) Effects of endothelin on blood vessels of the brain and choroid plexus. Brain Res 518:78-82 10. Kauser K, Rubanyi GM, Harder DR (1989) Endotheliumdependent modulation of endothelin-induced vasoconstriction and membrane depolarization in cat cerebral arteries. J Pharmacol Exp Ther 252:93-97 11. Kobayashi H, Hayashi M, Kobayashi S, Kabuto M, HandaY, Kawano H (1990) Effect of endothelin on the canine basilar artery. Neurosurgery 27:357-361 12. Koseki C, Imai M, Hirata Y, Yanagisawa Y, Masaki T (1989) Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol 256:R858-866 13. Koseki C, Imai M, HirataY, Yanagisawa M, Masaki T (1989) Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain. Neurosci Res 6:581-585 14. Lin HY, Kaji EH, Winkel GK, Ives HE, Lodish HF (1991) Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor. Proc Natl Acad Sci USA 88:3185-3189 15. MimaT, Yanagisawa M, ShigenoT, Saito A, Goto K,Takakura K, Masaki T (1989) Endothelin acts in faline and canine cerebral arteries from the adventitial side. Stroke 20:1553-1556 16. Nambi E Pullen M, Feuerstein G (1990) Identification of endothelin receptors in various regions of rat brain. Neuropeptides 16:195-199 17. Nishimura M, Takahashi H, Matsusawa M, Ikegaki I, Yoshimura M (1990) Intracerebroventricular injections of endothelin increase arterial pressure in conscious rats. Jpn Circ J 54:662-670 18. Nishimura M, Takahashi H, Matsusawa M, Ikegaki I, Sakamoto M, Nakanishi T, Hirabayashi M, Yoshimura M (1991) Chronic intracerebroventricular infusions of endothelin elevate arterial pressure in rats. J Hypertens 9:71-76 19. Rapoport SI (1976) Blood-brain barrier in physiology and medicine. Raven Press, New York 20. Saito A, Shiba R, Kimura S,Yanagisawa M, Goto K, Masaki T (1989) Vasoconstrictor response of large cerebral arteries of cats to endothelin, an endothelium-derived vasoactive peptide. Eur J Pharmacol 162:353-358 21. Schiffrin EL,Turgeon A,Tremblay J, Deslongchamps M (1991) Effects of ANR angiotensin, vasopressin, and endothelin on ANP receptors in cultured rat vascular smooth muscle cells. Am J Physiol 260:H58-H65
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