A Unique Serotonin Receptor in Choroid Plexus Is Linked To

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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4086-4088, June 1986 Neurobiology A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover (serotonin 5-HTjc site/serotonin 5-HT2 site/serotonin antagonists/cerebrospinal fluid/phosphoinositide hydrolysis) P. JEFFREY CONN*, ELAINE SANDERS-BUSH*t, BETH J. HOFFMANt, AND PAUL R. HARTIGt *Tennessee Neuropsychiatric Institute and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232; and tDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218 Communicated by Saul Roseman, January 23, 1986

ABSTRACT A novel serotonergic binding site, the 5-HTlc (11). These findings have been confirmed and extended in site, has been characterized recently in choroid plexus and brain (12-14) and other tissues (15-17). Serotonin-stimulated several brain regions. The biochemical and physiological roles phosphatidylinositol turnover in cerebral cortex is not sec- ofthis site have not been previously described. In this report we ondary to release of another neurotransmitter or of arachi- show that serotonin (5-hydroxytryptamine, 5-HT) stimulates donic acid (18), suggesting that the 5-HT2 receptor is directly phosphatidylinositol turnover in rat choroid plexus. The phar- coupled to phosphatidylinositol turnover. In the present macology of serotonin-stimulated phosphatidylinositol hydrol- studies, we have examined the effect of serotonin and ysis in choroid plexus was compared to the pharmacology in serotonin antagonists upon phosphatidylinositol turnover in cerebral cortex, where this response is mediated by the rat choroid plexus. The pharmacology of the response in serotonin 5-HT2 receptor. Serotonin increased phosphatidyl- choroid plexus was compared with that in cerebral cortex and inositol turnover in choroid plexus by 6-fold and in cerebral with the pharmacology ofthe 5-HT1c and 5-HT2 binding sites. cortex by 2.5-fold. Serotonin was >10-fold more potent in The present data support the hypothesis that serotonin- choroid plexus (EC_% = 46 nM) than in cerebral cortex (EC50 stimulated phosphatidylinositol turnover in choroid plexus is = 540 nM). The serotonin antagonists ketanserin, mianserin, mediated by the 5-HTjc binding site in this tissue. and spiperone inhibited the response in the two tissues with different potencies. In cerebral cortex all three exhibited METHODS nanomolar affinities consistent with their potencies at the Phosphatidylinositol Turnover. Phosphatidylinositol turn- 5-HT2 site. In choroid plexus, however, the rank order over in rat brain cerebral cortical slices was measured as (mianserin > ketanserin >> spiperone) and absolute potencies described (12) except that slices containing approximately 1 were consistent with binding to the 5-HT1c site. These data mg of protein were added to tubes containing 1 ,Ci (1 Ci = suggest that the 5-HT1c site in choroid plexus is a functional 37 GBq) of [3H]inositol and prelabeled for 3 hr. Agonists and receptor that utilizes phosphatidylinositol turnover as its bio- antagonists were added directly to the tubes in which chemical effector system. prelabeling occurred without washing away free [3H]inositol. Phosphatidylinositol turnover in choroid plexus was mea- The choroid plexus is the major site of cerebrospinal fluid sured in an identical manner except that a single rat choroid (CSF) production, but the mechanisms controlling produc- plexus [about 0.5 mg (wet weight) of tissue] was added to tion and secretion ofCSF are poorly understood (1). Whereas each tube and prelabeled for 90 min. the administration of either norepinephrine or serotonin Apparent Ki values of antagonists at phosphatidylinositol- decreases the secretion of CSF (2, 3), serotonin is the most linked receptors were estimated from the relationship: Ki = efficacious monoamine (3). Since the choroid plexus and 1C50/(1 + S/EC50), where IC50 = the concentration of walls of the ventricles contain serotonergic fibers (4-7), it is antagonist giving 50% of the maximal inhibition of the possible that endogenous serotonin is involved in the regu- response stimulated by S concentration of serotonin, and lation of CSF production. Therefore, it would be of consid- EC50 = the concentration of serotonin producing half- erable interest to find a receptor that mediates the effect of maximal stimulation. This equation is modified from the serotonin in the choroid plexus. We and others (8, 9) have Cheng and Prusoff equation (19) and assumes that the EC50 described a novel serotonin binding site on choroid plexus of serotonin equals its Ki. Since the presence of spare epithelial cells with characteristics distinct from those of receptors or threshold effects may alter this relationship, the 5-HT1A, 5-HT1B, and 5-HT2 sites. This site has been named apparent K1 values may not reflect true Ki values. the 5-HT1c site and can be labeled with [3H]serotonin or Radioligand Binding. Displacement of [3H]ketanserin bind- 125I-labeled lysergic acid diethylamide (125I-LSD). We have ing to the 5-HT2 site in cortex (12) and 125I-LSD binding to the recently reported the solubilization of this site from pig 5-HT1c site in choroid plexus (9) was measured as described choroid plexus (10). except that the buffer used for 125I-LSD binding was identical Identification of a biochemical effector system that is to that used for measurement of binding of [3H]ketanserin to linked to the 5-HT1c site would provide strong evidence that the 5-HT2 binding site (i.e., contained physiological salts, 10 this site serves as a functional receptor in choroid plexus and mM glucose, 10 ,uM pargyline, and 25 mM Tris HCl at pH other brain regions (8). Recent data have shown that another 7.4). This buffer was the same as that used for measurement serotonin receptor subtype is linked to phosphatidylinositol of phosphatidylinositol turnover except that 25 mM sodium turnover in the mammalian brain. Based upon the finding that bicarbonate replaced 25 mM Tris'HCl in phosphatidylinositol selective 5-HT2 antagonists inhibit serotonin-stimulated studies. We observed that the use of physiological concen- phosphatidylinositol turnover in cerebral cortex, we suggest- trations of salts in the 5-HT2 and 5-HTlc binding assays ed that the response is mediated by the 5-HT2 binding site Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); CSF, cere- The publication costs of this article were defrayed in part by page charge brospinal fluid; LSD, lysergic acid diethylamide; 123 I-LSD, 1251_ payment. This article must therefore be hereby marked "advertisement" labeled LSD. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4086 Downloaded by guest on September 28, 2021 Neurobiology: Conn et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4087 500 cies with apparent Ki values of 4.3 nM, 12 nM, and 23 nM for spiperone, ketanserin, and mianserin, respectively. In choroid plexus, however, spiperone was much less potent, 400 with an apparent Ki of 6.2 gM. Ketanserin was also less potent in choroid plexus (Ki = 130 nM), whereas the affinity 4)4 ofmianserin in the two tissues was nearly equal. The addition 300 of 1 ,uM muscarinic, a1-adrenergic or Hl-histaminergic 4n- tagonists (atropine, prazosin, or triprolidine) did not alter the release of [3H]inositol 1-phosphate induced by serotonin (data not shown). 200 - The apparent Ki value of serotonin at the 5-HT1c binding site (94 nM) was similar to its EC50 value at the phosphatidylinositol-linked receptor in choroid plexus but not in 100 cerebral cortex (Table 1). In the cerebral cortex, antagonist potencies at the phosphatidylinositol-linked receptor were l l similar to their potencies at the 5-HT2 binding site. In choroid 9 8 7 6 5 4 plexus, however, spiperone was much less potent in blocking serotonin-stimulated phosphatidylinositol turnover, which is Serotonin, -log M consistent with its low potency at the 5-HT1c binding site FIG. 1. Effect of increasing concentrations of serotonin upon (Fig. 2; Table 1). Furthermore, the rank order (mianserin > phosphatidylinositol turnover in choroid plexus (e) and cerebral ketanserin >> spiperone) and absolute potencies of the cortex (x). Serotonin-induced release of [3H]inositol 1-phosphate antagonists in blocking phosphatidylinositol turnover and was used as a measure ofphosphatidylinositol turnover. The data are 5-HT1c binding are in excellent agreement (Table 1). presented as percent stimulation above basal (3H]inositol 1-phos- Four lines of evidence support the hypothesis that phosphate radioactivity, which was 660 ± 29 cpm in cerebral cortex and phatidylinositol turnover in the choroid plexus is linked to the 1300 ± 119 cpm in choroid plexus. Maximum stimulation resulted in serotonin 5-HT1c site. First, the EC50 value of serotonin at 1807 ± 174 cpm and 7885 ± 832 cpm in cerebral cortex and choroid stimulating phosphatidylinositol turnover in choroid plexus is plexus, respectively. Each point is the mean of six determinations. similar to its Ki value for binding to the 5-HT1c site. Due to Vertical bars represent SEMs. the possibility that spare receptors may be present, an agonist EC50 value should be equal to or less than its Ki value for significantly lowered the apparent affinities of some seroto- binding to a given receptor. Furthermore, the potency of nergic ligands at these sites. Apparent Ki values were serotonin in choroid plexus is much higher than in the estimated from radioligand displacement data by the method cerebral cortex, where the response is mediated by the 5-HT2 of Cheng and Prusoff (19). binding site (11-14). Second, the apparent Ki values of spiperone, ketanserin, and mianserin at the 5-HTic site are RESULTS AND DISCUSSION similar to their apparent Ki values at the phosphatidylinositol- Serotonin elicited a concentration-dependent increase in linked serotonin receptor in choroid plexus but differ from [3H]inositol 1-phosphate release in choroid plexus and cere- their potencies at the 5-HT2, 5-HT1A, and 5-HT1B sites (20). bral cortical slices (Fig. 1). The effect of serotonin was much Third, selective antagonists of other phosphatidylinositol- more robust in choroid plexus (5- to 6-fold stimulation) than linked receptors (Hl-histaminergic, a1-adrenergic, or musca- in cerebral cortex (2.5-fold stimulation). Serotonin was also rinic) did not inhibit the response to serotonin. Fourth, the more potent in choroid plexus, with an EC50 of 46 nM as 5-HT1c site is the only serotonin binding site that has been compared to an EC50 of 540 nM in cerebral cortex. identified in choroid plexus. The 5-HT2, 5-HT1A, and 5-HTIB The putative serotonin antagonists ketanserin, mianserin, sites apparently are not present in this tissue (9, 10). and spiperone inhibited the response to serotonin (Fig. 2; Serotonin is by far the most potent neurotransmitter known Table 1), but their potencies in the two tissues differed. In to regulate a biochemical effector system in choroid plexus. cerebral cortex, all three antagonists had nanomolar poten- For example, norepinephnne stimulates /3-adrenergic recep, 120 A B

100 1 FIG. 2. Effect of mianserin (0) and spiperone (x) upon serotonin- 80- stimulated phosphatidylinositol turnover in cerebral cortex (A) E and of mianserin (e), spiperone (x), and ketanserin (o) upon se- ~60- l \ rotonin-stimulated phosphatidyl- 1 inositol turnover in choroid plexus (B). Increasing concentrations of 40 antagonists were added 15 min OF F~~~~~ before addition of 5 AM serotonin 1 to cerebral cortical slices (A) or 50 nM serotonin to choroid plexus \ (B). The data are presented as the Ipercent of maximum response oc- curring without antagonist added. k I 76 5 4 Each data point is the mean of six determinations. The vertical bars Antagonist, -log M represent SEMs. Downloaded by guest on September 28, 2021 4088 Neurobiology: Conn et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Potencies at serotonin receptors in cerebral cortex and choroid plexus Phosphatidylinositol response Radioligand binding Compound Cerebral cortex Choroid plexus 5-HT2 5-HTlc Ki, nM Ki, nM Mianserin 23 12 6.0 ± 0.6 5.1 ± 1.0 Ketanserin 12* 130 3.1 ± 0.8 1% ± 21 Spiperone 4.3 6200 2.0 ± 0.6 4600 ± 110 EC50, nM Serotonin 540 46 6200 ± 680t 94 ± 6 Apparent Ki values at phosphatidylinositol-linked receptors were estimated from the data in Fig. 2. Apparent Ki values at the two binding sites were estimated from radioligand displacement data by the method of Cheng and Prusoff (19). The binding data are presented as mean values ± SEM from three to five determinations. *Value obtained from Schild analysis of phosphatidylinositol turnover data in ref. 12. tValue obtained from competition binding data in ref. 12. tors linked to adenylate cyclase activation with an EC50 of 30 from Lilly Research Laboratories (P.J.C.) and the National Science AM in this tissue (21), which is lower by a factor of 650 than Foundation (B.J.H.). the potency of serotonin for the 5-HT1c-linked phosphatidyl- 1. Segal, M. B. & Pollay, M. (1977) Exp. Eye Res. Suppl. 25, inositol turnover system. In addition, the potency of seroto- 127-148. nin in the choroid plexus phosphatidylinositol system is 2. Lindvall, M., Edvinsson, L. & Owman, C. (1979) Exp. Neurol. approximately 10-fold higher than its potency in the 5-HT2- 64, 132-145. linked phosphatidylinositol turnover system in cerebral cor- 3. Maeda, K. (1983) Nihon Univ. J. Med. 25, 155-174. tex. It is interesting to note that ketanserin has a relatively 4. Aghajanian, G. K. & Gallagher, D. W. (1975) Brain Res. 88, high affinity for both the 5-HT2 and 5-HT1c receptors. 221-231. Because ketanserin binds with high affinity to the 5-HT2 site 5. Nakamura, S. & Moriyasu, N. (1978) Brain Nerve 30, 259-266. but not to the and sites, some investigators 6. Napoleone, P., Sancesario, G. & Amenta, F. (1982) Neurosci. 5-HTlA 5-HT1B Lett. 34, 143-147. have interpreted the finding that a single concentration or 7. Richards, J. G. & Guggenheim, R. (1982) Trends Neurosci. 5, dose ofketanserin blocks a response to serotonin as evidence 4-5. that the response is mediated by the 5-HT2 binding site. 8. Pazos, A., Hoyer, D. & Palacios, J. M. (1984) Eur. J. However, caution should be taken in making such a conclu- Pharmacol. 106, 539-546. sion because the 5-HT1c site is also blocked by ketanserin. 9. Yagaloff, K. A. & Hartig, P. R. (1985) J. Neurosci. 5, This study suggests that the 5-HT1c binding site on choroid 3178-3183. plexus epithelial cells is a functional receptor linked to 10. Yagaloff, K. A. & Hartig, P. R. (1986) Mol. Pharmacol. 29, phosphatidylinositol turnover. This system may be involved 120-125. on CSF 11. Conn, P. J. & Sanders-Bush, E. (1984) Neuropharmacology 3, in the known actions of serotonin production (3). 993-996. There are reports of indoleaminergic innervation of choroid 12. Conn, P. J. & Sanders-Bush, E. (1985) J. Pharmacol. Exp. plexus (5) and of low levels of serotonin in choroid plexus Ther. 234, 195-203. (22); however, the indoleaminergic innervation occurs most- 13. Conn, P. J. & Sanders-Bush, E. (1986) Fed. Proc. Fed. Am. ly on the walls of blood vessels (5, 6) rather than on the Soc. Exp. Biol. 45, 1637 (abstr.). epithelial cells, where the 5-HTlc site is localized (9). It is 14. Kendall, D. A. & Nahorski, S. R. (1985) J. Pharmacol. Exp. possible that serotonin is released into CSF where it exerts a Ther. 233, 473-479. hormone-like action on choroid plexus. Rich presynaptic 15. De Chaffoy de Courcelles, D., Leysen, J. E., De Clerck, F., stores of serotonin are present in a dense network of Van Belle, H. & Janssen, P. A. J. (1985) J. Biol. Chem. 260, on the surface of cells 7603-7608. serotonergic fibers present ependymal 16. Roth, B. L., Nakaki, T., Chuang, D.-M. & Costa, E. (1984) lining the walls of the ventricles (4). These supraependymal Neuropharmacology 23, 1223-1225. fibers do not form recognizable synapses with ependymal 17. Roth, B. L., Nakaki, T., Chuang, D.-M., Chernow, B. & cells (7) but may release serotonin into the CSF and activate Costa, E. (1985) Fed Proc. Fed. Am. Soc. Exp. Biol. 44, 1244 5-HT1c receptors in choroid plexus. There are many exam- (abstr.). ples of such hormone-like action of serotonin in other 18. Conn, P. J. & Sanders-Bush, E. (1986) Life Sci. 38, 663-669. systems (23-26). 19. Cheng, Y. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, Since the choroid plexus is a relatively simple tissue 3099-3108. containing a monolayer ofepithelial cells (27), these cells can 20. Sills, M. A., Wolfe, B. B. & Frazer, A. (1984) J. Pharmacol. and in culture This Exp. Ther. 231, 480-487. be readily dissociated grown (28). fact, 21. Nathanson, J. A. (1979) Science 204, 843-844. coupled with the high serotonin 5-HTic receptor density and 22. Moskowitz, M. A., Liebman, J. F., Reinhard, J. F., Jr., & the robust phosphatidylinositol response in this tissue could Schlosberg, A. (1979) Brain Res. 169, 590-594. make the choroid plexus a useful model system for studying 23. Kupfermann, I. (1979) Annu. Rev. Neurosci. 5, 207-227. the molecular mechanisms underlying the coupling of phos- 24. Aghajanian, G. K. (1981) in Serotonin Neurotransmission and phatidylinositol-linked receptors to phospholipase C. Behavior, eds. Jacobs, B. L. & Gelperin, A. (MIT Press, Cambridge, MA), pp. 156-185. The expert technical asnistance of Ms. Deborah Mayes and Ms. 25. Bloom, F. E. (1981) in Serotonin Neurotransmission and Be- Marsha is gratefully acknowledged. Also we thank Janssen havior, eds. Jacobs, B. L. & Gelperin, A. (MIT Press, Cam- Breeding bridge, MA), pp. 403-424. Pharmaceutica (Beerse, Belgium) for generously donating samples of 26. Gelperin, A. (1981) in Serotonin Neurotransmission and Be- spiperone and ketanserin. This work was supported by Alcohol, havior, eds. Jacobs, B. L. & Gelperin, A. (MIT Press, Cam- Drug Abuse and Mental Health Administration Research Grant MH bridge, MA), pp. 208-306. 34007 from the National Institute ofMental Health; National Science 27. McComb, J. G. (1983) J. Neurosurg. 59, 369-383. Foundation Grant BNS 84-07432; the Tennessee Department of 28. Crook, R. B., Kasagami, H. & Prusiner, S. B. (1981) J. Mental Health and Mental Retardation; and graduate fellowships Neurochem. 37, 845-854. Downloaded by guest on September 28, 2021