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 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 phosphati- dylinositol-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 phos- phate 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