Serotonin was >10-fold more potent in choroid plexus (EC_% = 46 nM) than in cerebral cortex (EC50. = 540 nM). The serotonin antagonists ketanserin, ...
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
(11). These findings have been confirmed and extended in brain (12-14) and other tissues (15-17). Serotonin-stimulated phosphatidylinositol turnover in cerebral cortex is not secondary to release of another neurotransmitter or of arachidonic acid (18), suggesting that the 5-HT2 receptor is directly coupled to phosphatidylinositol turnover. In the present studies, we have examined the effect of serotonin and serotonin antagonists upon phosphatidylinositol turnover in rat choroid plexus. The pharmacology of the response in choroid plexus was compared with that in cerebral cortex and with the pharmacology of the 5-HT1c and 5-HT2 binding sites. The present data support the hypothesis that serotoninstimulated phosphatidylinositol turnover in choroid plexus is mediated by the 5-HTjc binding site in this tissue.
A novel serotonergic binding site, the 5-HTlc ABSTRACT site, has been characterized recently in choroid plexus and several brain regions. The biochemical and physiological roles of this site have not been previously described. In this report we show that serotonin (5-hydroxytryptamine, 5-HT) stimulates phosphatidylinositol turnover in rat choroid plexus. The pharmacology of serotonin-stimulated phosphatidylinositol hydrolysis in choroid plexus was compared to the pharmacology in cerebral cortex, where this response is mediated by the serotonin 5-HT2 receptor. Serotonin increased phosphatidylinositol turnover in choroid plexus by 6-fold and in cerebral cortex by 2.5-fold. Serotonin was >10-fold more potent in choroid plexus (EC_% = 46 nM) than in cerebral cortex (EC50 = 540 nM). The serotonin antagonists ketanserin, mianserin, and spiperone inhibited the response in the two tissues with different potencies. In cerebral cortex all three exhibited nanomolar affinities consistent with their potencies at the 5-HT2 site. In choroid plexus, however, the rank order (mianserin > ketanserin >> spiperone) and absolute potencies were consistent with binding to the 5-HT1c site. These data suggest that the 5-HT1c site in choroid plexus is a functional receptor that utilizes phosphatidylinositol turnover as its biochemical effector system.
METHODS Phosphatidylinositol Turnover. Phosphatidylinositol turnover in rat brain cerebral cortical slices was measured as described (12) except that slices containing approximately 1 mg of protein were added to tubes containing 1 ,Ci (1 Ci = 37 GBq) of [3H]inositol and prelabeled for 3 hr. Agonists and antagonists were added directly to the tubes in which prelabeling occurred without washing away free [3H]inositol. Phosphatidylinositol turnover in choroid plexus was measured in an identical manner except that a single rat choroid plexus [about 0.5 mg (wet weight) of tissue] was added to each tube and prelabeled for 90 min. Apparent Ki values of antagonists at phosphatidylinositollinked receptors were estimated from the relationship: Ki = 1C50/(1 + S/EC50), where IC50 = the concentration of antagonist giving 50% of the maximal inhibition of the response stimulated by S concentration of serotonin, and EC50 = the concentration of serotonin producing halfmaximal stimulation. This equation is modified from the Cheng and Prusoff equation (19) and assumes that the EC50 of serotonin equals its Ki. Since the presence of spare receptors or threshold effects may alter this relationship, the apparent K1 values may not reflect true Ki values. Radioligand Binding. Displacement of [3H]ketanserin binding to the 5-HT2 site in cortex (12) and 125I-LSD binding to the 5-HT1c site in choroid plexus (9) was measured as described except that the buffer used for 125I-LSD binding was identical to that used for measurement of binding of [3H]ketanserin to the 5-HT2 binding site (i.e., contained physiological salts, 10 mM glucose, 10 ,uM pargyline, and 25 mM Tris HCl at pH 7.4). This buffer was the same as that used for measurement of phosphatidylinositol turnover except that 25 mM sodium bicarbonate replaced 25 mM Tris'HCl in phosphatidylinositol studies. We observed that the use of physiological concentrations of salts in the 5-HT2 and 5-HTlc binding assays
The choroid plexus is the major site of cerebrospinal fluid (CSF) production, but the mechanisms controlling production and secretion of CSF are poorly understood (1). Whereas the administration of either norepinephrine or serotonin decreases the secretion of CSF (2, 3), serotonin is the most efficacious monoamine (3). Since the choroid plexus and walls of the ventricles contain serotonergic fibers (4-7), it is possible that endogenous serotonin is involved in the regulation of CSF production. Therefore, it would be of considerable interest to find a receptor that mediates the effect of serotonin in the choroid plexus. We and others (8, 9) have described a novel serotonin binding site on choroid plexus epithelial cells with characteristics distinct from those of 5-HT1A, 5-HT1B, and 5-HT2 sites. This site has been named the 5-HT1c site and can be labeled with [3H]serotonin or 125I-labeled lysergic acid diethylamide (125I-LSD). We have recently reported the solubilization of this site from pig choroid plexus (10). Identification of a biochemical effector system that is linked to the 5-HT1c site would provide strong evidence that this site serves as a functional receptor in choroid plexus and other brain regions (8). Recent data have shown that another serotonin receptor subtype is linked to phosphatidylinositol turnover in the mammalian brain. Based upon the finding that selective 5-HT2 antagonists inhibit serotonin-stimulated phosphatidylinositol turnover in cerebral cortex, we suggested that the response is mediated by the 5-HT2 binding site
5-HT, 5-hydroxytryptamine (serotonin); CSF, cerebrospinal fluid; LSD, lysergic acid diethylamide; 123 I-LSD, 1251_ labeled LSD. tTo whom reprint requests should be addressed. Abbreviations:
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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FIG. 1. Effect of increasing concentrations of serotonin upon phosphatidylinositol turnover in choroid plexus (e) and cerebral cortex (x). Serotonin-induced release of [3H]inositol 1-phosphate was used as a measure of phosphatidylinositol turnover. The data are presented as percent stimulation above basal (3H]inositol 1-phosphate radioactivity, which was 660 ± 29 cpm in cerebral cortex and 1300 ± 119 cpm in choroid plexus. Maximum stimulation resulted in 1807 ± 174 cpm and 7885 ± 832 cpm in cerebral cortex and choroid plexus, respectively. Each point is the mean of six determinations. Vertical bars represent SEMs.
significantly lowered the apparent affinities of some serotonergic ligands at these sites. Apparent Ki values were estimated from radioligand displacement data by the method of Cheng and Prusoff (19). RESULTS AND DISCUSSION Serotonin elicited a concentration-dependent increase in [3H]inositol 1-phosphate release in choroid plexus and cerebral cortical slices (Fig. 1). The effect of serotonin was much more robust in choroid plexus (5- to 6-fold stimulation) than in cerebral cortex (2.5-fold stimulation). Serotonin was also more potent in choroid plexus, with an EC50 of 46 nM as compared to an EC50 of 540 nM in cerebral cortex. The putative serotonin antagonists ketanserin, mianserin, and spiperone inhibited the response to serotonin (Fig. 2; Table 1), but their potencies in the two tissues differed. In cerebral cortex, all three antagonists had nanomolar poten120
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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, with an apparent Ki of 6.2 gM. Ketanserin was also less potent in choroid plexus (Ki = 130 nM), whereas the affinity of mianserin in the two tissues was nearly equal. The addition of 1 ,uM muscarinic, a1-adrenergic or Hl-histaminergic 4ntagonists (atropine, prazosin, or triprolidine) did not alter the release of [3H]inositol 1-phosphate induced by serotonin (data not shown). 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 cerebral cortex (Table 1). In the cerebral cortex, antagonist potencies at the phosphatidylinositol-linked receptor were similar to their potencies at the 5-HT2 binding site. In choroid plexus, however, spiperone was much less potent in blocking serotonin-stimulated phosphatidylinositol turnover, which is consistent with its low potency at the 5-HT1c binding site (Fig. 2; Table 1). Furthermore, the rank order (mianserin > ketanserin >> spiperone) and absolute potencies of the antagonists in blocking phosphatidylinositol turnover and 5-HT1c binding are in excellent agreement (Table 1). Four lines of evidence support the hypothesis that phosphatidylinositol turnover in the choroid plexus is linked to the serotonin 5-HT1c site. First, the EC50 value of serotonin at stimulating phosphatidylinositol turnover in choroid plexus is similar to its Ki value for binding to the 5-HT1c site. Due to the possibility that spare receptors may be present, an agonist EC50 value should be equal to or less than its Ki value for binding to a given receptor. Furthermore, the potency of serotonin in choroid plexus is much higher than in the cerebral cortex, where the response is mediated by the 5-HT2 binding site (11-14). Second, the apparent Ki values of spiperone, ketanserin, and mianserin at the 5-HTic site are similar to their apparent Ki values at the phosphatidylinositollinked serotonin receptor in choroid plexus but differ from their potencies at the 5-HT2, 5-HT1A, and 5-HT1B sites (20). Third, selective antagonists of other phosphatidylinositollinked receptors (Hl-histaminergic, a1-adrenergic, or muscarinic) did not inhibit the response to serotonin. Fourth, the 5-HT1c site is the only serotonin binding site that has been identified in choroid plexus. The 5-HT2, 5-HT1A, and 5-HTIB sites apparently are not present in this tissue (9, 10). Serotonin is by far the most potent neurotransmitter known to regulate a biochemical effector system in choroid plexus. For example, norepinephnne stimulates /3-adrenergic recep,
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FIG. 2. Effect of mianserin (0) and spiperone (x) upon serotoninstimulated phosphatidylinositol turnover in cerebral cortex (A) and of mianserin (e), spiperone (x), and ketanserin (o) upon serotonin-stimulated phosphatidyll\ inositol turnover in choroid plexus (B). Increasing concentrations of antagonists were added 15 min 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 occurring without antagonist added. I k Each data point is the mean of six 76 5 4 determinations. The vertical bars represent SEMs.
1
F~~~~~ Antagonist, -log M
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Table 1. Potencies at serotonin receptors in cerebral cortex and choroid plexus Radioligand binding Phosphatidylinositol response 5-HTlc 5-HT2 Choroid plexus Cerebral cortex Compound nM Ki, Ki, nM 5.1 ± 1.0 6.0 ± 0.6 12 23 Mianserin 1% ± 21 3.1 ± 0.8 130 12* Ketanserin 4600 ± 110 2.0 ± 0.6 6200 4.3 Spiperone EC50, nM 94 ± 6 6200 ± 680t 46 540 Serotonin 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 AM in this tissue (21), which is lower by a factor of 650 than the potency of serotonin for the 5-HT1c-linked phosphatidylinositol turnover system. In addition, the potency of serotonin in the choroid plexus phosphatidylinositol system is approximately 10-fold higher than its potency in the 5-HT2linked phosphatidylinositol turnover system in cerebral cortex. It is interesting to note that ketanserin has a relatively high affinity for both the 5-HT2 and 5-HT1c receptors. Because ketanserin binds with high affinity to the 5-HT2 site but not to the 5-HTlA and 5-HT1B sites, some investigators have interpreted the finding that a single concentration or dose of ketanserin blocks a response to serotonin as evidence that the response is mediated by the 5-HT2 binding site. However, caution should be taken in making such a conclusion because the 5-HT1c site is also blocked by ketanserin. This study suggests that the 5-HT1c binding site on choroid plexus epithelial cells is a functional receptor linked to phosphatidylinositol turnover. This system may be involved in the known actions of serotonin on CSF production (3). There are reports of indoleaminergic innervation of choroid plexus (5) and of low levels of serotonin in choroid plexus (22); however, the indoleaminergic innervation occurs mostly on the walls of blood vessels (5, 6) rather than on the epithelial cells, where the 5-HTlc site is localized (9). It is possible that serotonin is released into CSF where it exerts a hormone-like action on choroid plexus. Rich presynaptic stores of serotonin are present in a dense network of serotonergic fibers present on the surface of ependymal cells lining the walls of the ventricles (4). These supraependymal fibers do not form recognizable synapses with ependymal cells (7) but may release serotonin into the CSF and activate 5-HT1c receptors in choroid plexus. There are many examples of such hormone-like action of serotonin in other systems (23-26). Since the choroid plexus is a relatively simple tissue containing a monolayer of epithelial cells (27), these cells can be readily dissociated and grown in culture (28). This fact, coupled with the high serotonin 5-HTic receptor density and the robust phosphatidylinositol response in this tissue could make the choroid plexus a useful model system for studying the molecular mechanisms underlying the coupling of phosphatidylinositol-linked receptors to phospholipase C. The expert technical asnistance of Ms. Deborah Mayes and Ms. Marsha Breeding is gratefully acknowledged. Also we thank Janssen Pharmaceutica (Beerse, Belgium) for generously donating samples of spiperone and ketanserin. This work was supported by Alcohol, Drug Abuse and Mental Health Administration Research Grant MH 34007 from the National Institute of Mental Health; National Science Foundation Grant BNS 84-07432; the Tennessee Department of Mental Health and Mental Retardation; and graduate fellowships
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