THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 269, No. 50, Issue of December 16, pp. 31850-31857, 1994 Printed in U.S.A.

Regulation of mRNA Encoding 5-HT, Receptors in P11 Cells Through a Post-transcriptional Mechanism Requiring Activation of Protein Kinase C*

(Received for publication, September 15, 1994)

Robert C. Ferry, Christopher D. Unsworthl, Roman P. Artymyshyn, and Perry B. Molinoff§ From the Denartment of Pharmacology. Universitv of Pennsylvania School of Medicine, Philadelphid, Pennsylvania 19104-6684

" .

Exposure of P11 cells to serotonin (5-HT) resulted in a transient increase in levels of 5-HT, receptor mRNA. Exposure to 5-HT for as short a time as 1 min was suf€i- cient to trigger a delayed increase in receptor mRNA. 5-HT-induced increases in receptor mRNA levels were not antagonized by the protein synthesis inhibitor cy- cloheximide. The increase in receptor mRNA levels was accompanied by a transient increase in the half-life of receptor mRNA; the rate of transcription of receptor mRNA was unchanged. Submaximal stimulation of phos- phinositide hydrolysis by partial agonists or B-fluoro- norepinephrine, an qadrenergic receptor agonist, also increased receptor mRNA levels. Exposure to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, mimicked these effects, whereas the protein kinase C inhibitor bisindolylmaleimide antagonized the effects of both 5-HT and PMA. When agonist-promoted increases in receptor mRNA were prevented, the rate of agonist-induced down-regulation was accelerated. These data suggest that levels of 5-HT, receptor mRNAare regu- lated by phospholipase C-coupled receptors via a protein kinase C-dependent, post-transcriptional mechanism and indicate that agonist-promoted increases in levels of 5-HT, receptor mRNA modulate receptor expression.

The neurotransmitter 5-hydroxytryptamine (5-HT)' inter- acts with multiple subtypes of receptors (1, 2) to elicit diverse effects. One of the first 5-HT receptor subtypes to be cloned was the 5-HT, receptor (3), formerly called the 5-HT2 receptor (1). This receptor is a member of the G protein-linked receptor superfamily and is coupled to the stimulation of PI hydrolysis. Numerous studies of the regulation of this receptor in the cen- tral nervous system have been carried out. Blackshear et al. (4) reported that administration of trifluoromethylphenylpipera- zine, a 5-HT, receptor agonist, resulted in a decrease in the density of 5-HT2, receptors in rat frontal cortex. Similar results

* This work was supported by United States Public Health Service Grants MH 48125 and NS 18591 and Mental Retardation Research Center Grant P30-HD-26979. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Institute for Dementia Research, Miles Inc., West Haven, CT 06516-4175 $To whom correspondence should be addressed: Bristol-Myers

Squibb Company, Pharmaceutical Research Institute, Central Nervous System Drug Discovery, 5 Research Parkway, Wallingford, CT 06492.

The abbreviations used are: 5-HT, 5-hydroxytryptamine (serotonin); DOB, (~)-2,5-dimethoxy-4-bromophenylisopropyl-arnine; DOI, (+)-2,5-

rine; LSD, lysergic acid diethylamide; PI, phosphoinositide; PMA, phor- dimethoxy-4-iodophenylisopropylamine; 6-FNE, 6-fluoronorepineph-

bo1 12-myristate 13-acetate.

Tel.: 203-284-6606; Fax: 203-284-6127.

have been observed after administration of other full or partial agonists, including quipazine (5), LSD, DOB, and DO1 (6). Paradoxically, antagonists of 5-HT, receptors (e.g. mianserin, ketanserin, metergoline, methysergide, and cyproheptadine) have also been observed to cause down-regulation of receptors in vivo (7, 8). However, problems of residual drug in binding assays and possible indirect effects of these agents have com- plicated interpretation of results of studies carried out in vivo using antagonists.

P11 cells, an immortalized cell line derived from transplant- able rat pituitary tumor 7315a (9), have been used as a model system to study regulation of 5-HT, receptors. 5-HTz, recep- tors in this cell line are rapidly desensitized following exposure to agonists (lo), an effect which appears to be mediated in part by a decrease in the density of receptors (10, 11). Agonists and partial agonists, but not antagonists, were found to down- regulate 5-HT, receptors in P11 cells (11). These findings sug- gested that agonist and antagonist-promoted receptor down- regulation in vivo may proceed through distinct mechanisms, with the effects of agonists likely to be the result of direct interactions with the receptors. However, mechanisms respon- sible for agonist-induced down-regulation of receptors have not been identified.

Levels of neurotransmitter receptors can be modulated through regulation of levels of mRNA encoding the receptors. Transcriptional regulation has been described for several G protein-linked receptors, including /3-adrenergic (121, cy-adre- nergic (13), dopamine (141, muscarinic acetylcholine (151, and thyrotropin-releasing hormone (16) receptors. The most widely studied of these receptors is the &-adrenergic receptor, for which a biphasic pattern of receptor mRNA regulation has been observed. Acute exposure to agonists or agents that elevate CAMP levels results in a 34-fold increase in levels of &-adre- nergic receptor mRNA (17). The increase has been shown to result from an increase in the rate of transcription of the &- adrenergic receptor gene, an effect mediated by a CAMP-re- sponse element in the proximal promoter region of the gene (12, 17). In contrast, chronic exposure to agonists results in a 50% decrease in &adrenergic receptor mRNA levels (17, 18). This decrease, which is due to a decrease in receptor mRNA stability (19), is thought to contribute to receptor down-regulation ob- served after prolonged exposure to agonists (12, 20).

Several studies of the regulation of 5-HT, receptor mRNA expression have been carried out, but inconsistent results have been obtained. Prolonged administration of antagonists to rats has been reported to cause an increase (21) or to elicit no change (22) in levels of 5-HTz, receptor mRNA. Results of stud- ies using primary cells in culture have shown that exposure to agonists results in an increase in levels of 5-HT, receptor mRNA (23, 24), whereas exposure to antagonists caused no change in levels of receptor mRNA (25). An extensive time course of changes in mRNA levels in response to drug treat-

31850

Regulation of 5-HT,, Receptor mRNA 31851

ment was not measured in these studies. In addition, intracel- lular mediators of the changes in levels of receptor mRNA were not investigated, making it difficult to determine whether the observed inconsistencies were due to cell type-specific mecha- nisms or were the result of artifacts of primary cell culture systems, which often contain multiple populations of cells. The objectives of the current study were to investigate transcrip- tional regulation of 5-HT, receptors and to identify mecha- nisms which regulate expression of 5-HT, receptor mRNA. An antisense riboprobe specific for 5-HT2, receptor mRNA was constructed and used in ribonuclease protection assays to quantify levels of 5-HT, receptor mRNA in P11 cells. Mecha- nisms responsible for regulating levels of 5-HT, receptor mRNA were explored and intracellular messengers participat- ing in these events were investigated.

EXPERIMENTAL PROCEDURES Materials-[my0-~H]Inositol(20 Ciimmol), [CX-~~PICTP (800 Ci/mmol),

'T-LSD (2200 Ci/mmol), and [a-32PlUTP (6000 Ci/mmol) were pur- chased from Dupont NEN. (~)-2,5-Dimethoxy-4-iodophenylisopro- pylamine hydrochloride, ketanserin tartrate, and 6-fluoronorepineph- rine hydrochloride were purchased from Research Biochemicals Inc. (Natick, MA). Bisindolylmaleimide (GF 109203X) was purchased from Calbiochem. Components of media for cell culture were purchased from Life Technologies, Inc., except fetal bovine serum, which was obtained from Hyclone (Logan, UT). Remaining drugs and chemicals were pur- chased from Sigma, except where otherwise indicated.

Cell Culture-PI1 cells (9) were grown in monolayer culture at 37 "C in high glucose Dulbecco's modified Eagle medium (supplemented with 100 units/ml penicillin, 100 pg/ml streptomycin, 2 mM L-glutamine, 150 pg/ml oxaloacetate, 50 pg/ml pyruvate, 0.2 unitdm1 insulin, 100 unitdm1 nystatin, and 10% charcoal-treated fetal bovine serum) in a humidified atmosphere containing 10% CO,. Cells were detached with a solution of 0.05% trypsin and 0.53 mM EDTA and were plated on 150-mm tissue culture plates at a density of 12,000-14,000 cellslcm'. Medium was replaced every third day. Treatment and harvesting of cells were initiated on days 4-6 when cells were approaching confluence.

Radioligand Binding-Binding assays using 'T-LSD to label 5-HT, receptors in membrane homogenates obtained from P11 cells were car- ried out as described previously (11). Nonspecific binding was defined using 1 ketanserin.

Measurement of PI Hydrolysis-Inositol phosphates were collected after addition of LiCl and analyzed as described previously (11).

Zsolation of RNA-P11 cells were detached from plates with trypsin, centrifuged for 10 min at 500 x g, and resuspended in phosphate- buffered saline. Samples were frozen in liquid nitrogen and stored at -80 "C until processed. RNA was isolated from frozen P11 cells and frozen tissue samples by LiCl precipitation essentially as described by Cathala et al. (26). Briefly, frozen cells from one 15-cm plate or 0.75 g of tissue were homogenized using a Brinkmann polytron (speed 7 for 1.5 min) in 5 ml of a solution containing 5 M guanidine isothiocyanate, 50 nw Tris, pH 7.5, and 10 mM EDTA, pH 8.0.4 M LiCl was then added to a final volume of 40 ml. After precipitation overnight at 4 "C, samples were centrifuged at 16,000 x g for 90 min, and the resulting pellets were resuspended in 5 ml of 3 M LiC1. Samples were centrifuged at 16,000 x g for 1 h and the resulting pellets resuspended in 2 ml of buffer con- taining 0.1% SDS, 1 mM EDTA, pH 8.0, and 10 mM Tris, pH 7.5. After extraction with phenolkhloroformhsoamyl alcohol (25:24:1, pH 5.2) and with chlorofodisoamyl alcohol (24:1), samples were precipitated over- night at -20 "C. RNA pellets were collected by centrifugation for 30 min at 16,000 x g, washed with 80% ethanol, and vacuum-dried. Samples were stored at -80 "C in RNase-free water containing 80 mM sodium acetate and 70% ethanol.

Construction of Recombinant Plasmids for Synthesis 0fRiboprobe.s- Using rat 5-HT, receptor cDNA as the template, a portion of the third intracellular loop corresponding to nucleotides 1380-1571 of the pub- lished sequence (3) was amplified by the polymerase chain reaction using forward (sense) primer, 5'-TCA CTT CAG AAA GAA TTC ACC lTG TGT GTG-3' and reverse (antisense) primer, 5'-TAG AAT ACT CAA GCT TGC ACG CCT TTT GCT CAT TGC-3'. Amplified cDNA was ligated into the EcoRI and HindIII sites of pGEM7Z-A+) (Promega Corp., Madison, WI) to generate a recombinant plasmid (5-HTU-i.J3B). This construct was used to transform Escherichia coli HBlOl cells. Transformed cells were grown on LB agar plates, single colonies

were expanded, and plasmid DNA was isolated using alkaline lysis followed by centrifugation through pZ523 columns (5 Prime-3 Prime, Inc., Boulder, CO). Sequencing of cloned cDNA inserts was carried out using a Sequenase Version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, OH). Preparation of cyclophilin clone plB15, containing a 700-base pair sequence of the coding region of a rat cyclo- philin gene inserted into pSP65, has been described previously (27).

Synthesis of Riboprobes-Plasmids containing a cDNA fragment en- coding a portion of the third intracellular loop of the 5-HT2, receptor (5-HT,-i3/3B) and a 700-base pair cDNA fragment encoding cyclophilin were linearized with EcoRI and HindIII, respectively. SP6 phage RNA polymerase (Promega Corp.) was used to synthesize 5-HT, receptor and cyclophilin antisense RNA transcripts of 228 and 728 bases, respec- tively. Conditions used were: 2 pg of template, 1 pl each of 10 mM ATP, GTP, and UTP, 2 pl of 0.1 M DTT, 40 units of recombinant RNase inhibitor (rRNasin; Promega Corp.), 40 units of RNApolymerase, 5 pl of transcription buffer (Promega Corp.), 80 pCi of [CX-~~PICTP (800 Cii mmol), and 0.5 pl of 0.1 mM CTP (5-HT, riboprobe) or 2 pl of 10 mM CTP (cyclophilin riboprobe) in a total volume of 25 p1. Reactions were incu- bated for 90 min at 38 "C, after which 2 units of RQ1 RNase-free DNase (Promega Corp.) were added to digest the template. Riboprobes were separated from unincorporated nucleotides by centrifugation through RNase-free Select D G-50 (RF) columns (5 Prime-3 Prime, Inc.). Tri- chloroacetic acid precipitation and liquid scintillation spectroscopy were used to measure the amount of label incorporated into the probe. The integrity of riboprobes was assessed by size fractionation of an aliquot of each riboprobe on a polyacrylamide gel followed by autoradiography.

Ribonuclease Protection Assay-An aliquot of total RNA (10 pg) from each sample was placed into a 1.5-ml microfuge tube and vacuum-dried. Hybridization buffer (40 pl of buffer consisting of 80% deionized form- amide with 400 mM NaCI, 40 mM Tris, pH 6.4, and 2 mM EDTA. pH 8.0) plus 500,000 counts of the 5-HTZ, riboprobe and 50,000 counts of the cyclophilin riboprobe were added to each tube, and samples were al- lowed to hybridize at 45 "C for 14-18 h. After hybridization, the mixture was digested with 2 pg of RNase A and 1900 units of RNase T1 (Pro- mega Corp.) for 90 min at room temperature in buffer containing 300 mM NaC1,20 mM Tris, pH 7.4, and 5 mM EDTA, pH 8.0. SDS (23 pl of a 10% solution) containing 60 pg of proteinase K (Boehringer Mannheim) was added to each tube, followed by incubation at 37 "C for 20 min. Following extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 5.21, samples were precipitated on dry ice for 30 min with 2 volumes of ethanol in the presence of 30 pg of yeast tRNA, which served as a carrier to facilitate precipitation. Samples were centrifuged, washed with 70% ethanol, and vacuum-dried. Samples were then dissolved in formamide loading buffer and size fractionated on a 7.8 mM urea, 5% polyacrylamide gel. Dried gels were apposed to phosphor storage screens for 5-20 h and then scanned and digitized using a Molecular Dynamics model 4005 PhosphorImager. The signal corresponding to the protected 5-HT, receptor riboprobe fragment was normalized to that of the cyclophilin signal from the same sample to control for experimental variability.

Nuclear Run-on Assays-Cells from one 10-cm plate (approximately 4 x lo7 cells) were used for isolation of nuclei according to the alternate protocol described by Greenberg and Bender (28). Briefly, cells were washed with phosphate-buffered saline and recovered by scraping with a rubber policeman. Cells were pelleted and resuspended in ice-cold lysis buffer, pelleted again, and resuspended in 1 ml of lysis buffer. An aliquot of Nonidet P-40 lysis buffer (1 ml) was added, and cells were homogenized using 15 strokes of a Dounce homogenizer. Pelleted nuclei were resuspended in 200 pl of glycerol storage buffer and stored at -80 "C. Purity of preparations of nuclei was assessed by phase-contrast microscopy.

Prepared nuclei (200 pl) were added to 200 pl of a reaction buffer containing (final concentrations): 5 mM Tris, pH 8.0,2.5 mM MgCl,, 0.15 M KC1, 2.5 II~M dithiothreitol, 0.5 mM unlabeled GTP, ATP, and CTP, and 250 pCi of tw3'P1UTP (6,000 Ci/mmol). The transcription mixture was incubated for 30 min at 30 "C, digested with RNase-free DNase and then with proteinase K, and extracted with phenolkhlorofodisoamyl alcohol (25:24:1, pH 5.2). Following trichloroacetic acid precipitation, transcripts were collected by filtration through Whatman GF/A filters. Radiolabeled RNA retained on the filters was recovered, treated again with DNase and proteinase K, extracted with phenollchloroford isoamyl alcohol (25:24:1, pH 5.21, and precipitated. Radiolabeled RNA was dissolved in diethylpyrocarbonate-treated water and heated to 90 "C for 5 min. Labeled RNA was hybridized to linearized plasmid (4 pg/slot) containing full-length rat 5-HT,, receptor cDNA (in vector pvL1393), cDNA encoding the third intracellular loop of the rat 5-HTZ,

31852 Regulation of 5-HT,, Receptor mRNA 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

c - - - - - - -700 bases -728 bases

0 -228 bases

j- - - .- - - -183 bases

5-HT Vehicle """M

P11 cells FIG. 1. Ribonuclease protection assay for 5-HT, receptor

mRNA. Autoradiograph of a polyacrylamide gel from a typical assay. Hybridization of 5-HT, receptor and cyclophilin riboprobes to total RNA from rat forebrain (lane 1 ), full-length sense 5-HT2, receptor RNA synthesized in vitro (lane 2), yeast tRNA (lane 31, and samples without RNA (lane 4 ) are shown. Undigested 5-HT, and cyclophilin riboprobes

with 10 PM 5-HT (lanes 7-9) or vehicle (lanes 10-12) for 1 h on receptor are shown in lanes 5 and 6, respectively. Lanes 7-12, effect of incubation

mRNA levels in P11 cells. Numbers represent length of riboprobes and riboprobes fragments.

receptor (in pGEM7Z-ff +), see Construction of Recombinant Plasmids for Synthesis of Riboprobes), or vector DNA immobilized on nitrocellu- lose membranes. Hybridizations using equivalent amounts of labeled RNA were carried out at 42 "C for 60 h after prehybridization for 6 h in hybridization solution lacking radiolabeled RNA. The hybridization so- lution contained (final concentrations): 6 x SSC (0.9 M NaCl, 0.1 M citric acid), 50% formamide, 100 pg/ml of sheared salmon sperm DNA, 5 x Denhardt's Solution, and 0.5% SDS. Membrane strips were washed in 2 x SSC, 0.5% SDS for 5 min at room temperature, 2 x SSC, 0.1% SDS for 15 min a t room temperature, 0.1 x SSC, 0.5% SDS for 30 min at 37 "C, and in 0.1 x SSC, 0.5% SDS for 30 min at 52 "C. Autoradiograms were generated by exposing membrane strips to film and intensifying screens for 5 days at -80 "C. For quantitation, dried membrane strips were apposed to phosphor storage screens for 20 h and then scanned and digitized using a Molecular Dynamics model 400s PhosphorImager.

Statistics-All experimental values are reported as means stand- ard errors. Individual experiments included three or more independent determinations and were carried out on two or more separate occasions. Individual determinations, either within a given experiment or from several independent experiments, are listed as number of determina- tions or = ." For binding experiments, data are shown as means 2 standard errors of three determinations each performed in triplicate. Comparisons of the data were made with a one-way analysis of variance (ANOVA) using StatView software for the Macintosh. The degree of certainty of statistically significant differences among data within a given experiment or between replicate experiments is indicated by a p value which is reported in the figure legends and in the text, respectively.

RESULTS Ribonuclease Protection Assay for 5-HTzA Receptor mRNA-

Based on the reported sequence of the 5-HT2, receptor, a 32P- labeled riboprobe which specifically recognized mRNA encod- ing the third intracellular loop of the 5-HT2, receptor was generated (see "Experimental Procedures"). Hybridization of this riboprobe to total RNAisolated from P11 cells, followed by digestion with single strand-specific ribonucleases and dena- turing polyacrylamide gel electrophoresis, yielded a protected riboprobe fragment of 183 bases (Fig. 1). Cohybridization with a 32P-labeled cyclophilin riboprobe yielded a second protected fragment of 700 bases. When compared to the amount of total RNA added to each tube (assessed by absorbance at 260 nM),

Time (h)

4 . , . , . I . I

0 2 4 6 8

Time of Exposure Lo 5-HT (3)

FIG. 2. Transient up-regulation of 5-HT, receptor mRNAafter exposure to of 5-HT. P11 cells were treated with 10 PM 5-HT and harvested at the indicated times. Total RNA was isolated and levels of 5-HT2, receptor mRNA were measured using a ribonuclease protection assay. Inset, effect of prolonged incubation with 5-HT on 5-HT, recep- tor mRNA levels. Data shown are combined from three independent experiments carried out on separate days and represent means -c stand- ard errors ( n = 3-6). *, p < 0.01 versus corresponding vehicle control; **, p < 0.005.

levels of cyclophilin mRNA were found to be unaffected by exposure to a variety of drugs, including those used in this study. The 5-HTzA receptor signal was normalized to that of the cyclophilin signal appearing in the same lane.

Full-length sense 5-HT2, receptor RNA synthesized in vitro and hybridized to the 5-HT2, receptor-specific riboprobe iden- tified the position of the protected riboprobe fragment and served as a positive control (Fig. 1, lane 2 1. A band in an iden- tical position was seen when the riboprobe was hybridized to total RNA from rat forebrain, a tissue rich in 5-HT2, receptors (Fig. 1, lane 1) . (Upper band in lane 1 represents protected cyclophilin riboprobe fragment.) Hybridization to non-homolo- gous RNA (Fig. 1, lane 3 and samples containing no RNA (Fig. 1, lane 4 ) served as negative controls.

Effects of 5-HT on Levels of 5-HTzA Receptor mRNA- Exposure to 5-HT resulted in an increase in 5-HT2, receptor mRNA levels (Fig. 1, lanes 7-9 and 10-12). The increase in mRNA after exposure to 5-HT was time dependent, with sig- nificant increases occurring within 30 min (Fig. 2). Levels of receptor mRNA were maximally increased in cells treated with 10 5-HT for 90 min (Fig. 2). In three experiments, mRNA levels in cells treated for 90 min were increased to 177-226% of control levels (195 f 4%, n = 11, p < 0.001). The increase in levels of receptor mRNA was dose dependent, with an EC,, of approximately 400 nM (data not shown). Levels of receptor mRNA were elevated only transiently and returned to control levels within 8-16 h (Fig. 2, inset). Continued exposure to 5-HT for up to 48 h caused no further change in the levels of receptor mRNA compared to control values. For this prolonged incuba- tion, 5-HT was added every 12 h to ensure that a sufficient concentration of 5-HT was present to fully occupy the receptors during the entire course of the experiment. This redosing schedule was based on the half-life of serotonin in cell culture, which was determined using high pressure liquid chromatog- raphy to be 13.6 h (data not shown).

Effects of Acute Exposure to 5-HT on Levels of 5-HTzA Recep- tor mRNA-Exposure to 5-HT for less than 15 min did not alter 5-HT2, receptor mRNA levels when measured immediately fol- lowing treatment. However, exposure for less than 15 min was sufficient to cause an increase in receptor mRNA levels when assayed long after exposure to 5-HT had ended. The time course for induction of the delayed increase in receptor mRNA was measured by incubating cells with 5-HT for defined periods of time, after which the 5-HT2, receptor antagonist ketanserin

Regulation of 5-HT,, Receptor mRNA

100 1 1

E

4 ** L. *** 3 0.6 . 0

d 0.4 - - 3 g 0.2

- v)

0.0 0

5-HT I

0 20 40 60

Duration of Exposure to 5-HT Before Adding Ketanserin (min)

receptor mRNA. P11 cells were incubated with 10 w 5-HT for t = 1, FIG. 3. Effects of brief exposure to 5-HT on levels of 5-HT,

5, 10,20,40, or 60 min, followed by incubation with 1 w ketanserin for (90 - t ) min. Samples were processed at t = 90 min, total RNA was isolated, and levels of receptor mRNA were measured using a ribonu- clease protection assay. Inset, effect of ketanserin (Ket) on levels of 5-HT,, receptor mRNA and on 5-HT-induced increases in levels of re- ceptor mRNA after incubation with drugs for 90 min. Data shown are means k standard errors ( n = 3). Similar data have been observed in two additional experiments. *, p < 0.01 compared to vehicle (Veh); **, p c 0.001.

(1 PM) was added and the incubation allowed to continue for a total of 90 min. Ketanserin prevented further activation of 5-HT, receptors without the need to include a wash step to remove 5-HT. Using this experimental paradigm, exposure to 5-HT for 1 min was observed to result in a 38% increase in levels of 5-HT2, receptor mRNA when measured 89 min after ketanserin was added (Fig. 3). In two independent experi- ments, a 1-min exposure caused a 25-38% increase in receptor mRNA levels (32 2 5%, n = 6, p < 0.005). This effect was time dependent: a 5-min exposure to 5-HT caused a 52% increase in receptor mRNA when measured 85 min after addition of ket- anserin, and a 20-min exposure to 5-HT caused a 68% increase when measured 70 min later (Fig. 3). In two separate experi- ments, a 5- and 20-min exposure to 5-HT resulted in delayed increases in receptor mRNA of 28-53% (38 2 6%, n = 6, p < 0.005) and 63-73% (69 12%, n = 6, p < 0.005), respectively. Ketanserin alone had no effect on receptor mRNA levels but completely antagonized the 5-HT-mediated increase (Fig. 3, inset).

The design of this experiment required that ketanserin im- mediately block further activation of 5-HTZA receptors by 5-HT. The ability of ketanserin to abolish the agonist activity of 5-HT was tested by adding ketanserin to cells that had been exposed to 10 p~ 5-HT for 5 min. Levels of inositol phosphates measured 1-25 min after the addition of ketanserin were the same as those seen after exposure to 5-HT for 5 min (data not shown). Because 5-HT causes a time-dependent increase in inositol phosphate levels (91, this indicated that ketanserin promptly antagonized the agonist activity of 5-HT.

Effects of Cycloheximide on Levels of 5-HTZA Receptor mRNA-To determine whether the increase in 5-HT, receptor mRNA levels after exposure to 5-HT required de novo protein synthesis, cells were treated with the inhibitor of protein syn- thesis cycloheximide (2.5 pg/ml) for 30 min prior to exposure to 5-HT for 60 min. This dose of cycloheximide inhibited protein synthesis by 96.9 2 0.3% in control cells and by 97.3 2 0.1% in 5-HT-treated cells as measured by incorporation of [14C]leucine (n = 4). Cycloheximide itself caused a small increase in levels of receptor mRNA but did not prevent 5-HT from elevating recep- tor mRNA levels (Fig. 4); levels of receptor mRNA in cells ex- posed to 5-HT for 1 h were increased to 158-170% of control levels (165 2 3%, n = 4, p < 0.001) in vehicle-treated cells and

2 0.8 t ***

31853

Veh 5-HT Veh 5-HT

Control + Cycloheximide

FIG. 4. Effects of cycloheximide on 5-HT-induced increases in levels of 5-HT, receptor mRNA. P11 cells were pretreated with cycloheximide (2.5 pg/ml) or vehicle (control) for 30 min and then incu- bated with 10 p~ 5-HT or H,O (Veh) for an additional 60 min. Total RNA was isolated and receptor mRNA levels were measured using a ribonu- clease protection assay. The data are expressed as the ratio of 5-HT, receptor mRNA to cyclophilin mRNA and are shown as means k

standard errors (n = 4). Similar results have been obtained in an inde- pendent experiment ( n = 4). ***, p < 0.001 uersus corresponding vehicle control; **, p < 0.001, uersus control vehicle.

147-157% (153 2 2%, n = 4 , p < 0.001) in cycloheximide-treated cells.

Effects of 5-HT on the Rate of Dunscription of the 5-HTZA Receptor Gene-To determine whether changes in the rate of transcription were responsible for 5-HT-induced changes in 5-HT, receptor mRNA levels, nuclear run-on assays were car- ried out after harvesting nuclei from cells treated with 5-HT for 45 min or 2.5 h. (Receptor mRNAlevels are increasing (45 min) or decreasing (2.5 h) at these times (Fig. 21.1 Newly elongated transcripts were hybridized to membranes containing immobi- lized full-length 5-HT, receptor cDNA or cDNA encoding the third intracellular loop of the 5-HT, receptor (Fig. 5A). The rate of SHT, receptor gene transcription was found to be unchanged at both times as measured using either cDNA as template (Fig. 5B). Similarly, no significant change in the rela- tive rate of transcription was observed after a 5- (0.90 2 0.08, versus control; n = 4) or 10- (0.95 2 0.13) min incubation with

Effects of 5-HT on the Stability of 5-HTZA Receptor mRNA- Because exposure to 5-HT did not alter the rate of 5-HT,, receptor gene transcription, turnover of receptor mRNA was studied in 5-HT-treated cells by measuring receptor mRNA levels at various times after inhibiting transcription with acti- nomycin D. Receptor mRNA turnover was examined after in- cubating cells with 5-HT for 45 min or 2.5 h, when levels of receptor mRNA are approximately equal (Fig. 2). (Receptor mRNA levels are increasing (45 min) or decreasing (2.5 h) at these times (Fig. 21.) Receptor mRNAin cells treated with 5-HT for 45 min decreased more slowly after the addition of actino- mycin D than in vehicle-treated cells (Fig. 6). A semi-logarith- mic plot of the data in this experiment revealed that 5-HT increased the half-life of 5-HTZA receptor mRNA by approxi- mately 75% (Fig. 6, inset A ) . In three independent experiments mRNA stability was increased from 73-112% (95 2 11%, n = 9, p < 0.001). The increase in receptor mRNA half-life was tran- sient; following a 2.5-h exposure to 5-HT, the half-life of recep- tor mRNA returned to control values (Fig. 6, inset B ). In three separate experiments (n = 9), no significant difference ( p > 0.2) in receptor mRNA half-life was observed in cells treated with 5-HT for 2.5 h (tla = 71-98 min; average tl,z = 82 2 8 min) compared to control values (65-76 min; 70 2 3 rnin). Incubation with actinomycin D for up to 180 min did not decrease levels of cyclophilin mRNA, indicating that turnover of cyclophilin

5-HT.

31854 FIG. 5. Relative rate of transcrip-

tion of the 5-HT, receptor gene fol- lowing exposure to 5-HT as assessed by nuclear run-on assays. A, PI1 cells grown for 6 days in 10-cm dishes were exposed to 10 p~ 5-HT for 45 min or 2.5 h. Cells were harvested, nuclei were iso- lated, and nuclear run-on assays were carried out in the presence of 250 pCi of [(r-R2PlUTP as described under "Experi- mental Procedures." Equal amounts of newly elongated RNA were hybridized to linearized plasmids (4 pgklot) containing full-length 5-HT, receptor cDNA ( I ) , cDNA encoding the third intracellular loop of the 5-HT2, receptor (3), or to plas-

(pvL1393 (2), or pGEM7Z-A +) (4), respec- mid lacking receptor cDNA inserts

tively) as controls. Blots were exposed to film and intensifying screens for 5 days at -80 "C. B, phosphor storage screens ap- posed to the blots shown inA for 24 h were scanned and digitized on a Molecular Dy- namics PhosphorImager. Background hy- bridization to plasmids not containing cDNA inserts was subtracted to account for nonspecific hybridization. Data shown are expressed relative to control levels and represent means * standard errors of duplicate determinations. Similar results have been obtained in two separate experiments.

Regulation of 5-HT,, Receptor mRNA

A. Control 10 PM 5-HT 10 VM 5-HT

45 min 2.5 hr

1 2 3 4 1 2 3 4 1 2 3 4

B.

-"-U Vehicle

o ; - . - . - . - . 0 100 200 300 400

Time After Addition of Actinomycin D (min)

FIG. 6. Changes in the stability of 5-HT, receptor mRNA after exposure to 5-HT. P11 cells were treated with 10 p~ 5-HT or vehicle (H,O) for 45 min or 2.5 h before the addition of actinomycin D (5 pg/ml). Cells were harvested at various times after the addition of actinomycin D, and receptor mRNA levels were measured using a ribonuclease pro- tection assay. Shown in the main panel are results obtained from cells that were treated with 5-HT for 45 min. Inset, semi-logarithmic plots of 5-HT,, receptor mRNA decay following inhibition of transcription with actinomycin D in cells treated with 5-HT for 45 min (A) or 2.5 h (B). Receptor mRNA half-lives (t,n) were calculated by linear regression of In (MJM,) versus time, where M, = receptor mRNA levels a t given time ( t ) , and M, = receptor mRNA levels at time zero. Data are shown as means * standard errors ( n = 3). This experiment has been repeated twice with similar results. *, p < 0.05 versus vehicle; **, p < 0.01; ***, p < 0.001.

mRNA is slower than that of 5-HT, receptor mRNA. Effects of LSD, DOI, and SFNE on Levels of 5-HTZA Receptor

mRNA-Compared to 5-HT, LSD and DO1 were partial ago- nists at stimulating PI hydrolysis (Fig. 7A) and increasing levels of 5-HTz, receptor mRNA (Fig. 7B). Incubation of P11 cells with 1 p~ LSD or 100 nM DO1 for 90 min resulted in 38 2 10% (25-58%, n = 3) and 33 e 3% (28-38%, n = 3) increases, respectively, in receptor mRNA levels. Stimulation of a,-adre- nergic receptors with 6-FNE, an a,-adrenergic receptor agonist which causes an increase in PI turnover similar in magnitude to that seen with LSD and DO1 (Fig. 7A), elicited a 21 5 1% (20-22%, n = 3) increase in the levels of 5-HTzA receptor mRNA (Fig. 7B). The effect of 6-FNE was not a consequence of a

1.25{ -,- Template: 0 Full-length

Third intracellular loop I I I , I

1.00 - I I

0.75 - 0.50 - 0.25 - O A

Control 45 min 2.5 hr

nonselective interaction with 5-HTz, receptors because homol- ogous desensitization of 5-HT2, receptors on P11 cells does not reduce the ability of 6-FNE to stimulate PI hydrolysis (10).

Effects of Modulators of Protein Kinase C on Levels of 5-HT,, Receptor mRNA-Treatment of P11 cells with 100 nM PMA for 105 min resulted in a 2-fold increase in 5-HTz, receptor mRNA levels (Fig. 8), an increase that was similar in magnitude to that observed after continuous exposure to 10 p~ 5-HT for 90 min (Fig. 8). Coincubation with 5-HT for 90 min did not lead to an additional increase in receptor mRNA, indicating that levels of mRNA were maximally increased by either treatment and suggesting that the effects of the drugs operate through com- mon mechanisms. Exposure of cells to the inactive phorbol ester 4a-phorbol 12,13-didecanoate did not alter receptor mRNA levels and did not interfere with the ability of 5-HT to increase receptor mRNA levels (Fig. 8). Bisindolylmaleimide (GF 109203X), a potent and selective inhibitor of protein kinase C (291, had no effect on 5-HTz, receptor mRNA levels, but fully antagonized the effects of PMA (Fig. 8). Bisindolylmaleimide also completely prevented 5-HT from increasing receptor mRNA levels (Fig. 8).

Rate of 5-HTM Receptor Down-regulation in the Presence or Absence of Increases in Levels of Receptor mRNA-If agonist- induced increases in levels of 5-HT, receptor mRNA are trans- lated into functional protein, the rate of receptor down-regula- tion in the absence of agonist-promoted increases in receptor mRNA would be expected to be faster than in cells in which the increase in receptor mRNA occurred. Actinomycin D, an inhib- itor of transcription, was used to prevent agonist-stimulated increases in levels of receptor mRNA. Exposure of P11 cells to actinomycin D prevented 5-HT from increasing receptor mRNA levels and resulted in a marked decrease in levels of 5-HT2, receptor mRNA (Table I) without affecting the density of recep- tors (after a 5-h incubation, receptor density was 99.5 * 2.9% of control levels; n = 3). Levels of receptor mRNA in cells treated with actinomycin D or actinomycin D and 5-HT were similar, approximately 30% of control levels, whereas levels of receptor mRNA in cells treated with 5-HT alone were increased by ap- proximately 75% over control levels. In cells treated with acti- nomycin D and 5-HT, the rate of receptor down-regulation was

Regulation of 5-HT,, Receptor mRNA 31855

A. PI Hydrolysis

l 2 I

I

0.0 vrh LSD Do1 6FNE 5.m

B. S-HT~A Receptor mRNA

200

3 150 b 8 100 *

50

Veb LSD DO1 6-FNE 5-HT

FIG. 7. Stimulation of PI hydrolysis and elevation of 5-HT, receptor mRNAlevels after activation of 5-J.-lT, and a,-adrener- gic receptors. Inositol phosphate production (A) and 5-HT, receptor

LSD, 100 n~ DOI, or 100 p~ 6-FNE for 45 (A) or 90 ( B ) min. PI data are mRNA levels (B) were measured after incubation with 10 p~ 5-HT, 1 PM

shown as means f standard errors ( n = 5) and are expressed as the percentage of membrane phospholipids converted to inositol phos- phates. Receptor mRNA levels were measured using a ribonuclease protection assay, and the data are expressed as means f standard errors ( n = 3). Similar results have been obtained in two additional groups of experiments. *, p < 0.05 compared to vehicle (Veh); **, p < 0.01; ***, p < 0.001.

0 Vehide Challenge: 5-HT

Untreated PMA 4a-phorbol Bisindolyl- maleimide

Pretreatment FIG. 8. Effects of activators and inhibitors of protein kinase C

on levels of 6-HT, receptor mRNG P11 cells were pretreated with 100 n~ PMA, an activator of protein kinase C, 500 n~ 4a-phorbol12,13- didecanoate (4a-phorbo1, an inactive phorbol analog), or 5 p~ bisindolyl- maleimide (an inhibitor of protein kinase C) for 15 min and then incu- bated for an additional 90 min with or without 10 p~ 5-HT or 100 n~ PMA. Levels of receptor mRNA were measured using a ribonuclease protection assay. Vehicle for 5-HT was H,O; vehicle for PMA, 4a-phor- bol, and bisindolylmaleimide was dimethyl sulfoxide (0.1% final con- centration). Data shown represent means standard errors ( n = 3). Similar results have been obtained in two additional independent ex- periments. *, p < 0.025, versus vehicle; **, p < 0.005.

greater than in cells treated with 5-HT alone (Fig. 9); the half- lives (t,) of receptors were 136 and 230 min, respectively (Fig. 9, inset). In three experiments (n = 91, actinomycin D acceler- ated 5-HT-induced down-regulation by 2641% (34 2 4%, p < 0.01).

Effects of Acute Exposure to 5-HT on the Density of 5-HT, Receptors-Receptor density on cells treated with 10 PM 5-HT decreased as a function of time of exposure to drug (Fig. 10, solid bars). Using a paradigm similar to that used to study

TABLE I Effects of actinomycin D on levels of 5-HT,, receptor mRNA

in P11 cells P11 cells were treated with vehicle (ethanol 0.25% final concentra-

tion) (1, 2) or actinomycin D (5 &ml) (3,4) for 30 min before exposing cells to H,O (1, 3) or 10 p~ 5-HT (2, 4) for an additional 90 min. Total RNA was isolated, and receptor mRNA levels were measured using a ribonuclease protection assay. Values shown represent means 2 standard errors of three determinations. Similar results have been observed in two additional experiments.

Treatment 5-HT, receptor RNA

% vehicle control

1. Vehicle 100.0 f 4.5

3. Actinomycin D 31.6 f 2.3" 4. Actinomycin D + 5-HT 32.0 f 2.0"

2. 5-HT 174.0 f 4.3"

" p < 0.001, versus vehicle.

t

c 3 Actinomycin D + 5-HT

0 0 100 200 300 400 500

Time (rnin)

of 5-HT, receptor mRNA on 5-HT-induced down-regulation of FIG. 9. Effects of preventing 5-HT-induced increases in levels

5-HT, receptors. P11 cells were treated with 10 p~ 5-HT or with the inhibitor of transcription actinomycin D (2.5 pg/ml) and 5-HT. Cells were harvested as indicated, membranes were prepared, and binding assays carried out using 1 n~ lZ5I-LSD. Inset, semi-logarithmic plot of data shown in main panel. The half-life (t,) of receptors was calculated from linear regression of ln(BJB,) uersus time, where B, = receptor density at a given time ( t ) , and Bo = receptor density at time zero. Results were calculated as a percentage of vehicle-treated controls, each assayed in triplicate, and are shown as means f standard errors (n = 3 or 4). This experiment has been repeated twice with similar results. *, p < 0.05, versus group receiving actinomycin D + 5-HT; **, p < 0.025.

effects of acute exposure to 5-HT on 5-HT, receptor mRNA levels (Fig. 3), the effects on receptor density of brief receptor stimulation were studied. Receptor down-regulation activated by a 15-min exposure to 5-HT (stippled bars) continued after the addition of the receptor antagonist ketanserin. The delayed receptor down-regulation proceeded to an extent similar to that seen in cells continually exposed to 5-HT. Cells treated with ketanserin had the same density of receptors as control cells confirming that the down-regulation observed in cells treated with both 5-HT and ketanserin was not due to the presence of residual ketanserin in the binding assay.

DISCUSSION

A major finding in the current study is that agonists elicit a biphasic change in levels of mRNA encoding 5-HT2, receptors. Acute exposure to 5-HT leads to an increase in receptor mRNA levels whereas longer exposure causes receptor mRNA levels to return to control values. Levels of cyclophilin mRNA were not altered indicating that the effects of 5-HT were not the result of a generalized change in the transcriptional activity of P11 cells. A transient increase in &-adrenergic (12) receptor mRNAlevels has also been observed after exposure to agonists. However, in contrast to the current results in which a decrease in levels of receptor mRNA to below control levels was not observed, pro- longed exposure to agonists has been reported to reduce &-

31856 Regulation of 5-HT,, Receptor mRNA

5-HT (10 pM) - 15 40 90 120 15 15 15 15 - Ketsnserin (1 phS) - _ . " - 25 75 105 15

Total incubation time . 15 40 90 120 15 40 90 120 15

Duration of Exposure (min)

5-HT, receptors. P11 cells were incubated with 10 5-HT (solid FIG. 10. Effects of acute exposure to 5-HT on the density of

bars), 5-HT and then 1 p ketanserin (stzppled bars), or ketanserin (open bars) for the times indicated. Total incubation times for cells treated with 5-HT or 5-HT and then ketanserin were identical. Cells were harvested as indicated, membranes were prepared, and binding assays carried out using 1 nM lz5I-LSD. Results were calculated as a percentage of radioligand specifically bound to membranes of vehicle- treated cells, each assayed simultaneously and in triplicate. Data are

errors (n = 8). Similar results have been obtained in a third experiment combined from two experiments and are shown as means * standard

(n = 5). * , p < 0.001, uersus untreated control (dotted bar).

adrenergic receptor mRNA levels to 50% of control values (17). Surprisingly, exposure to 5-HT for periods of time as short as

1 min was sufficient to cause a delayed increase in 5-HT,, receptor mRNA levels. The finding that prolonged exposure to 5-HT is not required to alter receptor mRNA levels demon- strates that increases in levels of receptor mRNA do not require prolonged stimulation of the receptors and indicates that expo- sure to 5-HT for short periods of time is sufficient to trigger increases in receptor mRNA. A process that requires only brief receptor stimulation might be expected to regulate receptor mRNA in vivo, where the duration of action of endogenous 5-HT is transient due to its reuptake and metabolism. In ad- dition to effects on receptor mRNA, stimulation of receptors for brief periods of time also has marked effects on receptor density (see below).

Arecently identifiedm-1-binding site in the promoter region of the 5-HTZA receptor gene (30) raised the possibility that immediate early genes such as c-fis participated in triggering the increase in levels of receptor mRNA. However, instead of an increase in the rate of transcription of the 5-HT,, receptor gene a transient increase in the stability of receptor mRNA was observed. Induction of receptor mRNA via a post-transcrip- tional mechanism which increases receptor mRNA stability is unique among non-peptide G protein-coupled receptors. Thus, for pz. (17) and a,,-adrenergic (13) receptors, as well as for 5-HT, receptors in uterine smooth muscle cells (24), enhanced rates of receptor gene transcription appear to account for ago- nist-induced increases in receptor mRNA.

Similar to the results of studies of &-adrenergic receptor mRNA (17), 5-HTZA receptor mRNA levels were elevated for only a short period of time after exposure to agonists, a finding that appears to be a consequence of a return of the half-life of receptor mRNA to control values during prolonged exposure to 5-HT. The decline in mRNA levels was not secondary to oxida- tion or metabolism of 5-HT because 5-HT, which has a half-life of approximately 13 h in medium, was added every 12 h. De- creases in the stability of receptor mRNA transcripts after pro- longed exposure to agonists have been documented in other receptor systems (19, 311, suggesting that changes in receptor mRNA stability are a common way to decrease levels of mRNA

I 120

120

encoding neurotransmitter receptors. A 32-kDa mRNA-binding protein, which has a binding spec-

ificity for repeats of the AUWA nucleotide pentamer, a con- sensus sequence that has been implicated in regulation of mRNA stability, has been described for PI- and &-adrenergic receptor mRNA (32). The expression of this protein varies in- versely with &-adrenergic receptor mRNA levels suggesting that it binds to, and destabilizes, &adrenergic receptor mRNA. Whether or not related stabilizing or destabilizing proteins that affects the stability of 5-HTz, receptor mRNA exist, and whether or not exposure to 5-HT would alter their activity, is not known at the present time.

Results from the current study contrast with results of two recent studies of the regulation of 5-HT,, receptor mRNA. In rat cerebellar granule cells (23) and in rat uterine smooth mus- cle cells (241, exposure to 5-HT causes a 2-4-fold elevation of 5-HTZA receptor mRNA levels that is sustained for 12 h or more, whereas in P11 cells receptor mRNA was elevated for only a brief period of time. The disparate results might be a conse- quence of distinct regulatory mechanisms. Inhibition of de novo protein synthesis with cycloheximide prevents agonist-induced increases in levels of 5-HT,, receptor mRNA in uterine smooth muscle cells (24) and in cerebellar granule cells (231, but did not prevent the increase in P11 cells. Furthermore, the increase in 5-HT, receptor mRNA in uterine smooth muscle cells appears to be the result of an increase in the rate of transcription of the receptor gene (24), rather than a change in the stability of receptor mRNA as was observed in P11 cells. Together these findings indicate that multiple mechanisms can participate in regulating levels of 5-HT, receptor mRNA and that these reg- ulatory mechanisms may be cell type-specific.

Hydrolysis of phosphoinositides appears to be a critical step in a pathway which regulates expression of 5-HTZA receptor mRNA in P11 cells. Partial agonists at stimulating PI hydrol- ysis were also partial agonists a t increasing 5-HT,, receptor mRNA. In addition, stimulation of PI turnover through activa- tion of a,-adrenergic receptors on P11 cells was found to in- crease levels of 5-HTZA receptor mRNA. Moreover, the effects of 5-HT on levels of receptor mRNA were dose dependent and comparable EC,, values for PI hydrolysis and mRNA increase (approximately 400 n~) were observed.'

The finding that stimulation of cyadrenergic receptors modulated 5-HTzA receptor mRNA expression indicates that cross-talk from a,-adrenergic to 5-HT, receptors occurs in P11 cells. Coupling to the PI hydrolysis cascade, a signaling path- way that is common to a variety of receptors including 5-HT2, and a,-adrenergic receptors, could explain the shared ability of these heterologous receptor systems to alter 5-HTzA receptor mRNA levels. Regulation of 5-HT,, receptor mRNA resulting from stimulation of qadrenergic receptors demonstrates that non-serotonergic systems coupled to PI turnover can alter ex- pression of 5-HT,, receptor mRNA and raises the interesting possibility that levels of 5-HT, receptor mRNA can be regu- lated through activation of heterologous receptor systems coupled to stimulation of phospholipase C. Potential effects of 5-HT,, receptor activation on regulating the expression of a,- adrenergic receptor mRNA were not investigated. Because ho- mologous regulation of mRNAencoding 5-HT,, receptors in P11 cells involves activation of protein kinase C (see below), 5-HT, receptor-mediated regulation of a,-adrenergic receptor mRNA is likely to occur since protein kinase C-mediated increases in a,,-adrenergic receptor mRNA levels have been reported else- where (13).

Protein kinase C is directly (via diacylglycerol) and indirectly (via inositol trisphosphate-induced calcium release) activated

R. C. Ferry, unpublished observations.

Regulation of 5-HTZA Receptor mRNA 31857

by metabolites of PI turnover. Stimulation of protein kinase C, which is required for agonist-promoted changes in levels of mRNA encoding thyrotropin-releasing hormone and a,,-adre- nergic receptors, resulted in a large increase in levels of 5-HTz, receptor mRNA, whereas inhibition of protein kinase C with a selective inhibitor blocked PMA- and 5-HT-induced increases in receptor mRNA levels. These findings indicate that the effects of 5-HT on receptor mRNA required activation of protein kinase C. Because activation of protein kinase C also leads to desen- sitization of 5-HT, receptors (33), it appears that protein ki- nase C is involved in control of 5-HT2, receptor-effector cou- pling, as well as expression of 5-HT, receptor mRNA.

Previous investigations (9, 11) have shown that exposure of P11 cells to 5-HT leads to down-regulation of 5-HT, receptors, a finding that contrasts with the 5-HT-induced increase in re- ceptor mRNA levels observed in the current study. Although a simple explanation for these findings is that changes in levels of receptor mRNA do not contribute to regulation of receptor density, we hypothesize that the increase in levels of receptor mRNA represented a productive counter-regulatory mecha- nism which modulates the density of receptors by opposing processes involved in receptor down-regulation. When agonist- induced receptor down-regulation was examined in cells that had been pretreated with actinomycin D to prevent 5-HT from elevating receptor mRNAlevels, 5-HT-promoted receptor down- regulation was observed to be faster than in cells treated with 5-HT alone (ie. in cells in which the increase in levels of re- ceptor mRNA took place). Because exposure to actinomycin D itself did not alter receptor density, the enhanced rate of down- regulation appears to be due to the ability of actinomycin D to prevent 5-HT from increasing receptor mRNA levels rather than from its ability to reduce receptor mRNA levels in 5-HT- treated cells to below control levels. These observations indi- cate that agonist-promoted increases in receptor mRNA levels attenuate receptor down-regulation, suggesting that the in- duced receptor mRNA is translated into functionally active protein.

Because exposure of P11 cells to 5-HT for very brief periods of time induced a delayed increase in 5-HT, receptor mRNA levels and since results of experiments with actinomycin D on receptor density are consistent with the increase in receptor mRNAbeing translated into new receptor protein, we speculate that the increase in 5-HTz, receptor mRNA levels may be part of a short term compensatory response designed to maintain receptor levels at or near control levels. In this model agonist- promoted increases in receptor mRNA levels represent a ho- meostatic mechanism by which short term decreases in recep- tor density are later counterbalanced by a delayed increase in receptor synthesis. Such a mechanism would help to ensure recovery of synaptic responsiveness following a period of synaptic activity that produced down-regulation of 5-HT, receptors.

The observation that exposure to 5-HT results in receptor down-regulation which continues well beyond the time that receptor activation has ceased (due to the addition of an an- tagonist) suggests that continued occupancy of the receptor is not necessary for receptor down-regulation. Thus, in a manner similar to that observed for levels of 5-HT, receptor mRNA, brief exposure to 5-HT appears to trigger events which lead to changes in receptor density. A regulatory mechanism such as this might be expected to have effects on receptor density in vivo where the duration of receptor occupancy is limited by the short half-life of the endogenous neurotransmitter.

In summary, the present results demonstrate that heterolo- gous receptor systems coupled to stimulation of PI hydrolysis

alter expression of 5-HT, receptor mRNA through a post-tran- scriptional mechanism requiring activation of protein kinase C, that brief receptor engagement has significant effects on recep- tor mRNA levels and receptor density, and that increases in levels of receptor mRNA have a modulatory effect on receptor expression. These findings suggest a novel possibility for regu- lation of 5-HTzA receptors by protein kinase C.

Acknowledgments-We are grateful to Dolan B. Pritchett for provid- ing full-length rat 5-HT, receptor cDNA, Behnam Ghasemzadeh for helpful discussions of slot-blotting and hybridization protocols, and Michael Robinson and Sina Djali for help in conducting high pressure liquid chromatography analyses. We also thank Lotte Gottschlich for a critical reading of the manuscript.

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