Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat c6 glioma cells

Pergamon

0306-4522(95)00323-l

/Veuroscience Vol. 69, No. 4, pp. I1 19-l 131, 1995 Elsevier Science Ltd Copyright 0 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

CHARACTERIZATION OF THE 5HYDROXYTRYPTAMINE,, RECEPTOR-ACTIVATED CASCADE IN RAT C6 GLIOMA CELLS J. M. ELLIOTT,*? N. R. NEWBERRY,? A. J. CHOLEWINSKI,t J. T. BARTRUP,_F S. J. BRIDDON,? J. E. CAREY,$ T. P. FLANIGAN,? R. A. NEWTON,? S. L. PHIPPS,t A. C. REAVLEY,? C. SMITH,? M. WIGMORE,t D. G. GRAHAME-SMITH? and R. A. LESLIE? tOxford University-SmithKline Beecham Centre for Applied Nemo-psychobiology, University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K. SBiotechnology Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K. Abstract-We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine,. receptor. The binding of [‘Hlketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine,. receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine,, receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = - 80 mV). Using caged molecules containing Ca’+ or inositol 1,4,5_trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca*+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+], and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine,. receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca*+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca’+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca*+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade. Key words: Ca’+, K+ channel, [‘Hlketanserin, desensitization,

*To whom correspondence should be addressed. b.p., base pair; CCCP, carbonyl m5-carboxyamidochlorophenyl-hydrazone; 5-CT, tryptaminei dbcAMP, dibutyryl cyclic adenosine monoohosahate: DMEM. Dulbecco’s Modified Eagle’s Medium; * DOI, 4-iodo-2,5-dimethoxyamphetan&e; EDTA, ethylenediaminetetra-acetic acid; EGTA, ethylene glycol-bis(/?-amino-ethyl ether) N,N,N’,N’-tetra acetic acid; FCS, fetal calf serum; fura-2/AM, furaacetoxymethylester; GppNHp, guanylylimidodiphosphate; Hank’s BSS, Hank’s balanced salt solution; HEPES, N-(2-hydroxyethyl) piperazine-N’-(2-ethanesulfonic acid; IP,, inbsitol 1,4,5_trisphosphate; 8-OHDPAT, 8-hydroxy-2(di-N-propylamino)-tetralin; 5-HT, 5-hydroxytryptamine; 5-MeO-DMT, 5-methoxy-N,Ndimethyltryptamine; 2-Me-5-HT, 2-methyl-5-HT; PI, phosphoinositide; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulphate.

Abbreviations:

down-regulation,

differentiation.

The mammalian 5-hydroxytryptamine (5-HT) receptor family currently comprises 14 distinct subtypesZ5 of which all except the 5-HT, receptor are functionally coupled to effector systems via guanine nucleotide binding proteins (G-proteins). The 5-HT, receptor sub-family differs from the remainder in coupling preferentially to the phosphoinositide second messenger system and comprises three further sub-types, identified as 5-HT,,, 5-HT,, and 5-HT,,.

Of these, the 5-HT,, receptor (formerly described simply as the 5-HT, receptor) has been best characterized and stimulation of this receptor in both neuronal and non-neuronal tissues causes the hydrolysis of phosphoinositides leading to the formation of inositol polyphosphates, the release of Ca2+ ions from intracellular stores and a subsequent

1119

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J. M. Elliott et al.

rise in cytosolic [Ca2+] (see Martin and Humphrey).*j The electrophysiological consequences of 5-HT2, receptor stimulation do not, however, show the same degree of uniformity. In the majority of cells studied, activation of the 5-HT,, receptor induces an inward current associated with a decrease in membrane conductance, most likely mediated by the closure of K+ channels.5v29In the C6 rat glioma cell line, however, activation of the 5-HT,* receptor induces an outward current associated with increased membrane conductance mediated by the opening of K+ channels.22~30The factors which determine the nature of the membrane conductance changes resulting from activation of ostensibly identical receptors in different cells are unclear. In the present study, we have carried out a detailed investigation of receptor-effecter coupling at the 5-HT,, receptor in the C6 rat glioma cell line. Since activation of the receptor in this cell results in the apparently atypical outward current response, our initial objective was to confirm the identity of the receptor with that characterized in neuronal tissue. Subsequently we delineated the elements of the functional cascade which constitute the biochemical and electrophysiological consequences of stimulation of the receptor by 5-HT. Finally we investigated the modulation of receptor function associated with desensitization induced by chronic agonist stimulation and with differentiation induced by raising intracellular CAMP levels. A preliminary account of some of the electrophysiological data has been presented elsewhere.3

EXPERIMENTAL

PROCEDURES

Materials The sources of reagents used were as follows: Furaacetoxymethylester (fura-2/AM), caged IP, and nitr-5, Calbiochem (Cambridge: U.K.): carbonvl m-chlorouhenvlhydrazone (CCCP), Idibutyryl cyclic -adenosine -monophosphate (dbcAMP), 5-hydroxytryptamine’HC1 (5-HT), ionomycin, poly+ornithine and all molecular biology reagents, Sigma Chemical Co. (Poole, Dorset, U.K.); Dulbecco’s Modified Eagle’s Medium (DMEM), and foetal calf serum (FCS), Flow laboratories (U.K.); glutamine, Gibco (U.K.); Hank’s balanced salt solution (Hank’s BSS), Imperial laboratories (Salisbury, U.K.); 4-iodo-2,5dimethoxyamphetamine L-hydroxy-2-(di-N(DGI), propylamino)-tetralin (8-OH-DPAT), quipazine dimaleate, 2-methyl-5-HT (2-Me-5-HT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and 5-carboxyamidotryptamine (5-CT), Research Biochemicals Inc. (U.S.A.). All other chemicals and reagents were of analytical grade from BDH (Dagenham, Essex, U.K.). The following compounds were kindly donated by the companies indicated: ketanserin tartrate, spiperone, pirenperone, ritanserin (Janssen, U.K.); pindolol, mesulergine, methysergide (Sandoz, Switzerland); granisetron (SmithKline Beecham, U.K.); ICI 170809 (ICI, U.K.); prazosin (Pfizer, U.K.); cyproheptadine (Merck Sharp & Dohme Ltd); mianserin (Organon, Holland); fluphenazine (Squibb); chlorpheniramine
rH]granisetron (sp. act. 61 Ci/mmol) were obtained from NEN-Du Pont (U.K.) and [‘Hlmesulergine (sp. act. 77 Ci/mmol) was obtained from Amersham International plc, (U.K.). Cell culture The rat C6 glioma cell line was obtained from the European Collection of Animal Cell Cultures, Porton Down, (Salisbury, Wiltshire, U.K.) and Dr Katherine Marsden, Institute of Psychiatry, (London). The cells were grown at 37°C in a humidified atmosphere of 5% CO, in air, in 75 cm2 or 600 cm2 plastic culture flasks or on poly-Lornithine-coated 13 mm diameter glass coverslips, in DMEM with 2 mM glutamine and 10% (v/v) FCS and used between passages 5267. Cells were differentiated by being cultured in DMEM with 2 mM glutamine, 2% (v/v) FCS and 1 mM dbcAMP (differentiation medium) and assayed after two to seven days in culture. Except where otherwise stated, studies were carried out using confluent differentiated cells. MessengerRNA

preparation

and northern blot analysis

Total RNA was prepared by a modification of the method of Auffray and Rougeon. Briefly, cultured cells and brain tissue were homogenized in 3 M LiCl, 6 M urea, 0.1% (w/v) sodium dodecvl sulohate (SDS). 0.1% (w/v) heoarin. 7 mM sodium acetate (PI-I 5.2) RNA’was precipitated at 4;C and pellets reprecipitated in 3 M LiCl 6 M urea. Pellets were resuspended in 10 mM Tris-HCl (PH 7.4) containing 1 mM EDTA, 0.1% (w/v) SDS and 400 pg/ml proteinase K incubated for 60 min at 37°C and RNA extracted with phenol and chloroform followed by ethanol precipitation. Poly(A)+ mRNA was prepared using Dynabeads (Dynal, U.K.) according to manufacturer’s instructions. RNA content was measured by optical density (260/280 nm). For northern blot analysis, RNA samples were denatured with glyoxal,‘* electrophoresed in 1.2% (w/v) agarose gels, transferred under vacuum to Hybond N membranes (Amersham) and cross-linked by UV irradiation. Membranes were incubated for 16 h at 55°C in prehybridization buffer consisting of 0.05 M Tri-HCl @H 7.4) 50% (v/v) deionized formamide, 10% (w/v) dextran sulphate, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) Ficoll-400, 0.1 “/(w/v) sodium pyrophosphate, 1% (w/v) SDS, 0.2% (w/v) bovine serum albumin (fraction V), 200 pg/ml bakers yeast tRNA, 200pg/ml salmon sperm DNA, 20pg/ml oolvadenvlic acid. 20 &ml oolvcvtidvlic acid and 1 M NaCl. Membranes were Then hybridized with radiolabelled probes to the 5-HT,, receptor or p-actin mRNAs for 20 h at 55”C, washed to final stringencies of 0.1 x saline sodium citrate (1 x SSC: 0.15 M NaCl, 0.015 M sodium citrate), 0.1% (w/v) SDS at 21°C and exposed to Kodak X-Omat film at -80°C in the presence of intensifying screens for appropriate periods. Plasmids containing rat 5-HT,, receptor*’ and human p-actin cDNAs (pBluescript SK-‘and pGEM-7Zf(+), resoectivelv) were radiolabelled with IGI-~~P~~CTP to aonroximate specific activities of 3 x lo*- c.p.m./pg DNA -using Amersham’s multiprime kit (according to manufacturer’s instructions) followine linearization with EcoRV (5-HT,, receptor) or BamHl (B-actin) digestion. Prior to addition to hybridization buffer, labelled DNA was separated from unincorporated label with Nick Columns (Pharmacia). Membranes were reprobed following stripping of previous signal in 0.1 x SSC, 0.1% (w/v) SDS at 65°C for approximately 2 h. The plasmid probes used in this study were kindly provided by Dr Lei Yu, Department of Medical and Molecular Genetics, Indianapolis, U.S.A. (5-HT,,) and Dr A. J. Wood, Department of Psychiatry, Oxford, U.K. (B-actin). I”

Linked reverse transcription-polymerase

chain reaction

Poly(A)+ mRNA was reverse transcribed for 60min at 42°C in a reaction volume of 10 ~1 consisting of 20 mM Tris

5-HT,, receptor cascade in C6 cells 1.25 U/p1 RNasin, 10 ng/nl specific anti-sense primers for the rat 5-HT,, sequence (complimentary to nucleotides 949-969 inclusive; GenEMBL accession number M30705) or human j-actin sequence (complimentary to nucleotides 1021-1041 inclusive; GenEMBL accession number X00351), and 1.25 U/n1 AMV reverse transcriptase (Pharmacia). The reaction was stopped by addition of 5 mM EDTA and the RNA template hydrolysed with 50 mM NaOH for 60 min at 65°C. Following neutralization with HCl to a final volume of lOOnI, 10~1 aliquots were frozen at -20°C. Polymerase chain reactions (PCR) were carried out in a Perkin Elmer Cetus DNA Thermal Cycler. RT products were added to a reaction buffer containing 1OmM Tris (PH 8.3), 50 mM KCl, 5 mM MgCl,, 0.8 mM each dNTP and 1 nM each of sense and anti-sense primers in a final reaction volume of 50 ~1, and overlaid with mineral oil to prevent evaporation. Anti-sense primers were as above, sense primers were homologous to nucleotides 667693 of the rat 5-HT, receptor sequence and nucleotides 561-581 of the human /I-actin sequence. After an initial denaturation step of 7 min at 94°C 1.25 U of AmpliTaq DNA polymerase (Perkin Elmer) were added. Amplification by PCR involved 30 cycles (C6 glioma cell mRNA) or 35 cycles (rat brain mRNA) of 1 min at 94”C, 30 s at 55°C and 1 min at 72°C. This was followed by a final extension step of 3 min at 72°C. A further 1.25 U of AmpliTaq were added every 10th cycle. Sequencing of polymerase chain reaction products

Appropriate PCR products were purified using Magic PCR Preps (Promega) and ligated into the pUCl8 vector at the SmaI site using the SureClone Ligation Kit (Pharmacia). The constructs were amplified in E. coli JMlOl. Double digest of the purified plasmids with BamHI/EcoRI revealed those constructs with the correct size of insert. These inserts were sequenced using Sequenase Version 2.0 DNA Sequencing Kit (USB). Kits were used according to manufacturer’s instructions. Radioligand binding

To prepare membranes for binding assay, cells from one megaplate were detached by scraping and sedimented by centrifugation (900 g, 10 min, 20°C) then homogenized in 5 mM Tris/EDTA (PH 7.4) using a Braun Teflon/glass homogenizer. The crude lysate was centrifuged at 15OOg, 5 min, 4°C and the pellet discarded. The membranes in the supernatant fraction were sedimented by centrifugation at 30,000 g, 15 min, 4°C washed twice by resuspension in the same medium and finally resuspended in incubation medium (50 mM Tris, 5mM MgCl,, 1 mM EGTA, pH 7.4). The final protein density was approximately 1 mg/ml. Binding assays were initiated by addition of 150 nl tissue to 100 ~1 radioligand in incubation medium. Non-specific binding was defined by the presence of methysergide (1 PM) for [lH]ketanserin and [3H]mesulergine, 5-HT (10 PM) for [‘H]8OH-DPAT and [‘HIS-HT and MDL-72222 (1OnM) for [3H]granisetron. Incubations were maintained for 60 min at 37°C and then terminated by filtration through Whatman GF-C glass-fibre filters using a Skatron cell harvester, after which each sample was washed with lOm1 cold incubation medium. Saturation binding of [3H]ketanserin and [3H]mesulergine was examined over the range O.lllOnM. For inhibition studies, binding of [rH]ketanserin or [3H]mesulergine (approximately 1.5 nM) was studied in the presence of 5 to 11 concentrations of unlabelled drug. In each case, radioactivity retained on the filters was estimated using a Beckman 5OOOCE scintillation counter. For all assays, samples were replicated in triplicate. Protein content was assayed according to the method of Peters0n.j’ Radioligand binding characteristics for [3H]ketanserin and [3H]mesulergine were determined from specific binding data by non-linear regression analysis.9 Preliminary Scatchard analysis of the data by linear regression generally exhib-

1121

ited correlation coefficient r > 0.95. Competition binding studies were analysed by non-linear regression using the EBDA/LIGAND program (Biosoft, U.K.). When studying agonist down-regulation, 5-HT was added to the standard medium bathing the cells and incubation maintained for 30 min-24 h. The medium was then removed and membranes prepared in the normal manner were examined by saturation binding of [3H]ketanserin as described above. In each assay, binding was studied at each of the three 5-HT concentrations studied together with control cells incubated in normal medium. Binding affinity for [3H]ketanserin was not altered by prior incubation with 5-HT at any concentration studied. The extent of 5-HT,, receptor down-regulation was therefore estimated as the B,, value for [3H]ketanserin expressed as a percentage of the control within the same assay. Phosphoinositide hydrolysis

For phosphoinositide hydrolysis studies, cells were grown in 24-well plates at an initial density of approximately 50,000 cells/cm2. After two to three days the growth medium was replaced by differentiation medium modified by using inositol-free DMEM supplemented with [‘Hlinositol (1 pCi/ml). Assays were carried out two days later. The labelling medium was removed and the cells were washed with DMEM then equilibrated with 450 ~1 DMEM containing 1OmM LiCl for 5min at 37°C. The reaction was initiated by addition of agonist (50 ~1) and maintained for 30 min at 37°C then terminated by removal of the incubation medium followed immediately by addition of 0.75 ml cold methanol, as described by Wilson et al.42 Cells were scraped from the plate and transferred to polythene tubes containing 0.75 ml chloroform. Each well was further washed with 0.75 ml HCl(2% v/v) which was also added to the polythene tube and the contents were thoroughly mixed. The tubes were then centrifuged (2000 g, 10 min, 4°C) and 750 ~1 aqueous phase was loaded onto Dowex AGl-x8 (formate form) columns. The columns were washed with 2ml water then 15 ml sodium formate (60mM)/sodium tetraborate (5 mM) and the inositol phosphates were eluted with 3 ml ammonium formate (0.8 M)/formic acid (0.1 M). Samples were mixed with 12 ml scintillation fluid and radioactive content estimated using a Beckman 5000CE scintillation counter. To estimate the incorporation of [3H]inositol into phospholipids, 300~1 chloroform sample was taken from each assay tube and evaporated to dryness. 100~1 methanol was added to resuspend the phospholipid and the samples counted in 3 ml scintillation fluid. Data were then expressed as percentage of [3H]phospholipid converted into [3H]inositol phosphates within each sample. Quantitation of phosphoinositide hydrolysis by 5-HT was estimated by analysis of the dose-response relationship according to a general logistic model. When studying desensitization effects, 5-HT was added to the cells in the presence of [‘Hlinositol and incubation maintained for 24 h. The 5-HT was removed together with the medium containing [‘Hlinositol and the cells were washed in the normal manner prior to maximal stimulation of the PI response by 10pM 5-HT. Basal responses were also measured following chronic incubation with 5-HT and the net response calculated as the difference between the basal and stimulated response when rechallenged with 5HT. The degree of desensitization was then estimated as a percentage of the control response seen in cells cultured in normal incubation medium. Measurement of intracellular [Ca*+]

Intracellular calcium ion concentrations, [Ca*+],, were measured with the fluorescent calcium indicator dye furausing the MCID Ml Image Analysis System (Imaging Research Inc., St. Catharines, Canada). C6 glioma cells were loaded with fura- by incubation in differentiation medium containing 10 PM fura-2/AM for 30 min at 37°C. After

J. M. Elliott et al.

1122

incubation, the coverslips were transferred to a laminar flow chamber (flow rate l-2 ml/min) on an inverted stage Nikon Diaphot microscope equipped __ with a x40 fluorescence objective (N.A. 0.8-1.3) and continuously perfused with Hank’s BSS containina 10 mM HEPES CDH 7.4). 2mM CaCl,. Cells were allo;ed to equilibrate &r 10 mm, until stable fluorescence ratios were attained, before measurements began. Fura- was excited alternately at 340 and 380 nm via a software-controlled LEP filter wheel positioned between a 150 W Xenon light source and the microscope. The emitted light was monitored at 510 nm and the cytosolic calcium concentration was calculated from the ratio of the fluorescent intensities at the two excitation wavelengths.” Minimum and maximum ratios were determined by exposing the fura-2-loaded cells respectively to 10pM ionomycin in a calcium-free buffer containing 10 mM EGTA, and to 10 PM ionomycin, 100 PM S-HT and 10 PM CCCP in the presence of 2 mM CaCl,. Drugs were applied by means of a “U-tube” positioned next to a group of cellsi This permitted fast application of the drug to a highly localized area of the coverslip. Experiments were carried out at room temperature (21-24°C). Electrophysiology C6 cells were grown on 19 mm polylysine-coated circular coverslips in Nunclon 6-well culture plates and were differentiated with 1 mM dibutyryl CAMP in DMEM, 1% glutamine and 2% FCS for two to nine days before use. For electrophysiological recordings, individual coverslips were transferred to an inverted stage microscope where they formed the base of a small bath (ca. 0.2ml) which was perfused with medium (2-3 ml/min) at 2426°C. The extracellular medium contained (mM): NaCl 140, KC1 5, MgCI, 1, dextrose 5, HEPES 10, CaCl, 2, (PH 7.4 with NaOH). Recordings were made with patch electrodes of 36 M resistance filled with an intracellular solution of (mM): KC1 125, MgCl, 1, HEPES 10, disodium adenosine 5’triphosphate - (Na,ATP) 2, disodium guanosine 5’trinhosnhate (Na,GTP) 1. (oH 7.4 with NaOH). brugs were‘applied to thl bath via the rapid superfusion system: 5-HT for 5-10 s, antagonists for l-5 min before the 5-HT. Occasionally, 5-HT (10 PM) was applied locally by pressure application (l&l5 p.s.i., 0.1-l s) from a drug pipette (tip diameter ca. 2 pm) situated ca. 50 pm from the cell. For caged compound experiments, caged. IP, (20 p M) or caged Ca2+ (2 mM nitr-S with 0.5 mM CaCl,) was added to the intracellular pipette solution. The caged molecules were released by a brief flash of ultraviolet (UV) light (369 nm, 0. l-l s) passed through the microscope lens using a Nikon xenon light source and an LEP filter wheel controller. Whole-cell voltage-clamp recordings were made at a holding potential of -50 mV, allowing for a junction potential of +4mV, using a List EPC-7 patch-clamp amplifier. The signal was filtered through a 3 kHz low pass filter and recorded on a Gould Windograf chart recorder with a further 15 Hz low pass filtering on recordings with voltage pulses. To determine the reversal potential of a response, a “staircase” pulse method was used whereby three sequential voltage pulses each 200ms, total duration 6OOms, were applied every 2 s. This gave current measurements at four holding potentials (-50, -65, -80 and -95 mV) during a single 5-HT application. The pulses were applied using a CED 1401 with VGEN software supplied by J. Dempster of Strathclyde University. All electrophysiological results are expressed as median and range. RESULTS

Radioligand

binding studies

Radioligand binding studies indicated binding of [3H]5-HT, [3H]8-OH-DPAT

no specific or [3H]-

granisetron in membranes from differentiated C6 cells whereas both [3H]ketanserin and [WImesulergine exhibited substantial specific binding. Further investigation demonstrated that the binding of [3H]ketanserin was saturable within the range 0.1-10 nM (Fig. 1) and of high affinity (Kd = 0.45 nM; pK, = 9.42 f 0.05 M) and limited capacity (B,,,,, = 96 f 7 fmol/mg protein, mean f SE., n = 18). Nonspecific binding of [3H]ketanserin in these assays was low, comprising 20% of total binding at 1 nM. Binding capacity remained consistent when assayed repeatedly over 10 consecutive passages (coefficient of variation = 12%). Binding of [3H]mesulergine was also saturable with slightly lower affinity (Kd= 2.17 nM; pK, = 8.70 f 0.06, n = 7) and similar capacity (B,,, = 93 + 14 fmol/mg protein) to that of [3H]ketanserin. Paired comparison of [3H]ketanserin and [3H]mesulergine binding in seven membrane samples indicated a significant correlation in binding capacity revealed by the two ligands (r = 0.93; P < 0.005). Inhibition of [3H]ketanserin binding by serotonergic antagonists indicated a typical 5-HT,, receptor profile with Hill coefficients close to unity (Table 1). Comparison of the affinities of these compounds in C6 cells with those of the SHT,, receptor in rat frontal cortex labelled by [3H]ketanserin’3 revealed a significant positive correlation (r = 0.73; P < 0.01; n = 11). In contrast, comparison with affinities of the same compounds for the 5-HT,, receptor in rat choroid plexus tissue labelled with [3H]mesulergine’2 demonstrated no significant correlation (r = 0.03; P > 0.05; n = 9). In addition to labelling 5-HT,, receptors, [3H]ketanserin also binds to a,-adrenoceptors and tetrabenazine-sensitive sites.‘9,20 In the present study both prazosin (a potent cc,-adrenoceptor

T

0

1

2

4

5

[‘HI KETANSERIN

W)

3

6

7

Fig. 1. Specific binding of E3H]ketanserin to membranes of differentiated and undifferentiated rat C6 glioma cells. C6 cells were cultured in DMEMjlOX FCS and differentiated by addition of 1 mM dibutyryl CAMP, as described in Experimental Procedures. Specific binding of [‘Hlketanserin to differentiated (solid circle) and undifferentiated (open circle) cell membranes was defined using 1 PM methysergide. Data shown are means of triplicate estimates in a single representative assay. Inset shows Scatchard transformation of the same data.

5-HT,,

receptor cascade in C6 cells

antagonist) and tetrabenazine proved weak inhibitors of [3H]ketanserin binding (Table 1). The binding of [3H]mesulergine to membranes of C6 cells was inhibited by spiperone, ketanserin and mianserin with affinities and Hill slopes similar to those revealed using [3H]ketanserin. Of the endogenous monoamine neurotransmitters, S-HT proved the most potent inhibitor of [3H]ketanserin binding (Table 1). Displacement by serotonergic agonists including 5-HT occurred with a shallow slope. Addition of the stable GTP analogue guanylylimidodiphosphate (GppNHp; 10 PM) reduced the affinity of 5-HT and increased the mean Hill coefficient from 0.72 to 1.10. MessengerRNA

analyses

To investigate the presence of 5-HT,, receptor mRNAs in these cells Northern blots and RT-PCR were performed. Poly(A)+ mRNA was isolated from differentiated C6 cells and probed for the presence of S-HT,, receptor mRNA. ‘*P-labelled plasmid-derived probe to the rat 5-HT,, receptor hybridized specifically to transcripts, under conditions of high stringency, between 5 and 6 kb (Fig. 2A). The probe also detected bands in poly(A)+ mRNA from whole rat brain in this size range. In addition, hybridization with 32P-labelled human /I-actin cDNA probe indicated the relative level of mRNA in each lane. Further analysis of the poly(A)+ mRNA from these cells by RT-PCR with rat 5-HT,, receptor specific oligonucleotide primers revealed a 303 base pair (b.p.) product following ethidium bromide agarose gel electrophoresis (Fig. 2B). A similar product is also detectable in mRNA isolated from rat brain. The signal derived from the C6 glioma cell mRNA was dependent on the inclusion of reverse transcriptase in the reactions (Fig. 2B). To confirm the identity of the 303 b.p. RT-PCR product from C6 glioma cell mRNA, the DNA fragment was sub-

1123

cloned and partially sequenced. This sequence proved identical to that of nucleotides 756-960 of the rat 5-HT,, receptor. Measurement of phosphoinositide turnover

The functional coupling of the 5-HT,, receptor in C6 cells to the phosphoinositide second messenger system was investigated by prelabelling the cells with [3H]inositol. Stimulation by 5-HT in the presence of 10 mM LiCl caused a concentration-dependent increase in the production of inositol phosphates (Fig. 3) with EqO = 0.14 PM (pECSo = 6.85 + 0.08 M; mean f S.E., n = 9). Maximal response occurred at 10pM 5-HT and constituted 2.8 * 0.4% [3H]phosphoinositide content, which approximated twice the basal turnover. Sequential elution of the inositol phosphates from the Dowex columns according to the method of Berridge et aL4 indicated that inositol monophosphate constituted the major product with small amounts of both the bisphosphate and trisphosphate. Stimulation of phosphoinositide turnover by 5-HT (50 PM) was effectively blocked by 1 PM spiperone or ketanserin and inhibited to a lesser degree by mesulergine but no inhibition was evident at 1 PM concentrations of atropine, prazosin or chlorpheniramine. Incubation of the cells with pertussis toxin (100 ng/ml) for 24 h prior to assay did not alter the PI response to 5-HT (data not shown). Measurement of intracellular Ca*+

The mean basal levels of [Ca*+], measured in single C6 cells were 287 * 25 nM (n = 63). When 5-HT was applied for a duration of 40 s, maximal [Ca2+li levels were attained by 10 s followed by a gradual decline to basal values by 40 s and the magnitude of the response varied between individual cells (Fig. 4A). In all subsequent experiments applications of 5-HT were pulsed on for 10 s and the maximal [Ca*+], levels then recorded. Following a brief (10 s) application of 5-HT

Table 1. Ligand profile for inhibition of [‘Hlketanserin binding to membranes of C6 rat glioma cells PK,

nH

Methysergide

9.22

Pirenperone Spiperone Ketanserin Cyproheptadine Mesulergine Mianserin Ritanserin ICI 170809 Fluphenazine Chlorpheniramine Prazosin

9.18 9.06 8.79 8.70 8.43 8.32 8.19 8.19 7.71 5.67 4.89

1.04 0.98 1.05 1.03 1.02 0.97 0.97 1.13 1.07 1.04 1.11 0.94

DO1 Quipazine mCPP SmethoxyDMT 5-HT 5-HT + GppNHp Noradrenaline Dopamine Histamine Carbachol Tetrabenazine

PIC,~

nH

7.14 6.58 6.23 6.01 5.43 4.62

0.69 0.58 0.90 0.17 0.72 1.10 <5 <5 <5 15 15

Binding of t3H]ketanserin (1.5 nM) to membranes of C6 cells was determined in the presence of five to 11 concentrations of competing ligand and data analysed by a non-linear regression program (EBDA). Ligand binding affinity was estimated from the n+, value and expressed as pm,, for agonists and pK, for antagonists, calculated according to the method of Cheng and Prusoff.7 Each value represents the mean of two to seven separate determinations.

J. M. Elliott et al.

1124

A GACTIN,

48s

S-HT~A: a

b

Fig. 2. 5-HT,, receptor mRNA in C6 glioma cells. (A) Northern blot of poly(A)+ mRNA from whole rat brain (a) and differentiated C6 cells (b). Samples were probed for /I-actin and 5-HT,, receptor mRNAs. 18 s and 28 s ribosoma1 RNA are as indicated. (B) RT-PCR analysis of poly(A)+ mRNA from C6 cells (lanes a, b and c) and whole rat brain (lanes d and e) with oligonucleotides specific for 5-HT,, receptor (a, b and d) and j-actin (c and e) mRNAs. Numbers on the right indicate molecular weights in base pairs. Reverse transcriptase was omitted from reactions shown in lane a.

a rapid rise in [Ca2+], was observed in the majority of cells (> 85%). The log-concentration-response curve for 5-HT, measured at the peak of the response, exhibited a steep slope with an EC5,, value of 0.5 /*M. In order to evaluate which 5-HT receptor subtype was responsible for the observed elevations of [Ca2+]i levels, various 5-HT antagonists and agonists were tested. The response to 10 PM 5-HT in C6 cells was blocked by 10 nM concentrations of the 5-HT, receptor antagonist ketanserin and also by 10 nM spiperone, which has a lOOO-fold higher affinity for the 5-HT,, receptor compared with the 5-HT,, receptor (Fig. 4B). In all cells responding to 5-HT, the response was inhibited in the presence of ketanserin or spiperone. Mesulergine (10 nM) was unable to block the 5-HT-mediated response but was effective at higher (50 nM and 1 PM) concentrations (Fig. 4B). 1 PM pindolol and 1 ,uM granisetron had no effect on the 5-HT-mediated increases in [Ca’+],. DOI, a 5HT,, receptor partial agonist, at 1 PM concen-

trations had no effect on [Ca2+], but at 10pM induced a small increase in [Ca2+li (Fig. 4C). The responses to DO1 were seen in only a small subpopulation of cells (< 27%). When DO1 was tested at 1 PM it was able to inhibit a 5-HT evoked response, (from 640 f 115 nM [lOpM 5-HT] to 27 + 12nM [lOpM 5-HT f 1 PM DOI], mean f SE., n = 4). Quipazine, a 5-HT, receptor partial agonist and a 5-HT, receptor antagonist (1 PM) also caused a small increase in [Ca2+li as did 10pM 5-MeO-DMT (Fig. 4C). 5-CT, a 5-HT, receptor agonist (1 PM), &OH-DPAT, a 5-HT,, receptor agonist (1 p M), and 2-Me-5-HT, a 5-HT3 receptor agonist (10 PM), each had no detectable effects on [Ca’+], levels when applied to C6 cells (Fig. 4C). The ability of 5-HT to mobilize calcium was examined in calcium-free medium to determine whether elevated [Ca2+], levels were a result of calcium entry from the extracellular medium or release from intracellular stores. When calcium was removed from the medium, 5-HT was still able to elicit a response in C6 cells by elevating [Ca2+li to levels similar to those seen in the presence of extracellular calcium (1115 + 271 nM over basal levels; mean + S.E., n = 3). Depletion of intracellular calcium stores by prior application of 1 PM ionomycin abolished this response. Electrophysiology C6 cells had a median apparent input resistance of 280 MR (17-3500 Mn) (n = 204) and in currentclamp they had a median resting membrane potential of -44 mV ( - 19 to - 69 mV). Superfused 5-HT (1 PM, 5 s) produced an outward current of 180 pA

0

1

,I

Basal

/

I 0.01

0.1

1

[ 5-HT ]

10

100

WI

Fig. 3. Stimulation of inositol phosphate production by 5-HT in differentiated and undifferentiated rat C6 glioma cells. Differentiated (solid circle) and undifferentiated (open circle) cells were cultured as described in Experimental Procedures and incubated with inositolfree DMEM/lO% FCS containing [3H]inositol (1 pCi/ml) for two days prior to assay. Production of [3H]inositol phosphates ([‘HIIP,) was initiated by addition of 5-HT in the presence of LiCl (10mM) and expressed as % conversion from [‘Hlinositol phospholipid. Data shown are means k SE. of quadruplicate estimates in a single representative assay.

S-HT,,

receptor

cascade

in C6 cells

1125

0

0 0 ??

0

2 s s ft ii I!! .= 5 8

loo-

5

IO

15 20 25 30 Time (seconds)

Cell Cell Cell Cell

35

1 2 3 4

40

*

80 60-

7 *

4020 O-

Fig. 4. Measurement of [Ca*+], in C6 cells in response to serotonergic receptor agonists and antagonists. (A) Time course of response of C6 cells to a continuous application of 10 PM 5-HT (application beginning at time 0). Maximal increases in [Caz+], were usually attained by 10 s and responses returned to basal levels by approximately 40 s. Individual cells respond to varying degrees, both in maximal response and time course of response, to the same concentration of 5-HT. This figure is representative of one experiment repeated four times in different cells with similar results. (B) Effects of serotonergic receptor antagonists on 5-HT-mediated increases in [Ca*+], in C6 cells. Results are expressed as mean k SE. of four separate experiments in each instance, and indicate the percentage of the initial response to 10 PM 5-HT; all 5-HT applications were at this concentration. 5-HT was applied for 10 s and the maximal response recorded. After a 10 min recovery period, either 5-HT alone was once again applied, or 5-HT in combination with the indicated antagonist, on the same group of cells. In all antagonist studies, compounds were added to the perfusion buffer 5 min prior to the application of the second 5-HT pulse. *P < 0.05 versus control (Student’s paired t-test). (C) Effects of 5-HT receptor agonists on calcium mobilization in C6 cells. Results are expressed as an increase in [Ca*+], over basal levels for three to five experiments (mean f S.E.). Drugs were applied to cells for 10 s and the maximal response recorded. *P < 0.05 versus basal level (Student’s paired t-test).

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(15-1200 PA) lasting 34 s (9-108 s) in 134 out of 204 cells studied. This outward current was associated with an increased membrane conductance likely mediated by the opening of potassium ion selective channels since the response had a reversal potential of -8OmV (-73 to -88mV, n = 14). 10pM 5-HT produced no larger response and 0.1 PM 5-HT was virtually ineffective. There were no apparent differences in resting potential, input resistance or morphology between those cells that did and did not respond to 5-HT. The outward currents induced by superfused or pressure-applied 5-HT were reduced by ketanserin (10-30 nM, n = 3; Fig. 5A) or spiperone (10 nM, n = 3) confirming that this response was likely mediated by 5-HT,, receptors. In a given cell, the response evoked by pressure-applied 5-HT was similar to that evoked by superfused 5-HT. Replacing the calcium ions in the superfusate with magnesium ions did not reduce the 5-HT in-

duced outward current (n = 4) but when EGTA (10 mM) was included in the intracellular pipette solution to chelate intracellular calcium ions the median response to 5-HT was significantly smaller (0 pA, CL150 PA, n = 6) compared with that in cells recorded on the same coverslips with the standard internal solution (160 pA, 5&410 pA, n = 5, data not shown). Prolonged 5-HT applications induced oscillating responses, as seen in our calcium imaging experiments. Caffeine (10 mM) which can evoke intracellular calcium release in neurons24 evoked little

A) CONTROL

B)

i\

KETANSERIN

‘n’

Cl

WASH

A 100 pA 20 set

Fig. 5. Electrical changes induced by 5-HT on C6 glioma cells. (A) The 5-HT induced outward current on C6 cells voltage clamped at - 50 mV was abolished by perfusion of the SHT, receptor antagonist ketanserin. 5-HT was pressure applied at the points indicated by solid circles (500 ms pulse, 10 p.s.i., 10 PM 5-HT in drug pipette). Ketanserin (30nM) was bath applied for 4min before 5-HT was reapplied. Washout was for 22 min. (B) Bath application of 5-HT (1 FM, 5 s) onto a C6 glioma cell recorded using a pipette solution containing caged IP, (20pM) induced an outward current of 360 pA. On the same cell 3 min later, a 100 s flash of UV light (arrow) triggered an outward current resembling that seen with 5-HT. (C) The pipette solution used to r&ord from this cell contained cagedckcium (2 mM nitr-5 with 0.5mM CaCl,). A 100 ms flash of UV light (arrow) triggered a rapid and prolonged outward current together with an increase in conductance, as indicated by the downward current deflections. Scale bar applies to all three figures.

or no outward current (median = 10 pA, range &50, n =9). In order to determine whether the electrophysiological response to 5-HT in C6 cells was directly related to the hydrolysis of phosphoinositides and subsequent rise in intracellular [Ca2+], we studied the effects of photolytic release of IP, and Ca*+ from caged molecules using UV light. When recording from 12 cells with pipettes cbntaining caged IP3 (Fig. 5B), a 1 s flash of UV light through the microscope lens resulted in a large outward current in 10 cells of 250 pA (60-820 PA) lasting 21 s (10-50 s). 5-HT was bath applied to eight of the 10 cells: it induced responses of 135 pA (7&600 PA) lasting 26 s (1240 s). The reversal potential for the inositol 1,4,5trisphosphate (IP,)-induced response was determined in three cells as -77 mV (- 76 to - 78 mV), compared with -77 mV (-73 to -77 mV, n = 4) for a 5-HT induced response in these experiments. 5-HT induced an outward current in the two IP,-filled cells that did not give a response to a UV flash. When recording from six cells with pipettes containing caged Ca2+, (Fig. 5C) we needed shorter UV pulses (100 ms) to evoke smaller (83 pA; 15-250 pA, n = 6) but more prolonged outward currents (duration 79 s; 20-320 s). Repeated application of UV pulses induced similar responses when studied in individual cells. The reversal potential for this response was determined in two cells to be -73 mV and -75 mV. In these cells, 5-HT did not evoke a response, probably because of the calcium buffering afforded by the nitr-5. The onset of the calcium induced response (ca. 60 ms) was faster than the onset of the response induced by photolytically released IP, (ca. 600ms) and the local pressure application of 5-HT to cells (ca. 2 s). UV light evoked no response in cells recorded with the standard intracellular solution alone. Comparison of 5-hydroxytryptamine,, receptor characteristics in d@erentiated and undlferentiated C6 cells C6 cells grown in medium in the absence of dibutyryl CAMP differed in morphology from the differentiated cells in that they were more rounded and exhibited fewer neurite processes. Binding studies using [3H]ketanserin (Fig. 1) indicated no difference in radioligand affinity between undifferentiated and differentiated cells (p& = 9.39 + 0.10 M and 9.31 f 0.15 M respectively, n = 4) but there was a significantly higher binding capacity in the undifferentiated cells (B,,, = 165 + 13 vs 116 f 13 fmol/mg protein, n = 4, P < 0.05, Student’s unpaired t-test).

Comparison of the phosphoinositide (PI) response to 5-HT revealed a markedly higher maximal response in undifferentiated cells (E,,,, = 11.9 f 2.7%, n = 5) than in differentiated cells (E,,, = 2.6 + 0.6%, n = 5; P < 0.001, unpaired t-test). This was not due to any difference in the incorporation of [3H]inositol into the cells, since the total dpm in each well was similar

5-HT,,

receptor cascade in C6 cells

when differentiated and undifferentiated cells were seeded at similar density, nor to any difference in basal turnover of [3H]phosphoinositides, which was 2.2 f0.2% total incorporated tritium in undifferentiated cells and 2.1 + 0.2% in differentiated cells. The potency of 5-HT to stimulate the production of [3H]inositol phosphates was significantly lower in undifferentiated cells (pECso = 6.51 f 0.07) than in differentiated cells (PEGS,, = 6.89 + 0.14; P < 0.05, unpaired t-test) but the magnitude of this difference was outweighed by the difference in maximal response, hence the net stimulation of phosphoinositide hydrolysis was greater at all concentrations of 5-HT in undifferentiated cells than in differentiated cells (Fig. 3). In contrast to the increased phosphoinositide response observed in undifferentiated cells, we found that fewer cells (< 50%) responded to 10 PM 5-HT with increased [Ca’+] in undifferentiated cells compared with differentiated cells (> 85%). Similarly the 5-HT induced outward current was observed in only two out of 15 undifferentiated cells (80 and 200pA) compared with responses in 12 out of 17 differentiated cells recorded over the same period (140 pA, @-1200pA; median, range). We also found that the membrane potential of undifferentiated cells was significantly higher (- 61 mV, -44 to - 72 mV) than differentiated cells (-44 mV, -28 to 61 mV) but the apparent input resistance was not significantly different: 150 MR (S&500 MR) in undifferentiated cells compared with 250 MR (30-1000 MQ) in differentiated cells. Desensitization and 5-hydroxytryptamine,, down -regulation by 5-hydroxytryptamine

receptor

Continued exposure of differentiated C6 cells to 5-HT reduced the binding capacity of [3H]ketanserin without altering the binding affinity. The extent of 5-HT, receptor down-regulation was dependent on the concentration and duration of exposure to 5-HT (Fig. 6A). Following 30min incubation with 5-HT (10 PM) the maximum degree of receptor loss observed was 30% whereas after 24 h incubation almost 90% of the receptors had been lost. Chronic exposure of the cells to 5-HT for 24 h was also found to reduce the subsequent PI response without altering the EC,, value for 5-HT (Fig. 6B). The degree of desensitization observed was dependent on the concentration of 5-HT employed in a similar fashion to the loss of binding, but the extent of desensitization was consistently greater than the loss in receptor number (Fig. 6A). Electrophysiological studies of this desensitization phenomenon were less extensive, but did demonstrate that the outward current induced by 10 FM 5-HT diminished in ampli$ude with repeated 10 s applications at 5 min intervals and usually became undetectable after 2040 min. Bath applications of 1 PM 5-HT for 5 s at 5 min intervals gave the most reproducible responses with the second

A

.30

min

2h

24 h

-.

0

5-HT

Incubation

Cow.

(PM)

0-I Basal

0.01

0.1

1

[ 5-HT ]

10

100

(PM)

Fig. 6. Desensitization of 5-HT,, receptors in C6 rat glioma cells. (A) Time course of 5-HT,, receptor desensitization and down-regulation following exposure to 5-HT. Differentiated C6 cells were incubated with 5-HT for 30 min-24 h and then washed and assayed for receptor number using [3H]ketanserin (solid symbols) or phosphoinositide response (open symbols). Values shown are the means of three to four experiments with SE. < 10% in each case. (B) Effect of desensitization on phosphoinositide response induced by 5-HT. Cells were labelled with [3H]inositol as described in Experimental Procedures and then incubated with 10 PM 5-HT CoDen circle) or saline vehicle (solid circle) for 2 h. After washing, phosphoinositide hydrdlysis was initiated by addition of 5-HT and [3H]inositol phosphate production expressed as % conversion from [‘Hlinositol phospholipid. Data shown are means + S.E. of quadruplicate estimates in a single experiment representative of three such assays. response having a median reduction of 16% (n = 23). Local pressure application of 5-HT also gave reproducible responses, but the concentration reaching the cell was unknown. Hence the desensitization of the conductance changes mediated by the 5-HT,, receptor appeared to be more pronounced with longer application of high concentrations of 5-HT. DISCUSSION

A principal finding of this study is that the endogenous 5-HT receptor on C6 glioma cells is biochemically identical to the rat brain 5-HT, receptor, as demonstrated by a wide range of methods. Firstly, C6 cells contain mRNA which appears identical to that for the rat brain 5-HT,, receptor. Secondly,

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[3H]ketanserin binds to this cell line in a manner which is pharmacologically indistinguishable from the way it binds to S-HTzA receptors in rat frontal cortex. Thirdly, as in the rat brain, 5-HT receptor activation on C6 cells results in increased phosphatidyl inositide turnover. Finally, as generally observed with receptors coupled to phosphatidyl inositide turnover, activation of the receptor in C6 cells by 5-HT stimulates an increase in cytosolic free calcium ion concentration released from intracellular stores. In contrast, the electrophysiological response to stimulation of the 5-HT,, receptor in C6 cells (hyperpolarization associated with increased membrane conductance) differs markedly from that observed at the 5-HT,, receptor in rat brain tissue (depolarization associated with decreased conductance). In keeping with the biochemical evidence, however, we have shown that the electrophysiological response to 5-HT in C6 cells likely results directly from IP, production, the release of intracellular calcium ions and the opening of calcium-activated potassium channels. Northern blot analysis of C6 cell poly(A)+ mRNA with a rat 5-HT,, receptor cDNA probe identified transcripts between 5 and 6 kb. The absence of a discrete 5-HT2, receptor transcript in this study is in agreement with that of Julius et ~1.‘~ and Mengod et aI.26 who described two 5-HT,, receptor mRNAs migrating between 5 and 6 kb in rat cortex as well as other CNS regions. In contrast, Roth and Ciaranello3’ and Roth et ~1.~~using a synthetic oligonucleotide probe to the 5-HT,, receptor, detected a single band in rat brain. It is unclear whether this latter result reflects differences in electrophoretic parameters between these studies or differences in specificity between the probes used. Further evidence for the presence of the rat 5-HT,, receptor transcripts in these cells was provided by RT-PCR and sequence analyses. RT-PCR using rat 5-HT,, receptor specific primers yielded a product predicted from the rat 5-HT,, receptor sequence. Omission of reverse transcriptase from these reactions indicates that this product is derived from mRNA rather than genomic DNA. Sequencing of this RT-PCR product revealed complete homology with a portion of the sequence encoding the third cytoplasmic loop of the rat 5-HT,, receptor.16 This region shows the least homology with other G-protein coupled seven transmembrane domain receptors. These data support the presence of the rat 5-HT,, receptor mRNA in C6 glioma cells. Previous studies using [3H]spiperone37 and [3H]5HP’ have indicated the presence of 5-HT receptors on C6 cells but were unable to specify the sub-type. In this study [3H]ketanserin clearly labelled a single site at which the binding affinities of other serotonergic ligands correlated closely with values reported for the 5-HT,, receptor in rat brain tissue.13 This

confirms the identity of the site labelled by [‘251]iodoLSD in C6 cells* as being the 5-HT,, receptor. In several species, including man and pig, [3H]mesulergine binds selectively to the 5-HT,, rather than 5-HT,, receptor 32 but this selectivity is much weaker in the rat due to a single amino acid difference within the receptor sequence.” In C6 cells [‘Hlmesulergine labelled the same population of receptors as [3H]ketanserin and the potent inhibition revealed by spiperone and ketanserin confirmed binding to the 5-HT,, rather than 5-HT,, receptor. Inhibition of [3H]ketanserin binding by 5-HT revealed a shallow displacement curve which was steepened and shifted to the right by addition of the stable GTP analogue GppNHp, characteristic of an interaction between a receptor and guaninenucleotide binding protein (G-protein). The identity of the relevant G-protein(s) is undefined at present but the ineffectiveness of pertussis toxin to modify the PI response to 5-HT excludes the G,/G, subfamilies. The characteristics of phosphoinositide turnover induced by 5-HT are similar to those previously reported in C6 cells,’ in rat pituitary Pl 1 cellsI and in rat brain slices. I8 The potency of 5-HT to induce phosphoinositide turnover and calcium mobilization in the C6 cells was similar and both responses were potently inhibited by spiperone and ketanserin, confirming mediation by the 5-HTzA receptor. The lack of effect of removing extracellular Ca*+, together with the abolition of response to _5-HT following depletion of Ca*+ stores by ionomycin, is in agreement with earlier reports implicating receptor-stimulated release of IP, in the release of calcium from intracellular stores.** In electrophysiological experiments it was found that exogenously applied 5-HT and intracellularly released IP, and Ca*+ all evoked outward currents in C6 cells. This response to 5-HT was also mediated by activation of 5-HT,, receptors, since the response was blocked by ketanserin and spiperone and was reduced by buffering intracellular calcium levels but not by lowering the extracellular calcium ion concentration, confirming the findings of Manor and Moran.23 Our evidence further indicates that the intracellular source of the calcium ions is probably not from caffeine-sensitive stores since this evoked little or no response itself. The photolytic release of IP, intracellularly mimicked the 5-HT-induced response in duration and reversal potential, which strongly suggests that they share a common mechanism of action. The response to intracellularly released calcium ions had a similar reversal potential but was much slower; this may have been due to the inherent calcium ion buffering properties of nitr-5. There were clear differences in the onset of these responses. The response to intracellular calcium ions was the fastest (ca. 60 ms) followed by the response to intracellular IP, (ca. 600 ms) while the response delay following direct application of 5-HT

5-HT,,

receptor

to the cell via a pressure pipette measured approximately 2 s. These factors taken together are consistent with the idea that SHT,, receptors in C6 cells mediate their response via a sequential cascade of events comprising the production of IP,, causing a rise in intracellular calcium ion levels, which then activates calcium-activated potassium channels in the membrane. In contrast, the activation of 5-HT,, receptors generally leads to a depolarization of central neurons by the closing of potassium channels.5,29 However, it cannot be assumed that the same biochemical route is involved in the generation of these depolarizing responses. In NG108-15 cells transformed to express Ml muscarinic receptors, which are also functionally coupled to phosphoinositide hydrolysis, acetylcholine evokes an outward current followed by a slower inward current. The outward current is mediated by the opening of calciumactivated potassium channels and the inward current by the closing of potassium channels. Only the outward current, however, appears to be mediated via IP, .34 Interestingly, a 5-HT,, receptor-mediated hyperpolarization of cultured neonatal hippocampal pyramidal cells has been observed4’ and appears to be mediated by IP,. It is likely, therefore, that activation of the 5-HTZA receptor induces different electrophysiological responses via distinct biochemical routes, possibly involving mediation via different G-proteins. The studies discussed above were all carried out using differentiated cells. When repeated using undifferentiated cells, significant changes were observed in both binding and functional characteristics of the 5-HT,, receptor. These changes were not, however, consistent with a simple, sequential cascade in receptor activation, since receptor density and maximal extent of phosphoinositide turnover induced by 5-HT were increased in undifferentiated cells whereas the proportion of cells exhibiting a rise in intracellular Ca2+ and in membrane conductance was decreased. Differentiation is associated with numerous changes in gene expression which may underlie the effects seen in C6 cells in terms of altered levels of the 5-HT,, receptor and inositol phosphate response and additional unknown proteins which regulate Ca2+ release and membrane conductance. Such comparison of functional responses are complicated, however, by the differences in time-scale of these measurements, being seconds in the case of Ca2+ mobilization and electrophysiological changes and several minutes in the case of phosphoinositide hydrolysis. These data do, though, warn against the elaboration of a simple cascade hypothesis to explain the mechanism of 5-HT,, receptor activation and indicate the important role played by currently unidentified regulatory factors. Prolonged exposure of C6 cells to the endogenous agonist 5-HT induced desensitization of all

cascade

in C6 cells

1129

three responses mediated by the 5-HT,, receptor as well as down-regulation of receptor number. Similar characteristics have been reported for the 5-HT2* receptor in rat brain, following administration of serotonergic.agonists in Coo6 and in Pll rat pituitary” and vascular smooth muscle3’ cell lines. Previous reports have indicated tachyphylaxis in the stimulated Ca2+ rise and electrophysiological response in C6 cells following repeated administration of 5-HT.30 Our data suggest that these effects are mediated at an early stage in the receptor cascade, probably due initially to decreased efficiency in coupling between the receptor, G-protein and phospholipase C, whilst continued exposure to agonist then leads to a reduction in the number of receptors. This chain of events could be investigated in greater detail using an agonist radioligand or low-temperature binding of an antagonist radioligand to intact cells. The ability to examine each component response suggests that the C6 cell line provides an excellent model for detailed examination of this phenomenon in relation to the 5-HT,, receptor. Indeed a recent study found that down-regulation of the receptor by 5-HT is associated with an equivalent decrease in the level of receptor mRNA,39 indicating that regulation of receptor activity probably occurs at multiple points in the metabolic cycle of the receptor protein. The physiological role of 5-HT receptors on glial cells is unclear. Glial cells are traditionally seen as buffers and support cells for the more electroactive neuronal cells. Evidence is increasing, however, for a more active role for glial cells since a wide range of neurotransmitter receptors are found on them and postsynaptic potentials have been demonstrated in them.27 Rises in intracellular calcium ions in astrocytes can spread, via gap junctions between them, in slowly moving waves and, indeed, this type of signal can be transmitted to neighbouring neurones.28 CONCLUSION

In conclusion, this study demonstrates that the rat C6 glioma cell line endogenously expresses a 5-HT,, receptor which is biochemically identical to that identified in rat brain. The functional cascade of events following receptor activation the appears to be clearly defined, although consequences of cell differentiation indicate that additional unknown factors play an important part in defining the final response. This cell line therefore constitutes an excellent model with which to examine the detailed mechanism of action of the rat 5-HT,, receptor. Acknowledgements-We would like to thank the Medical Research Council (RAN) and the Wellcome Trust (SJB) for financial support.

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REFERENCES 1. Ananth U. D., Leli U. and Hauser G. (1987) Stimulation of phosphoinositide hydrolysis by serotonin in C6 glioma cells. J. Neurochem. 48, 253-261. 2. Auffray C. and Rougeon F. (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumour RNA. Eur. J. Biochem. 107, 303-314. 3. Bartrup J. T. and Newberry N. R. (1994) 5-HT,, receptor-mediated outward current in C6 glioma cells is mimicked by intracellular IP, release. NeuroReport. 5, 1245-1248. 4. Berridge M. J., Dawson R. M. C., Downes C. P., Heslop J. P. and Irvine R. F. (1983) Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem. J. 212, 473482. 5. Bobker D. H. and Williams J. T. (1990) Ion conductances affected by 5-HT receptor subtypes in mammalian neurons. Trends Neurosci. 13, 169-173. 6. Buckholtz N. S., Zhou D. and Freedman D. X. (1988) Serotonin, agonist administration down-regulates rat brain serotonin, receptors. Life Sci. 42, 2439-2445. I. Cheng Y. C. and Prusoff W. H. (1973) Relationship between the inhibition constant (K,) and the concentration of inhibitor which causes 50 per cent inhibition (vQ of an enzymatic reaction. Biochem. Pharmac. 22, 3099-3018. 8. Ding D., Toth M., Parks C., Hoffman B. J. and Shenk T. (1993) Glial cell-specific expression of the serotonin 2 receptor gene: selective reactivation of a repressed promoter. Molec. Brain Res. 20, 18lll91. Analyt. Biochem. 110, 9-18. 9. Duggleby R. G. (1981) A nonlinear regression program for small computers. study of bovine chromaffin cells and of their sensitivity 10. Fenwick E. M., Marty A. and Neher E. (1982) A patch-clamp to acetylcholine. J. Physiol. 331, 5777597. 11. Grynkiewicz G., Poenie M. and Tsien R. (1985) A new generation of Car+ indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440-3450. 12. Hoyer D., Engel G. and Kalkman H. 0. (1985) Molecular pharmacology of 5-HT, and 5-HT, recognition sites in rat and pig brain membranes: radioligand binding studies with [‘HIS-HT, [‘H]8-OH-DPAT, ( -)[‘251]iodocyanopindolol, [‘Hlmesulergine and [3H]ketanserin. Eur. J. Pharmac. 118, 13-23. Characterization 13. Hoyer D., Pazos A., Probst A. and Palacios J. M. (1986) Serotonin receptors in the human brain-II. and autoradiographic localisation of 5-HT,, and 5-HT, recognition sites. Brain Res. 376, 97-107. 14. Ivins K. J. and Molinoff P. B. (1990) Serotonin-2 receptors coupled to phosphoinositide hydrolysis in a clonal cell line. Molec. Pharmac. 37, 622430. and down-regulation of 5-HT, receptors in PI 1 cells. J. Pharmc. 15. Ivins K. J. and Molinoff P. B. (1991) Desenzitisation exp. Ther. 259, 423429. 16. Julius D., Huang K. N., Livelli T. J., Axe1 R. and Jesse11 T. M. (1990) The S-HT, receptor defines a family of structurally distinct but functionally conserved serotonin receptors. Proc. nntn. Acad. Sci. U.S.A. 87, 928-932. 17. Kao H.-T., Adham N., Olsen M. A., Weinshank R. L., Branchek T. A. and Hartig P. R. (1992) Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT, receptor from a human to a rat pharmacology. Fedn. Eur. biochem. Sots Letf. 307, 324-328. S. R. (1985) 5-Hydroxytryptamine-stimulated inositol phospholipid hydrolysis in rat 18. Kendall D. A. and Nahorski . .__ _ cerebral cortex slices: pharmacological characterization and effects ot- antidepressants. J. Pharmac. exp. Ther. 233, 473479. 19. Leysen.J. E., Niemegeers C. J. E., van Neuten J. M. and Laduron P. M. (1982) [3H]Ketanserin (R41 468), a selective ‘H-ligand for serotonin, receptor binding sites. Molec. Pharmac. 21, 301-314. 20. Leysen J. E., Eens A., Gommeren W., van Gompel P., Wynants J. and Janssen P. A. J. 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inositol