Characterization of a Novel 3H-5-Hydroxytryptamine Binding Site Subtype in Bovine Brain Membranes

Home , Corynanthine, Cyanopindolol, Iodocyanopindolol, Mesulergine

The Journal of Neuroscience, March 1997, 7(3): 894-903

Richard E. Heuring and Stephen J. Peroutka Departments of Neurology and Pharmacology, Stanford University Medical Center, Stanford, California 94305

3H-5-Hydroxytryptamine (5HT) binding sites were analyzed terized: 5-HT, siteslabeled by 3H-5-HT and 5-HT, siteslabeled in bovine brain membranes. The addition of either the 5-HT,,- by 3H-spiperone(Peroutka and Snyder, 1979). More recently, selective drug 8-OH-DPAT (100 nM) or the 5-HT,,-selective 5-HT, binding sites labeled by 3H-5-HT have been shown to drug mesulergine (100 nM) to the assay rkulted in a 5-l 0% be heterogeneous(Pedigo et al., 1981; Schnellmanet al., 1984; decrease in specific 3H-5-HT binding. Scatchard analysis Asarch et al., 1985; Hoyer et al., 1985b; Peroutka, 1986). To revealed that the simultaneous addition of both drugs de- date, 3 distinct subtypesof the 5-HT, binding site have been creased the B,,,,, of 3H-5-HT binding by lo-15% without af- identified. 5-HT,, sitescan be directly labeled with 3H-8-hy- fecting the & value (1.8 f 0.3 nM). Competition studies us- droxy-N,iV-dipropyl-2-aminotetralin (8-OH-DPAT) (Gozlan et ing a series of pharmacologic agents revealed that the sites al., 1983; Hall et al., 1985; Peroutka, 1985,1986), 3H-ipsapirone labeled by 3H-5-HT in bovine caudate in the presence of 100 (Dompert et al., 1985) or 3H-l-(2-(4-aminophenyl)ethyl)-4-(3- nM 8-OH-DPAT and 100 nM mesulergine appear to be ho- trifluoromethylphenyl)piperazine (PAPP) (Ransomet al., 1986) mogeneous. 5-HT,, selective agents such as 8-OH-DPAT, and display nanomolar affinity for 8-OH-DPAT, ipsapirone, ipsapirone, and buspirone display micromolar affinities for and buspirone.The 5-HT,, site appearsto be limited to rat and these sites. RU 24989 and (-)pindolol are approximately mouse brain and displays high affinity for RU 24969 and 2 orders of magnitude less potent at these sites than at (-)pindolol (Hoyer et al., 1985a; Heuring et al., 1986). The 5-HT,, sites which have been identified in rat brain. Agents 5-HT,, site is denselypresent in the choroid plexus but hasalso displaying nanomolar potencies for 5-HT,, sites such as been identified in rat and porcine cortex (Pazos et al., 1984; mianserin and mesulergine are 2-3 orders of magnitude less Yagaloff and Hartig, 1985; Peroutka, 1986). This site displays potent at the 3H-5-HT binding sites in bovine caudate. In relatively high affinity for mesulergine,mianserin, and meth- addition, both 5-HT,- and 5-HT,-selective agents are essen- ysergide.Although all 5-HT, binding sitescan be labeledby 3H- tially inactive at these binding sites. These 3H-5-HT sites 5-HT, each of the known subtypesdisplays a unique pharma- display nanomolar affinity for 5-carboxyamidotryptamine, cological profile. 5-methoxytryptamine, metergoline, and 5-HT. Apparent K, The bovine caudatecontains a high density of 3H-5-HT bind- values of 1 O-l 00 nhl are obtained for &LSD, RU 24969, me- ing sites (Whitaker and Seeman, 1978; Peroutka and Snyder, thiothepin, tryptamine, methysergide, and yohimbine, 1981). Preliminary studiesin this laboratory indicated that the whereas /-LSD and corynanthine are significantly less po- specific 3H-5-HT binding in this region contained non-5-HT,, tent. In addition, these 3H-5-HT labeled sites are regulated binding sites(Peroutka, 1985). Therefore, the present study was by guanine nucleotides and calcium. Regional studies in- performed in order to characterize thesesites more extensively. dicate that this class of sites is most dense in the basal The data show that the majority of 3H-5-HT binding sitesin ganglia but exists in all regions of bovine brain. These data bovine caudateare homogeneousand distinct from the 5-HT,,, therefore demonstrate the presence of a homogeneous class 5-HT,,, and 5-HT,, sitesthat have been describedpreviously. of 5-HT, binding sites in bovine caudate that is pharmaco- Moreover, the site appearsto exist in all bovine brain regions logically distinct from previously defined 5-HT,,, 5-HT,,, and is the most common 5-HT, binding site subtype in bovine 5-HT,,, 5-HT,, and 5-HT, receptor subtypes. We therefore brain. suggest that this class of sites be designated the 5-HT,, subtype of binding sites labeled by 3H-5-HT. Materials and Methods Receptorbinding assays were performed according to the methodsof Radioligand binding studies have identified multiple 5-hy- Peroutka(1985). Briefly, adultbovine brains were obtained from a local droxytryptamine (5HT) receptorsin the mammalian CNS. Ini- slaughterhouse,dissected, immediately frozen, and storedat -70°C tially, 2 main types of central 5-HT binding siteswere charac- until needed.On the day of study, the tissuewas defrosted and ho- mogenizedin 20 vol of 50 mM Tris-HCl (pH 7.7 at 25°C)using a Brinkmann Polytron and then centrifugedin an IEC B20A centrifuge at 49,000x g for 10min. The supematantwas discarded, and the pellet Received June 16, 1986; revised Sept. 8, 1986; accepted Sept. 25, 1986. wasresuspended in the samevolume of Tris-HClbuffer and incubated This work was supported in part by the John A. Hartford Foundation, the Alfred P. Sloan Foundation, and NIH Grant NS 12 15 l- 11. We would like to at 37°C for 10 min prior to a second centrifugation at 49,000 x g for thank Faith Smith for help in preparation of the manuscript, Christina M. De- 10 min. The final pellet was resuspended in 80 vol of Ttis-HCl buffer mopulos for excellent technical assistance, and Dr. Charles Weitz for assistance containing10 WM pargyline,4 mM calciumchloride and 0.1%ascorbic with HPLC experiments. acid. For experimentsconcerning the effectsof Ca2+,membranes were Correspondence should be addressed to Stephen J. Peroutka at the above ad- suspendedin Caz+-freebuffer. Ascorbicacid wasadded to all assays dress. becauseof its recentlydemonstrated effects on 3H-5-HTstability (Per- Copyright 0 1987 Society for Neuroscience 0270-6474/87/030894-10$02.00/O outka et al., 1986~).The suspensionswere immediatelyused in the The ;lournal of Neuroscience, March 1987, 7(3) 895

* 5-HT

u 8-OH-DPAT

m MESULERGINE

m - LOGeRUG]

Figure 1. 5-HT, 8-OH-DPAT and mesulergine competition studies with 80 )H-5-HT binding in bovine caudate. M 5-HT Increasing concentrations of 5-HT, 8-OH-DPAT, or mesulergine were in- 40- - S-OH-DPAT a cubated with 2.0 nM 3H-5-HT and bovine caudate membranes as described m MESULERGINE in Materials and Methods. Data shown 20- are the results of a single experiment performed in triplicate. Each experiment was repeated 3-6 times. A, Com- petition curves under standard assay conditions. B, Competition curves in the presence of 100 nM 8-OH-DPAT + - LOGbRUG] 100 nM mesulergine.

binding assay. In addition, the stability of the 3H-5-HT (i.e., >98% MO); quipazine (Miles Scientific, Naperville, IL); RU 24969 (Roussel recovery) under standard assay conditions was documented by high- Uclaf, Paris); ipsapirone (Troponwerke, Cologne); methysetgide, me- pressure liquid chromatographic analysis as described previously (Per- sulergine, (-)pindolol (Sandoz, East Hanover, NJ); mianserin (Organon, outka et al., 1986~). West Orange, NJ); (-)propranolol (Ayerst, New York, NY); d-LSD, Binding assays consisted of 0.1 ml ‘H-ligand (final concentration, I-LSD (National Institute on Drug Abuse, Bethesda, MD); m-chloro- 1.6-2.0 nM ‘H-5-HT), 0.1 ml buffer or displacing drug and 0.8 ml tissue phenylpiperazine (mCPP) (Aldrich, Milwaukee, WI); metergoline (Far- suspension. Following incubation at 25°C for 30 min, the assays were mitalia, Milan); buspirone (Bristol Myers, Evansville, IN); haloperidol rapidly filtered under vacuum through #32 glass fiber filters (Schleicher (McNeil Pharmaceuticals, Spring House, PA); chlorimipramine (Ciba- and Schuell; Keene, NH) with two 5 ml washes using 50 mM Tris-HCl Geigy, Summit, NJ); prazosin (Pfizer, New York, NY); GppNHp (Phar- buffer (pH 7.4). Radioactivity was measured by liquid scintillation spec- macia, Piscataway, NJ); MDL 72222 (Merrel-Dow, Strasburg, France); troscopy in 5 ml of 3a70 scintillation cocktail (Research Products In- methiothepin (Hoffmann-La Roche, Nutley, NJ). WB 4 10 1 was the gift ternational, Mount Prospect, IL) at 56% efficiency. Specific binding was of Dr. David A. Greenberg, 5-carboxyamidotryptamine (5-CT) was the defined using 10 PM 5-HT for all experiments and represented 70-80% gift of Dr. Roger Whiting, and yohimbine was the gift of Dr. Julian of total binding for ‘H-5-HT. Davidson. Drugs were obtained from the following sources: ‘H-5-HT (19.8 Ci/ Drug displacement studies were analyzed by computer-assisted non- mmol; Amersham, Arlington Heights, IL); 8-OH-DPAT, l-(m-trifluo- linear regression analysis based on the methods of Munson and Rodbard romethylphenyl)piperazine (TFMPP) (Research Biochemicals, Inc., (1980) and McPherson (1983). Drug concentrations ranged from lo-l1 Waltham, MA); spiperone, ketanserin (Janssen Pharmaceuticals; Beer&; to 1O-4 M. All data were initially analyzed based on an assumption of cyproheptadine, 5-methoxytryptamine, corynanthine, metoclopramide, a l-site model of “H-l&and binding. The data were then analyzed ac- 5-HT, tryptamine, N,N-dimethyltryptamine (N,N-DMT), GTP, GDP, cording to a 2-site model, and the results of the curve fitting were GMP, ATP, dopamine, (-)epinephrine (Sigma Chemical Co., St. Louis, statistically compared to the 1-site study by an F test. The 2-site model 896 Hewing and Peroutka l 3H-5-HT Binding in Bovine Caudate

Table 1. Computer-assisted nonlinear regression analysis of 5-HT, S-OH-DPAT, and mesulergine interactions with “H-5-HT binding in bovine caudate

‘H-5-HT + 100 nM &OH-DPAT + )H-5-HT 100 nM mesulergine DIW K, K, K 5-HT 2.9 f 2 (100%) - 3.2 f 2 I-OH-DPAT 1.7 f 0.9 (7%) 1100 f 200 (93%) 700 + 80 tiesulergine 0.9 1 + 0.1 (8%) 10,000 + 2000 (92%) 6500 + 500 Competition studies were performed as described in Materials and Methods using 2 nM IH-5-HT. Computer-assisted analysis of the competition curves was performed according to the method of Munson and Rodbard (1980) and McPherson (1983) and was used to determine K, values and relative concentrations of each binding site. Each curve was fit according to both a I-site and a 2-site model. When the 2-&e model gave a statistically better fit (p < 0.05), 2 K, valuesare given, with the percentage of each site reported in parentheses. Data given are the means + SE of 3 separate experiments, each performed in triplicate. was accepted if the observed “fit” was significakly better @ < 0.05) ditions (Fig. 1A). By contrast, neither 8-OH-DPAT nor mesu- than the “fit” observed using a l-site model of 3H-ligand binding. All lergine displaced any specific 3H-5-HT binding at concentra- other statistical comparisons were made using the standard t test. tions below lo-’ M. Above concentrations of 2 x lo-’ M, Results monophasiccompetition curveswere observedwith both drugs. Computer-assistediterative curve fitting analysisconfirmed that 5-HT, S-OH-DPAT, and mesulerginecompetition studieswith all 3 drug competition curves are adequately describedby a 3H-5-HT in bovine caudate 1-site model of ‘H-5-HT binding (Table 1). Drug competition studieswith ‘H-5-HT were performed in bovine caudateas shown in Figure 1A. 5-HT competesfor specific 3H-S-HT binding in a monophasicmanner. It beginsdisplacing Saturation analysisof 3H-5-HT binding in bovine caudate specific3H-5-HT binding at concentrations above lo-lo M and Saturation studieswere performed with 3H-5-HT under stan- displaces50% of binding at approximately 3 x 1O-9 M. All dard assay conditions or in the presenceof 100 nM 8-OH- remaining specific binding is displacedby a concentration of DPAT + 100 nM mesulergine.3H-5-HT displayedmonophasic 3 x lo-’ M, and no further displacementis observed at con- saturation in both the absenceand presenceof 100 nM g-OH- centrationsas high as 1O-4M. Therefore, 1O-5M 5-HT was used DPAT + 100 nM mesulergine. For the experiment shown in to define nonspecific binding in all subsequentexperiments. Figure 2A, Scatchardanalysis demonstrated a singlehigh-affinity Computer-assistednonlinear regression analysis of the data shows (KD = 1.1 nM) component of 3H-5-HT binding (Fig. 2B). How- that the 5-HT competition curve is adequately describedby a ever, in the presenceof 100 nM 8-OH-DPAT + 100 nM me- l-site model with a K, value of 2.9 + 1 nM (Table 1). sulergine,3H-5-HT labeled approximately 10% fewer binding In contrast, the 5-HT,,-selective drug 8-OH-DPAT and the sites(B,,, = 8.2 pmol/g tissue)than under standardassay con- 5-HT,,-selective drug mesulerginecompete with 3H-5-HT in a ditions (9.0 pmol/g tissue), despite the fact that the KD value biphasic manner (Fig. 1A). Each drug competes for a small (1.5 nM) was not significantly different. Basedon an analysisof percentageof sitesat nanomolar concentrations with a plateau 4 independent saturation studiesin bovine caudate, ‘H-5-HT observedat concentrationsbetween 1O-8 and 2 x 1O-’ M. g-OH- displayed a KD value of 1.5 -t 0.2 nM and a B,,, of 8.7 f 0.3 DPAT displacesremaining specific 3H-5-HT binding at con- pmol/g tissue under standard assayconditions. In the presence centrationsabove 2 x lo-’ M. Similarly, mesulerginecompetes of 100 nM 8-OH-DPAT + 100 nM mesulergine,the K, was for all but approximately 10% of specific )H-5-HT binding at essentiallyunchanged (1.8 f 0.5 nM). However, the B,,, value concentrations as high as 1O-4 M. Computer-assistediterative (7.7 f 0.3 pmol/g tissue)was reduced significantly compared curve-fitting analysisconfirms the biphasicnature of the curves with control values (p < 0.05, t test). This 11% decreasein B,,, by best fitting each with a 2-sitemodel (p < 0.0 1). 8-OH-DPAT is consistent with the competition studiesshown in Figure lA, recognizes7 + 3% of total ‘H-5-HT siteswith high affinity (K, = which suggestthat lO-15% of ‘H-5-HT labeled sitesin bovine 1.7 k 1 nM) and the remaining 93 f 3% of siteswith relatively caudate are displaced by 100 nM 8-OH-DPAT + 100 nM me- low affinity (K, = 1100 + 200 nM). Similarly, mesulerginerec- sulergine. ognizesa low percentage(8 f 2%) of high-affinity sites (K, = 0.91 ? 0.1 nM) and the remaining sites with relatively low Effect of nucleotideson -‘H-5-HT binding in bovine caudate affinity (K, = 10,000 f 2000 nM). Sincethese data suggesta low Nucleotide [GTP, GDP, Gpp(NH)p, GMP, ATP] interactions percentageof 5-HT,, and 5-HT,, sites in bovine caudate, all with specific 3H-5-HT binding in the presence of 100 nM g-OH- subsequentcompetition experiments were performed in the DPAT + 100 nM mesulerginewere examined in bovine caudate presenceof 100 nM 8-OH-DPAT + 100 nM mesulergine.These membranes.GTP, GDP, and the nonmetabolizableGTP ana- drug concentrations are able to selectively block the low per- log, Gpp(NH)p, significantly decreasedspecific 3H-5-HT bind- centage of putative 5-HT,, and 5-HT,, sites without signifi- ing (p < 0.01) at a concentration of 1O-3 M. Each nucleotide cantly affecting the remaining 3H-5-HT-labeled sites. displaced 40-42% of specific 3H-5-HT binding in the bovine Under this condition, each of the 3 drugs appearsto recognize caudate. By contrast, 1O-3M ATP and GMP had no significant a homogeneouspopulation of specific 3H-5-HT binding sites effect on specific 3H-5-HT binding. A statistically significant (Fig. 1B) in bovine caudate. The competition curve for 5-HT difference exists between the effect ATP and those of GTP, GDP, is virtually identical to the curve observed under standard con- or GppNHp (p < 0.0 1 for each comparison).Thus, modulation The Journal of Neuroscience, March 1987, 7(3) 897

;; 11 z 10

iil;:::--‘=-;‘I / 2 5 0 E 4 H 3H-5-HT+100 nM 8-OH-DPAT 0 3 +ioonM MESULERGINE k!

Ii I I I I I I I I I ml OO 3 8 9 12 15 18 21 24 3H-5-HT FREE (nM)

2 1”9 v1.1 9.0 M 3H-5-HT ti

Figure 2. ‘H-5-HT saturationstudies 81 \ 1.5 8.2 m 3H-5-HT + 100 nM 13-OH-DPAT in bovine caudate. Increasing concen- + 100 nM MESULERGINE trations (0.95-46 nM) of ‘H-5-HT were incubated with bovine caudate membranes under standard assay (0) conditions or in the presence of 100 nM &OH-DPAT + 100 nM mesuletgine (B) 4- as described in Materials and Methods. Data shown are the results of a single 3- experiment performed in triplicate. Each experiment was repeated 4 times. Scat- 2- chard data were analyzed by linear- lb regression analysis. The K, and B,,, values given are for this single experiment, which was performed in tripli- 0 1 2 3 4 5 8 7 8 9 cate. A, Specific )H-5-HT binding. B, 3H-5-HT BOUND/FREE (X lo21 m4 Scatchard analysis of data shown in A. of specific 3H-5-HT binding sites in bovine caudate showsa Ca*+. This effect was significantly greater (p < 0.01) than the rank order of sensitivity as previously demonstratedwith other effect observed in the presenceof 4 mM Ca*+ (i.e., 60 + 1% of 5-HT binding site subtypes:GppNHp = GTP = GDP > GMP = control values). By contrast, 1O-3 M ATP effects on ‘H-5-HT ATP (Peroutka et al., 1979; Mallat and Hamon, 1982; Battaglia binding were unaltered in the absenceof Ca*+ (100 + 3% of et al., 1984; Schlegeland Peroutka, 1986). control values). This pattern of Ca2+ sensitivity has been describedpreviously for other 5-HT binding site subtypes(Mallat Efect of 4 mM Caz+ on specific3H-5-HT binding in bovine and Hamon, 1982; Hall et al., 1985; Blurton and Wood, 1986). caudate In the presenceof 100 nM 8-OH-DPAT + 100 nM mesulergine Regional analysis of ‘H-.5-HT binding in the absenceor in bovine caudate membranes,4 mM Ca2+ modulated specific presenceof 100 nM 8-OH-DPAT and 100 nM mesulergine XH-5-HT binding. At half-maximal 3H-5-HT concentrations(2.0 A concentration of 2.0 nM 3H-5-HT was usedto define regional nM), the addition of 4 mM Caz+ to the binding assaycaused an differencesin total 3H-5-HT binding versus )H-5-HT binding increasein total binding and a decreasein nonspecificbinding, in the presenceof 100 nM 8-OH-DPAT + 100 nM mesulergine. yielding an increasein specific3H-5-HT binding. For example, As noted in previous studies(Peroutka and Snyder, 198l), total 4 mM Ca*+ increasedtotal binding by 7 * 1% and decreased 3H-5-HT binding was most densein the substantianigra, cau- nonspecificbinding by 23 f 1% compared with values obtained date, hippocampus, globus pallidus, and putamen (Table 2). usinga Ca*+-freebuffer. The net result was a significant increase However, a distinct difference existed between regions in the (17 f 1%; p < 0.01) in the specific binding of 3H-5-HT to amount of specific3H-5-HT binding that remained in the pres- bovine caudate membranesin the presenceof 100 nM g-OH- ence of 100 nM 8-OH-DPAT + 100 nM mesulergine.In both DPAT + 100 nM mesulergine.In addition, the sensitivity of caudate and globuspallidus, approximately 80% of specific)H- 3H-5-HT binding to 1O-3M GTP was significantly increasedin 5-HT binding remained under this condition. By contrast, the the absenceof Ca*+. For example, 1O-3M GTP decreased3H- majority of specific)H-5-HT binding in hippocampuswas dis- 5-HT binding to 48 + 4% of control values in the absenceof placed by 100 nM 8-OH-DPAT + 100 nM mesulergine,with 898 Heuring and Peroutka l 3H-5-HT Binding in Bovine Caudate

Table 2. Regional distribution of specific 3H-5-HT binding in the absence or presence of 100 nM 8-OH-DPAT and 100 nM mesulergine

Specific)H-5-HT binding (100= 3.8 + 0.3 pmoVgtissue) Percentage >H-5-HT+ of total 3H-5-HT 100nM 8-OH-DPAT + binding Region (total) 100nM mesulergine remaining Substantianigra 129 dz 20 85 + 5 66 Caudate 100* 9 83 + 0.3 83 Hippocampus 93 -+ 10 28 + 4 30 Globuspallidus 88 f 10 70+ 10 80 Occipitalcortex 83 + 1 50 k 2 60 Putamen 81 +6 54 * 4 67 Superior colliculus 78 + 20 61 f 10 78 Inferior colliculus 76 zk 10 60 + 9 79 Frontd cortex 75 zk 4 58 + 8 77 Temporalcortex 72 k 4 41 + 2 57 Cingulatecortex 63 f 10 40 IL 1 63 Thalamus 59 f 6 49 f 2 83 Hypothalamus 55 + 6 34 * 4 62 Pulvinar 29 + 1 26 f 1 90 Choroidplexus 24 +- 2 11 f 1 46 Pons 21 + 4 19 + 4 90 Cerebellarnuclei 16 k 4 16 + 4 100 Medullaoblongata 15 f 4 5 + 0.4 33 Cerebellarcortex 7t2 822 110 Binding assays were performed as described in Materials and Methods. Values given are the mean + SE of triplicate assays. Each experiment was repeated 3-4 times. Data are described as the percent of total specific binding observed using 2.0 nM )H-5-HT, where 100% = 3.8 + 0.3 pmol/g wet weight tissue. only 30% of the original amount of specific 3H-5-HT binding WB 4 101, buspirone and ipsapirone display nanomolar affinity remaining. for 5-HT,, sites in rat frontal cortex (Peroutka, 1986), they In cortical areas,total specific3H-5-HT binding rangedfrom compete for specific 3H-5-HT binding in bovine caudate at 63 to 83% of caudate values. The amount of specific binding micromolar concentrations. Moreover, all 3 competition curves remaining in the presenceof 100 nM 8-OH-DPAT + 100 nM are monophasicbelow concentrations of 1O-4 M. The Hill slope mesulergineranged from 57% in the temporal cortex to 77% in of WB 4101 competition with ‘H-5-HT binding under these the frontal cortex. While the midbrain, brain stem, and cere- conditions was 1.1 + 0.02. K, values for eachagent are presented bellar regions contained relatively low densitiesof total 3H-5- in Table 3. HT binding sites, they were similar in that they contained a By contrast, certain 5-HT,,-selective drugs were more potent very high percentageof sitesnot displacedby 100 nM 8-OH- competitors of ‘H-5-HT binding sitesin bovine caudate (Fig. DPAT + 100 nM mesulergine.The single exception was the 3B). Computer analysisof RU 24969 interactions with the spe- medulla oblongata, in which only 35% of the specific3H-5-HT cific 3H-5-HT binding indicates that the data are adequately binding remained in the presenceof 8-OH-DPAT and mesu- explained by a singlepopulation of binding siteswith a K, value lergine. of 25 f 1 nM. The piperazinesTFMPP and mCPPalso displaced The choroid plexus was the only other brain region in which ‘H-5-HT in a monophasicmanner, with Hill slopesof 0.90 f the majority of ‘H-5-HT binding wasdisplaced by 100 nM 8-OH- 0.1 and 0.99 f 0.03, respectively. In addition, 2 relatively potent DPAT + 100 nM mesulergine.This finding is of interest since inhibitors of 5-HT,, sites in rat brain (Hoyer et al., 1985), the choroid plexus is the region in which 5-HT,, binding R sites (-)pindolol and (-)propranolol, were more than loo-fold less were first identified and characterized (Pazos et al., 1984; potent at these ‘H-5-HT binding sitesin bovine brain (Table Yagaloff and Hartig, 1985). 3). Similarly, drugs reported to display relatively high affinity for Drug interactions with ‘H-5-HT binding sitesin bovine the 5-HT,, binding site subtype (Pazos et al., 1984; Hoyer et caudate in the presenceof 100 nM 8-OH-DPAT + 100 nM al., 1985b) were markedly less potent in competing for 3H-5- mesulergine HT binding in bovine caudate (Fig. 3C). Methysergide is the To further characterize specific3H-5-HT binding sitesin bovine most potent of the 5-HT,,-selective agents,with an IC,, value caudate, the interactions of a seriesof drugs were determined of approximately 100 nM and a Hill slopeof 0.92 f 0.06. Mian- in competition studieswith ‘H-5-HT in the presenceof 100 nM serin was slightly lesspotent (K, = 480 nM; Hill slope= 1.04 + 8-OH-DPAT + 100 nM mesulergine.Figure 3A showsthat sev- 0.03), while ketanserinand mesulerginewere essentiallyinactive eral potent 5-HT,,-selective drugs are all relatively weak in at the 3H-5-HT binding sitesin bovine caudate. competing for 3H-5-HT binding in bovine caudate. Although The most potent agentsat this classof 3H-5-HT binding sites The Journal of Neuroscience, March 1987, 7(3) 899

ii I I JlOO- $2 - ii z 5 8o ; : : 80- I; + a ‘- H WB 4101 0 : 40- iL 0 L ISAPIRONE 3 ’ E g - BUSPIRONE 20- m CL : :, z 0 I I I I I I \ ic z 9 8 LO’G [DRUGI 5 4 - m r

B -

-TFMPP

I I I I I I I 1 Oat------L c 1 Figure 3. Competition studies of 5-HT,,-, SHT,,-, and S-HT,,-selective drugs with ‘H-5-HT binding in bovine caudate membranes in the presence of 100 nM 8-OH-DPAT + 100 nM mesulergine. Competition studies with ‘H-5-HT (1.6-2.0 nM) binding in the presence of 100 nM I-OH-DPAT + 100 4 MMETHYSERGIDE nM mesulergine were performed as described in Materials and Methods. The dMIANSERIN data given are the results of single ex- 20- *KETANSERIN periments performed in triplicate. Each experiment was repeated 3-6 times. A, Competition studies with 5-HT,,-selective drugs. B, Competition studies with 5-HT,,-selective drugs. C, Com- petition studies with 5-HT,,-selective LOG [DRUG] drugs. 900 Heuring and Peroutka 0 3H-5-HT Binding in Bovine Caudate

Table 3. Drug interactions with 3H-5-HT binding in bovine caudate membranes

K K DWZ 6-4 Dt-e (n@ SHT,, selective drugs Tryptamines WB 4101 590 + 60 5-CT 0.75 + 0.1 I-OH-DPAT 700 TL 50 5-Methoxytryptamine 2.5 + 1 Ipsapirone 16,000 k 2000 5-HT 3.2 f 1 Buspirone > 100,000 Tryptamine 36 k 6 SHT,, selective drugs N, N-DMT 190 f 10 RU 24969 25 k 1 SHT, selective drugs TFMPP 280 + 30 Cyproheptadine 1200 + 100 mCPP 1400 k 200 Haloperidol 8200 zk 600 (-)Pindolol 7000 k 700 Spiperone > 100,000 (-)Propranolol 7200 + 5000 SHT, selective drugs 5-HT,, selective drugs Metoclopramide 9000 Ik 300 Methysergide 120 + 20 MDL 72222 > 100,000 Mianserin 480 + 20 Others Mesulergine 6500 f 500 Ketanserin 53,000 iz 10,000 Methiothepin 36 + 6 Yohimbine 59 f 6 Ergot derivatives Quipazine 800 + 100 Metergoline 4.2 + 1 Corynanthine 23,000 + 1000 d-LSD 24 f 2 Dopamine 36,000 + 5000 I-LSD > 100,000 Epinephrine > 100,000 Prazosin > 100,000

Binding assays using 1.6-2.0 no 3H-5-HT in the presence of 100 no 8-OH-DPAT + 100 no mesulergine were performed as described in Materials and Methods. IC,, values were determined by log-logit analysis. K, values were calculated according to the equation K, = IC,J( 1 + [‘H-5-HTIIK,). The KD value for ‘H-5-HT (1.8 nM) was derived from saturation experiments. Data given are means K, f SE of 3-6 experiments, each performed in triplicate. were tryptamine derivatives (Table 3). 5-CT had a K, of 0.75 f that thesesites be designated5-HT,, binding sites. 0.1 nM, while 5-HT and 5-methoxytryptamine were slightly less The initial studies of 3H-5-HT binding suggestedthat the potent. Ergot derivatives were also potent agents.Metergoline, radioligand labeleda heterogeneouspopulation of binding sites d-LSD, and methysergidehad K, values of 4.2 f 1,24 I~I2, and (Bennett and Snyder, 1976; Fillion et al., 1978). Nelson and 120 ? 20 nM, respectively. In addition, yohimbine, a putative colleagueswere the first to suggestthat at least 2 distinct subtypes alpha,-adrenergicagent, was moderately potent at this classof could be identified using3H-5-HT (Pedigoet al., 1981; Schnell- 3H-5-HT binding sites(K, = 59 + 6 nM). The binding site also man et al., 1984). The 5-HT,, site was defined as having high displayedstereoselectivity, as demonstratedby the inactivity of affinity for spiperone,while the 5-HT,, site wasdefined as being l-LSD and corynanthine. Drugs that are potent agents at the relatively insensitive to this neuroleptic. More recently, the de- putative 5-HT, receptor in the periphery (MDL 72222 and me- velopment of highly potent and binding site subtype selective toclopramide) were essentially inactive at this central 5-HT agentshas greatly facilitated the analysisof 5-HT, binding site binding site. Finally, the biogenic amine neurotransmittersepi- subtypes(Middlemiss and Fozard, 1983). nephrineand dopamine were inactive at this binding site. To date, the 5-HT,, site has been the most extensively characterized subtype of the 5-HT, classof binding sites(Table 4). Discussion This site can be directly labeledby 3H-8-OH-DPAT (Gozlan et The major finding of the present study is that 3H-5-HT labels al., 1983; Hall et al., 1985;Peroutka, 1985,1986), 3H-ipsapirone a population of binding sitesin bovine caudate that is distinct (Dompert et al., 1985), 3H-PAPP (Ransomet al., 1986), or 3H- from previously defined 5-HT,,, 5-HT,,, and 5-HT,, binding WB 4 10 1 (Norman et al., 1985). Unlike the 5-HT, binding site site subtypeslabeled by ‘H-5-HT. The binding of 3H-5-HT in subtype describedin the presentreport, the 5-HT,, site displays bovine caudate is saturable, stereoselective,and displays high nanomolar affinity for 8-OH-DPAT, WB 4 101, and ipsapirone. affinity (KD = 1.8 + 1 nM). Computer-assistedcurve-fitting anal- In addition, the site is denselypresent in the hippocampus(Pa- ysis suggeststhat thesesites are homogeneousin bovine caudate. zos and Palacios, 1985). The most potent agents at these sites are tryptamines, ergots, A number of functional correlates have been proposed for RU 24969, and yohimbine. The sitesare modified by guanine this site. Neurophysiologically,5-HT and 5-HT,, selectiveagents nucleotidesand calcium. Regional studiessuggest that this class inhibit intrinsic raphe cell firing (VanderMaelen and Wilder- of sitesis present in all brain regionsand that it representsthe man, 1984; Sprouseand Aghajanian, 1985; Trulson and Ar- predominantform of 5-HT, binding site subtypein bovine brain. asteh,1985) and the population spikegenerated by hippocampal Since these pharmacologicalcharacteristics are distinct from CA1 cells (Beck and Goldfarb, 1985; Peroutka et al., 1987). previously described 5-HT, binding site subtypes, we suggest In addition, both forskolin-induced stimulation of adenylate The Journal of Neuroscience, March 1987, 7(3) 901

Table 4. Characteristics of 5HT receptor subtypes

5-HT,, 5-HT,, 5-HT,, 5-HT,, 5-HT, 5-HT, Radiolabeled by ‘H-5-HT 3H-5-HT ‘H-5-HT )H-Spiperone - ‘251-CYP ‘H-Mesulergine ‘H-Mesulergine (Rat and 1251-LSD 1251-LSD mouse only) 3H-WB 4101 ‘H-Ketanserin 3H-Buspirone 3H-Ritanserin ‘H-PAPP ‘H-Mianserin 1Z51-Methyl-LSD High-density Raphe nuclei Substantia Choroid plexus Basal ganglia Layer IV Peripheral regions nigra cortex neurons Hippocampus Globus pallidus Drug potencies 1000 nM Mianserin Mianserin Spiperone Mesulergine RU 24969 Methysergide Spiperone 8-OH-DPAT Spiperone 5-HT Ketanserin Mesulergine I-OH-DPAT 8-OH-DPAT Potential Raphe and Synaptosomal Phospatidyl- ? Kidney Head twitches Excitation of functional hippocampal “autoreceptor” inositide perfusion sympathetic correlates inhibition turnover Vascular neurons Basilar artery ? Rat fundus contractions contractions contractions

cyclase in guinea pig hippocampus (De Vivo and Maayani, 1986) Palacios, 1985). Extensive pharmacologicalstudies have dem- and drug-induced contractions of the canine basilar artery (Per- onstrated that synaptosomalrelease of 5-HT can be modulated outka et al., 1986b; Taylor et al., 1986) appearto be modulated by 5-HT,, receptorsin the rat (Engelet al., 1986; Raiteri et al., by 5-HT,, receptors.The 5-HT,, selective agents8-OH-DPAT 1986). Interestingly, the 5-HT,, site appearsto be present in and 5-MeDMT produce the “5-HT behavioral syndrome” and only rat and mousebrain and not in other species,such as cow, at least 3 specificcompolients of the syndrome (reciprocal fore- guinea pig, frog, chicken, turtle, or human (Heuring et al., 1986). paw treading, head-weaving, and tremor) have been attributed The 5-HT,, site was first characterized in membranesfrom to stimulation ofthe 5-HT,, receptor (Tricklebank, 1985; Smith pig choroid plexus and cortex (Pazos et al., 1984). The site has and Peroutka, 1986). been labeledby 3H-5-HT, )H-mesulergine,and lZSI-LSDin the The 5-HT,, site has been labeled in rat brain with both 3H- rat choroid plexus and may also be present in rat cortex (Pazos 5-HT (Sills et al., 1984; Blurton and Wood, 1986; Peroutka, et al., 1984; Yagaloff and Hartig, 1985;Blurton and Wood, 1986; 1986)and 1251-cyanopindolol(Hoyer et al., 1985a). The 5-HT,, Peroutka, 1986). Recent studiesusing rat choroid plexus have site reportedly displaysextremely high affinity (0.38 nM) for RU shown that the 5-HT,, site is linked to phosphatidylinositol 24969 and relatively low affinity for d-LSD and 8-OH-DPAT turnover (Conn et al., 1986).Pharmacologically, the 5-HT,, site (Peroutka, 1986). The site also has relatively high affinity (p& displays nanomolar affinity for 5-HT, mesulergine,methyser- values < 100 nM) for (-)pindolol and (-)propranolol (Hoyer et gide, and mianserin. By contrast, the sitesdescribed in the pres- al., 1985a),drugs that are more than loo-fold lesspotent at the ent report have significantly lower affinity for mesulergine, sitesdescribed in the present report. Conversely, mianserin is methysergide,and mianserin. more than 2 orders of magnitude lesspotent at the 5-HT,, site Previous investigators have also suggestedthat other binding than at the 3H-5-HT site describedin the present report (Hoyer sitescan be labeledby 3H-5-HT. However, a classof siteswith et al., 1985a; Peroutka, 1986). The highest densitiesof 5-HT,, the pharmacologicalcharacteristics described in the presentre- sitesin rat brain are found in the caudate, superior colliculus, port hasnot yet beenreported. For example, Robaut et al. (1986) lateral geniculate, subiculum, and substantia nigra (Pazos and identified a 3H-5-HT binding site in rat brain with a K, of 13- 902 Heuring and Peroutka - 3H-5-HT Binding in Bovine Caudate

15 nM that was insensitive to ergot derivatives and GTP. Sim- rat stomach fundus, while tyryptamine and N,N-DMT were 32- ilarly, solubilized 5-HT, binding sites apparently do not rec- and 11 O-fold, respectively, less active than 5-HT. Similar rank- ognize the isomers of LSD (Allgren et al., 1985). order potencies exist for these analogs at the putative 5-HT,, In addition, 2 other major types of 5-HT receptor have been site (Table 3). More recently, Clineschmidt et al. (1985) con- described (Table 4). The 5-HT, receptor was first identified in cluded that the fundic 5-HT receptor resembled the “5-HT,” brain membranes (Leysen et al., 1978) and displays high affinity binding site but that the heterogeneous nature of the radioligand for classical 5-HT antagonists and many neuroleptics (Peroutka binding precluded a more definitive correlation. Of note is the and Snyder, 1979). The receptor mediates head twitches in rats fact that yohimbine was a potent antagonist in this system, in and mice, as well as 5-HT-induced contractions in many vas- agreement with its high affinity for the putative 5-HT,, site. cular systems (Leysen et al., 1984; Peroutka, 1984). Finally, a Clearly, more detailed future studies are needed to define 5-HT, receptor has been identified in the PNS (Bradley et al., functional correlates of the putative 5-HT,, binding site sub- 1986). This putative serotonergic site has not been radiolabeled, type. However, the present data demonstrates that 3H-5-HT and no correlate has been identified in the CNS. Nonetheless, labels a fourth subpopulation of binding sites that are distinct the receptor displays high affinity for 5-HT and related analogs from the previously described 5-HT,,, 5-HT,,, and 5-HT,, sites. in physiological studies, and, unlike previously described 5-HT The characterization of this homogeneous population of 3H-5- receptors, it can be blocked potently by drugs such as MDL HT labeled sites should facilitate the correlation of physiological 72222, metoclopramide, and (-)cocaine (Fozard, 1984). actions with radioligand binding data. The identification and Thus, the pharmacological characteristics of the homoge- characterization of all physiologically relevant 5-HT receptors neous class of binding sites labeled by 3H-5-HT in bovine cau- should, ultimately, elucidate the role of 5-HT in the CNS and date are distinct from the previously defined subtypes of the PNS. 5-HT, class of binding sites, as well as from 5-HT, and 5-HT, receptors. The 3H-5-HT sites in bovine caudate display high References affinity for 5-HT, are saturable, and display stereoselectivity for Allgren, R. L., M. M. A. Kyncl, and R. D. Ciaranello (1985) Phar- the isomers of LSD and yohimbine. The most potent agents at macological characterization of solubilized 5-HT, serotonin binding these sites are tryptamine and ergot derivatives, as well as RU sites from bovine brain. Brain Res. 348: 77-85. 24969. When 5-HT,, and 5-HT,, sites are pharmacologically Asarch, K. B., R. W. Ransom, and J. C. Shih (1985) 5-HT,, and blocked by the addition of 100 nM 8-OH-DPAT + 100 nM 5-HT,, selectivity of two phenylpiperazine derivatives: Evidence for mesulergine, this class of sites can be demonstrated in all regions 5-HT,, heterogeneity. Life Sci. 36: 1265-1273. Battanlia. G.. M. Shannon. and M. Titeler (1984) Guanvl nucleotide of bovine brain, with highest densities observed in the basal anddivalent cation regulation of cortical S, receptors. J. Neurochem. ganglia. The extensive pharmacological characterization of this 43: 1213-1218. homogeneous and unique class of binding sites in the bovine Beck, S. G., and J. Goldfarb (1985) Spiperone differentiates multiple caudate enables us to suggest that this class of sites be designated 5-hydroxytryptamine responses in rat hippocampal slices in vitro. Eur. J. Pharmacol. 116: 195-197. the 5-HT,, subtype of binding sites labeled by 3H-5-HT. Rather Bennett, J. P., Jr., and S. H. Snyder (1976) Serotonin and lysergic acid than being a relatively localized binding site subtype (as are the diethylamide binding in rat brain membranes: Relationship to post- 5-HT,, and 5-HT,, sites), this class of sites appears to exist in synaptic serotonin receptors. Mol. Pharmacol. 12: 373-389. high density in many brain regions. Indeed, this class of sites is Blurton, P. A., and M. D. Wood (1986) Identification of multiple binding sites for [3H]5-hydroxytryptamine in the rat CNS. J. Neu- the predominant subtype of 5-HT, sites in bovine brain. rochem. 46: 1392-l 398. To date, 2 specific physiological effects of 5-HT have been Bradley, P. B., G. Engel, W. Feniuk, J. R. Fozard, P. P. A. Humphrey, described that may correlate with the novel 5-HT binding site D. N. Middlemiss, E. J. Mylecharane, B. P. Richardson, and P. R. described in the present report. In the first system, Charlton et Saxena (1986) Proposals for the classification and nomenclature of al. (1986) described an inhibitory prejunctional “5-HT,-like” functional receptors for 5-hydroxytryptamine. Neuropharmacology 25: 563-576. receptor in the isolated perfused rat kidney. This 5-HT receptor Charlton, K. G., R. A. Bond, and D. E. Clarke (1986) An inhibitory inhibited the stimulus-induced release of 3H-noradrenaline fol- prejunctional5-HT,-like receptor in the isolated perfused rat kidney. lowing sympathetic periteronal nerve stimulation to the kidney. Naunyn Schmiedebergs Arch. Pharmacol. 332: 8-l 5. 5-CT was the most potent agonist (IC,, = 1.8 nM), whereas 5-HT Clineschmidt, B. V., D. R. Reiss, D. J. Pettibone, and J. L. Robinson (IC,, = 45 nM) and RU 24969 (IC,, = 250 nM) were slightly less (1985) Characterization of 5-hydroxytryptamine receptors in rat stomach fundus. J. Pharmacol. EXD. Ther. 235: 696-708. potent agonists. 8-OH-DPAT was totally inactive in this system. Cohen, M. L., and L. A. Wittenauer -( 1986) Further evidence that the The effects of these agonists could be antagonized by methioth- serotonin receptor in the rat stomach fundus is not 5-HT,, or 5-HT,,. epin, metergoline, and methysergide but not by cyproheptadine, Life Sci. 38: 1-5. ketanserin, mesulergine, (-)propranolol, or (-t)pindolol. The Conn, P. J., E. Sanders-Bush, B. J. Hoffman, and P. R. Hartig (1986) A unique serotonin receptor in choroid plexus is linked to phos- authors concluded that the 5-HT receptor conformed to the phatidylinositol turnover. Proc. Natl. Acad. Sci. USA 83: 4086-4088. general criteria of a “5-HT,-like” receptor but could not be De Vivo, M., and S. Maayani (1986) Characterization of the correlated to either the 5-HT,,, 5-HT,,, or 5-HT,, subtypes. 5-hydroxytryptamine,. receptor-mediated inhibition of forskolin- The effect of 5-HT and related agents in the isolated perfused stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther. 238: 248-253. rat kidney system is therefore quite similar to the drug potencies Dompert, W. U., T. Glaser, and J. Traber (1985) )H-TVX Q 7821: at the putative 5-HT,, binding site described here. Identification of 5-HT, binding sites as target for a novel mutative 5-HT-induced contraction of the rat stomach fundus is a sec- anxiolytic. Naunyn Schmiedebergs Arch. Pharmacol. 328: 467-470. ond physiological effect that does not appear to be mediated by Engel, G., D. Hoyer, E. Schlicker, and K. 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