at-1015, a newly synthesized 5-ht2 receptor antagonist, dissociates slowly from the 5-ht2 receptor...

Journal of

JPP 2002 54 1123ndash1128 2002 The AuthorsReceived February 13 2002Accepted April 9 2002ISSN 0022-3573 AT-1015 a newly synthesized 5-HT2 receptor antagonist

dissociates slowly from the 5-HT2 receptor sites inrabbit cerebral cortex membrane

Mamunur Rashid Masatomo Watanabe Mikio Nakazawa

and Takafumi Nagatomo

Abstract

The purpose of this study was to investigate the association and dissociation kinetics of [3H]AT-

1015 from 5-HT2 receptors in rabbit cerebral cortex membranes using a radioligand binding

assay method and to make a comparison with those of [3H]ketanserin binding Scatchard

analysis of [3H]AT-1015 binding in rabbit cerebral cortex membranes indicated the existence of

a single class of binding sites (dissociation constant Kd macr 218 nM) The speci c binding of

[3H]AT-1015 increased slowly with time and the association rate constant of [3H]AT-1015

binding (k1 macr 01229 minshy 1 nMshy 1) was two times slower than that of [3H]ketanserin binding

(k1 macr 02451 minshy 1 nMshy 1) The dissociation rate constant of [3H]AT-1015 binding (t 12macr

3703 min) was six times slower than that of [3H]ketanserin binding (t 12macr 629 min) when the

addition of excess unlabelled ligands were AT-1015 and ketanserin respectively The

dissociation rate constant of [3H]AT-1015 was slowed to a greater degree (t 12macr 16340 min and

t 12macr 19812 min) by the addition of ketanserin and sarpogrelate as excess unlabelled ligands

than was that of [3H]ketanserin (t12macr 1776 min and t 1

2macr 1845 min) by the addition of AT-1015

and sarpogrelate as an excess unlabelled ligand respectively These ndings on the dissociation

kinetics of [3H]AT-1015 have con rmed and supported previously reported evidence of the

slower dissociation of AT-1015 from 5-HT2 receptors

Introduction

It has been reported (Kihara et al 2000) that AT-1015 (N-[2-[4-(5H-dibenzo[ad] cyclohepten - 5 - ylidene) - piperidino]ethyl] - 1 - formyl - 4 - piperidinecarboxamidemonohydrochloride monohydrate) is a potent 5-HT2A receptor antagonist It hasalso been suggested that it might have a slow dissociation from the 5-HT2 receptorbinding sites in rat aorta and its insurmountable antagonism might be relevant toits therapeutic potential in peripheral vascular disease AT-1015 selectively inhibited5-HT2A-receptor-mediated platelet aggregation in-vitro and this inhibitory activityof AT-1015 was almost equivalent to that of ketanserin and 100 times more potentthan that of sarpogrelate We also reported (Gong et al 2000) that AT-1015 was astrong noncompetitive 5-HT2 antagonist which inhibited the maximum contractionresponses in porcine coronary arteries without endothelium induced by 5-HT and

-methylserotonin On the other hand ketanserin induced rightward shifts ofcontraction response without signireg cant inhibition of maximum response

In our previous studies (Rashid et al 2001a) it was revealed that AT-1015 had highbinding a nity to 5-HT2 receptors in rabbit cerebral cortex membranes and theblockade of specireg c [3H]ketanserin binding sites in the rabbit cerebral cortex membranesinduced by AT-1015 was not readily reversed by washing However the inhibition of[3H]ketanserin binding induced by ketanserin and sarpogrelate was readily reversed bywashing It was also suggested in our report that AT-1015 might strongly bind to 5-HT2

receptor sites with the pretreatment of the membrane fractions and was not readily

Department of PharmacologyNiigata College of Pharmacy5-13-2 Kamishinei-cho Niigata950-2081 Japan

Mamunur Rashid TakafumiNagatomo

Department of Public HealthNiigata College of MedicalTechnology 5-13-3 Kamishinei-cho Niigata 950-2076 Japan

Masatomo Watanabe

Department of MedicalTechnology School of HealthSciences Faculty of MedicineNiigata University Niigata951-8518 Japan

Mikio Nakazawa

Correspondence T NagatomoDepartment of PharmacologyNiigata College of Pharmacy5-13-2 Kamishinei-cho Niigata950-2081 Japan E-mail nagatomoniigata-pharmacjp

Funding This research wassupported by a grant from thePromotion and Mutual AidCorporation for Private Schoolof Japan

1123

1124 Mamunur Rashid et al

dissociated from bound 5-HT2 receptors after washoutunlike ketanserin and sarpogrelate Recently Kihara et al(2001) reported that AT-1015 was a potent and long actingoral antithrombic agent with a low risk of bleeding-timeprolongation in a photochemically induced arterial throm-bosis model in the rat femoral artery The duration of theantithrombic eŒect of AT-1015 was much longer than thatof sarpogrelate The author also suggested that this eŒectof AT-1015 might be elicited by its potent and long-actinginhibition of vasoconstriction through the 5-HT2A receptor

The purpose of this study is to investigate the as-sociation and dissociation kinetics of the binding of[3H]AT-1015 a radioligand of AT-1015 to 5-HT2 recep-tors in rabbit cerebral cortex membranes and make acomparison with those of [3H]ketanserin binding Theinvestigation may provide further evidence of its slowerdissociation that has been reported previously in wash-out experiments using the method of centrifugationresuspension and radioligand binding assay The studywas also performed to compare the dissociation of[3H]AT-1015 caused by addition of excess unlabelledAT-1015 with that induced by the addition of excessunlabelled other 5-HT2A selective antagonists such asketanserin and sarpogrelate

Materials and Methods

Materials

[3H]Ketanserin (633 Ci mmolshy 1) was purchased fromNEN Life Science Products Inc (Boston MA) [3H]AT-1015 (181 TBq mmolshy 1) was obtained from ADMETOX Research Institute Daiichi Pure Chemicals CoLtd Ibaraki Japan AT-1015 (N-[2-[4-(5H-dibenzo[ad]cyclohepten - 5 -ylidene) - piperidino]ethyl] - 1 - formyl -4-piperidinecarboxamide monohydrochloride monohy-drate) used in the study was synthesized and donated byAjinomoto Co Ltd Tokyo Japan Ketanserin wasobtained from RBI (Research Biochemical Incorpor-ated Natick MA) and sarpogrelate was obtained fromMitsubishi Chemical Corporation Tokyo Japan

Preparation of membrane-enriched fraction

Membrane-enriched fraction of rabbit cerebral cortexwas prepared by the method described previously(Hosohata et al 1995) In brief the brain was removedfrom a male rabbit (Japanese white 3plusmn 4 kg) after therabbit had been anaesthetized with sodium pentobar-bital (25 mg kgshy 1 iv) and exsanguinated The cerebralcortex was then separated from the whole brain andminced in 10 volumes of sucrose buŒer (10 mm Tris-HCl 025 m sucrose pH 74) and was homogenized byusing a glass homogenizer The homogenate was thencentrifuged at 40000 g for 30 min at 4 deg C The super-natant was discarded and the resultant pellet was resus-pended in the incubation medium containing 120 mm

Tris-HCl and 40 mm MgCl2 (pH 74) The membrane-enriched fraction was then immediately frozen in liquidnitrogen and stored at reg 80 deg C until used Membraneprotein concentrations were determined by the methodof Bradford (1976) with bovine serum albumin used asstandard

Radioligand binding studies

5-HT

receptor binding assays

The radioligand binding assay for 5-HT2 receptor sub-types was performed according to the method describedpreviously (Nagatomo et al 1988) Experiments wereperformed at a reg nal volume of 200 L of assay buŒer(60 mm Tris-HCl 20 m m MgCl2 01 ascorbic acidand 10 m pargylline pH 74) with 50 L of [3H]AT-1015 100 L of membrane preparation (01 mg of pro-tein) and 50 L of vehicle or test solution containingvarious concentrations of the test compounds Theconcentration of [3H]AT-1015 ranged from 003 n m to60 n m for the saturation binding experiments After60 min incubation at 23deg C the reaction mixture wasterminated by the addition of 20 mL of ice-cold buŒer(50 mm Tris-HCl pH 74) and rapidly reg ltered undervacuum through a Whatman GFC glass reg ber reg lterpre-soaked in 01 polyethyleneimine (PEI) The reg lterswere washed reg ve times with 20 mL of ice-cold buŒer(50 mm Tris-HCl pH 74) The retained radioactivity inthe vial that contained the reg lter with 2 mL of toluene-trinitron based scintillation macr uid was determined using aliquid scintillation counter (Packard 2200 Tri-CarbScintillation Analyzer Packard Instrument Co IncMariden CT) The specireg c binding was determined bysubtracting the remaining nonspecireg c binding in thepresence of 10 m unlabelled AT-1015 from the totalbinding

Kinetic studies of [$H]AT-1015 and [$H]ketanserinbinding to 5-HT

receptors

Kinetic studies of [3H]AT-1015 and [3H]ketanserinbinding to 5-HT2 receptors were carried out accordingto the method of Ojima et al (1997) with slight modireg ca-tion In association experiments the amount of specireg cbinding of [3H]AT-1015 (10 n m ) and [3H]ketanserin(125 n m ) to the rabbit cerebral cortex membranes weredetermined at a reg nal volume of 02 mL of assay buŒer(60 mm Tris-HCl buŒer 20 m m MgCl2 01 ascorbicacid and 10 m pargylline pH 74) with 50 L of[3H]AT-1015 and [3H]ketanserin 100 L of membranepreparation (01 mg of protein) and 50 L of vehicle ornonspecireg c agent after various time intervals between 1and 90 min and 1 and 60 min respectively at 23deg CAfter incubation at the various time intervals at 23deg Cthe reaction mixture was terminated by the addition of20 mL ice-cold buŒer (50 m m Tris-HCl pH 74) andrapidly reg ltered under vacuum through a WhatmanGFC glass reg ber reg lter pre-soaked in 01 PEI Thereg lters were washed reg ve times with 20 mL of ice-cold

1125Slow dissociation of AT-1015 from 5-HT2 sites

buŒer (50 m m Tris-HCl pH 74) The retained radio-activity in the vial that contained the reg lter with 2 ml oftoluene-trinitron based scintillation macr uid was deter-mined using a liquid scintillation counter (Packard 2200Tri-Carb Scintillation Analyzer Packard InstrumentCo Inc Mariden CT)

In dissociation experiments [3H]AT-1015 (10 n m ) and[3H]ketanserin (125 n m ) were incubated to steady statewith rabbit cerebral cortex membranes (01 mg protein)at a reg nal volume of 02 mL of 60 mm Tris-HCl buŒer(pH 74) containing 20 m m MgCl2 01 ascorbic acidand 10 m pargylline for 60 and 30 min respectivelyat 23 deg C Then dissociation was induced by the additionof excess unlabelled ligands (10 m ) Afterincubation with excess unlabelled ligands at varioustime intervals the reaction mixture was terminated by theaddition of 20 mL of ice-cold buŒer (50 m m Tris-HClpH 74) and rapidly reg ltered under vacuum through aWhatman GFC glass reg ber reg lter pre-soaked in 01 PEIThe reg lters were washed reg ve times with 20 mL of ice-coldbuŒer (50 m m Tris-HCl pH 74) The retained radio-activity in the vial that contained the reg lter with 2 mL oftoluene-trinitron based scintillation macr uid was determinedusing a liquid scintillation counter (Packard 2200 Tri-Carb Scintillation Analyzer Packard Instrument Co IncMariden CT)

Data analysis

Data were given as the means sup3 sd The dissociationconstant (Kd) and the density of 5-HT2-receptor bindingsites (Bmax) in the membrane preparation of the rabbitcerebral cortex were determined by linear regressionanalysis of the saturation binding curves transformed toScatchard plots The dissociation rate constant (k

shy 1)was determined from the reg rst-order plot of ln (Bt

Beq)versus time where Beq and Bt are the amount of specireg cbinding at equilibrium and time t (the time after theaddition of unlabelled ligand) using Sigma Plot Program(Jandel Scientireg c San Rafael CA) The half-life of theligandplusmn receptor complex (t 1

2) was calculated using t 1

2macr

0693kshy 1 The observed rate constant (kobs) was de-

termined from the pseudo-reg rst-order plot of the specireg cbinding data versus time kobs was calculated usingnonlinear regression to reg t the specireg c binding data tothe one-phase exponential association equation Y macrYmax(1 reg eshy kobst) where Ymax is the maximum specireg cbinding and Y is the specireg c binding that increases to amaximum plateau (at equilibrium ) equal to Ymax Theassociation rate constant (k1) was calculated from k1 macr(kobs reg k

shy 1)[L] where [L] is the concentration of theradioligand

Statistical analysis

Data were given as mean sup3 sd Statistical signireg cance ofthe data was evaluated using Mannplusmn Whitney U-test forthe comparisons of two groups and one-way analysis of

variance followed by Tukeyrsquo s test for comparison ofmore than three groups kobs was calculated by nonlinearregression curve reg t and one-phase exponential associ-ation equation using GraphPad Prism (GraphPad Soft-ware Inc San Diego CA)

Results

Saturation analysis of [3H]AT-1015

The specireg c binding of [3H]AT-1015 to rabbit cerebralcortex membranes was saturable in the range 003plusmn60 n m Specireg c binding represented 60plusmn 80 of the totalradioactivity bound to the cerebral cortex membranesA Scatchard analysis of saturation binding data yieldeda linear plot suggesting the presence of a single popu-lation of binding sites The Kd value for specireg c [3H]AT-1015 binding was 218 sup3 084 nm and the Bmax was126961 sup3 22847 fmol (mg protein)shy 1 (n macr 6)

Kinetic studies of [3H]AT-1015 and[3H]ketanserin binding

The time-course of association and dissociation of [3H]AT-1015 (10 n m ) and [3H]ketanserin (125 n m ) bindingto rabbit cerebral cortex membranes is depicted inFigures 1 and 2 respectively The specireg c binding of[3H]AT-1015 increased slowly with incubation time andreached equilibrium after approximately 60 min Thisequilibrium was maintained at a steady level for up to180 min of incubation The specireg c binding of [3H]ketanserin increased very rapidly and reached equi-librium after approximately 30 min of incubation Theassociation rate constants (k1) of both radioligands were

Figure 1 Time-course of the association and dissociation of [3H]AT-

1015 binding to rabbit cerebral cortex membranesAfter [3H]AT-1015

(10 nm ) binding to membranes had reached equilibrium (at 60 min)

dissociation was induced by the addition of unlabelled AT-1015

(10 m D) ketanserin (10 m U) or sarpogrelate (10 m ^) The

specireg c binding is expressed as a percentage of the specireg c binding at

equilibrium (maximal binding) Data are the means ( sup3 sd) of 4 or 5

experiments each performed in duplicate


Figure 2 Time-course of the association and dissociation of [3H]

ketanserin binding to rabbit cerebral cortex membranes After [3H]

ketanserin (125 n m ) binding to membranes had reached equilibrium

(at 30 min) dissociation was induced by the addition of unlabelled

ketanserin (10 m +) AT-1015 (10 m _) or sarpogrelate (10 m

D) The specireg c binding is expressed as a percentage of the specireg c

binding at equilibrium (maximal binding) Data are the means ( sup3 sd)

of 4 or 5 experiments each performed in duplicate

Figure 3 Association kinetics of the specireg c binding of [3H]AT-1015

and [3H]ketanserin to rabbit cerebral cortex membranes Pseudo-reg rst-

order kinetic plots of initial binding of [3H]AT-1015 (10 n m D) and

[3H]ketanserin (125 n m E) to membranes The observed rate con-

stant (kobs) was calculatedusing nonlinear regression to reg t the specireg c

binding data to the one-phase exponential association equation The

second-order association rate constant (k1) was calculated from k1 macr(kobs reg k

shy 1)[L] where kshy 1 is the dissociation rate constant and [L] is

the concentration of the radioligand

Table 1 Association kinetics of specireg c [3H]AT-1015 and [3H]

ketanserin binding to 5-HT2 receptors in rabbit cerebral cortex

membrane

Radioligand Observed rate constant

(kobs) (minshy 1)

Association rate constant

(k1) (minshy 1 n m shy 1)

[3H]AT-1015 01418sup3 00128 01229sup3 00128

[3H]Ketanserin 04199sup3 00209 02451sup3 00166

Data are the means ( sup3 sd) of 4 or 5 experiments each performed

induplicateP 005vs [3H]AT-1015determinedbyMannplusmn Whitney

U-test

Figure 4 Dissociation kinetics of specireg c binding of [3H]AT-1015

(10 nm A) and [3H]ketanserin (125 nm B) to rabbit cerebral cortex

membranes A After [3H]AT-1015 (10 n m ) binding to membranes

reached equilibrium (at 60 min) dissociation was induced by the

addition of unlabelled AT-1015 (10 m E) and also induced by the

addition of unlabelled ketanserin (10 m ) or sarpogrelate (10 m

^) B After [3H]ketanserin (125 n m ) binding to membranes reached

equilibrium (at 30 min) dissociation was induced by the addition of

unlabelled ketanserin (10 m E) and also induced by the addition of

unlabelled AT-1015 (10 m ^) or sarpogrelate (10 m ) On the

ordinate Beq and Bt are binding at equilibrium and time t (time after

addition of the unlabelled ligand) The slope of the reg rst-order plot is

the dissociation rate constant (kshy 1) Data are the means ( sup3 sd) of 4 or

5 separate experiments each performed in duplicate

calculated from these studies (Figure 3) The associationrate constants (k1) of [3H]AT-1015 and [3H]ketanserinwere found to be 01229 and 02451 minshy 1 n m shy 1 re-spectively (Table 1)

When the specireg c binding of [3H]AT-1015 (10 n m )and [3H]ketanserin (125 n m ) reached a steady level orequilibrium then dissociation was induced by the ad-dition of 10 m unlabelled AT-1015 and ketanserinrespectively (Figure 4a b) Table 2 summarizes thedissociation rate constant (k

shy 1) and half-life (t 12) of both


Table 2 Dissociation kinetics of specireg c [3H]AT-1015 and [3H]ketanserin binding to 5-HT2 receptor in

rabbit cerebral cortex membrane

Addition of

unlabelled ligand

Dissociation rate constant (kshy 1) (minshy 1) Half-life (t 1

2) (min)

[3H]AT-1015 [3H]Ketanserin [3H]AT-1015 [3H]Ketanserin

AT-1015 00189sup3 00019 00399sup3 00063gg 3703sup3 382 1776sup3 303ggKetanserin 00043sup3 00003 01141sup3 00252 16340sup3 985 629sup3 126

Sarpogrelate 00037sup3 00012 00377sup3 00021gg 19812sup3 4802 1845sup3 110gg

Values are mean ( sup3 sd) of 4 or 5 experiments each performed in duplicate P 0001 vs AT-1015 when

the radioligand was [3H]AT-1015 and ggP 0001 vs ketanserin when the radioligand was [3H]ketanserin

(one-way analysis of variance followed by Tukeyrsquos test)

radioligands AT-1015 produced slow release of [3H]AT-1015 from the [3H]AT-1015plusmn receptor complex with adissociation rate constant (k

shy 1) and half-life (t 12) of

00189 minshy 1 n m shy 1 and 3703 min respectively The dis-sociation of [3H]AT-1015 was further slowed down (5plusmn 6times) with a t 1

2of 16340 and 19812 min by the addition

of ketanserin and sarpogrelate as an excess unlabelledligand respectively In contrast the dissociation of[3H]ketanserin caused by the addition of excess unlabelledketanserin was very rapid with a dissociation rate con-stant (k

shy 1) of 01141 minshy 1 n m shy 1 and a t 12

of 629 minThe dissociation of [3H]ketanserin was slightly slowed(t 1

2macr 1776 and 1845 min) by the addition of AT-1015

and sarpogrelate as an excess unlabelled ligand re-spectively Table 2 shows that the dissociation rate of[3H]AT-1015 from rabbit cerebral cortex membrane wassix times slower than that of [3H]ketanserin

Discussion

We investigated the association and dissociation kineticsof [3H]AT-1015 binding a radioligand of AT-1015using rabbit cerebral cortex membranes and made acomparison with those of [3H]ketanserin binding Thispreparation was used because 5-HT2 receptor subtypeswere found in high concentration in the cerebral cortexblood vessels and gastrointestinal and uterine smoothmuscles (Conn amp Sanders-Bush 1987 Zifa amp Fillon1992) the cerebral cortex being the most highly enrichedin 5-HT2A receptor (Pazos et al 1985 Roth et al 1987)In our previous reg ndings it was observed that AT-1015had demonstrated high binding a nity in both therabbit cerebral cortex membranes and platelet mem-branes using [3H]ketanserin as a radioligand (Rashid etal 2001a b) AT-1015 was a potent 5-HT2A receptorantagonist and it selectively inhibited 5-HT2A-receptor-mediated platelet aggregation (Kihara et al 2000) It wasalso reported that AT-1015 was a strong non-com-petitive 5-HT2 antagonist and it reduced the maximalcontraction in a 5-HT-induced vasoconstriction in bothrat thoracic artery (Kihara et al 2000) and porcinecoronary artery (Gong et al 2000) whereas ketanserin

inhibited the contraction without signireg cant eŒect onmaximum response Several hypothetical mechanismshave been proposed to explain insurmountable antag-onism one of which is that it is thought to be producedby slow dissociation of the antagonistplusmn receptor complex(Robertson et al 1992 Wienen et al 1992 Panek et al1995) To examine this hypothesis we had previouslyinvestigated the dissociation ability of AT-1015 andcompared it with other 5-HT2 antagonists AT-1015slowly dissociated from 5-HT2 receptors in rabbit cerebralcortex membranes using washout experiments In thisstudy we have investigated the association and dis-sociation rate constants and half-life of [3H]AT-1015binding to the rabbit cerebral cortex membranes andemphasized further evidence of its slower dissociationfrom 5-HT2 receptor sites

In saturation binding studies Scatchard- and Hill-plot analyses of [3H]AT-1015 binding indicated thepresence of a single population of binding sites (Kd macr218 n m Bmax macr 126961 fmol (mg protein)shy 1) and theabsence of negative or positive cooperation In pre-liminary experiments we also used a very large concen-tration range (0025plusmn 17 n m ) of [3H]AT-1015 for examiningits probable two sites of binding to rabbit cerebralcortex membranes and the saturation curve did not matchwith the two-site binding We also observed that [3H]AT-1015 binding in rabbit cerebral cortex membranes reachedequilibrium within 60 min incubation at 23deg C using thereaction mixture mentioned in the Materials and Methodssection We also used a concentration of 10 n m of [3H]AT-1015 for the association and dissociation kinetics studiesbecause receptor occupancy was high and equilibriumbinding was reached at the same incubation time as insaturation binding

In our kinetic studies [3H]AT-1015 associated slowlyand time dependently with an association rate constantof 01229 minshy 1 n m shy 1 in rabbit cerebral cortex membranesand its association rate constant was two times slowerthan that of [3H]ketanserin (k1 macr 02451 minshy 1 n m shy 1) aknown 5-HT2 radioligand of rapid association with therat prefrontal cortex (Leysen et al 1981) In dissociationkinetics [3H]ketanserin dissociated rapidly with an initialhalf-life of dissociation (t 1

2) of 629 min in rabbit cerebral


cortex membranes which is almost similar to the results ofother authorsrsquo reports (t 1

2of [3H]ketanserin macr 35 and

58 min) (Leysen et al 1981 de ChaŒoy de Courcelles et al1986) On the other hand the dissociation rate of [3H]AT-1015 was six times slower than that of [3H]ketanserin Thekinetic data (ie dissociation rate constant and half-life) of[3H]AT-1015 have given a quantitative value for the slowerdissociation of AT-1015 which was previously reportedqualitatively Therefore this investigation correlateswith our previous report and provides further evidence ofits slower dissociation from 5-HT2 receptors in rabbitcerebral cortex membranes Kihara et al (2001) also re-ported the long-lasting antithrombic eŒect of AT-1015 andthis eŒect was much longer than that of sarpogrelate

Another possible explanation for this insurmountableantagonism is the allosteric modireg cation of the receptors(Wienen et al 1992 Panek et al 1995) To evaluate thishypothesis we examined the dissociation of [3H]AT-1015 from 5-HT2 binding sites of the rabbit cerebralcortex induced by the addition of excess unlabelledketanserin and sarpogrelate The rate of dissociation of[3H]AT-1015 from 5-HT2 receptors after the addition ofexcess unlabelled AT-1015 was signireg cantly diŒerentfrom that following the addition of excess unlabelledother 5-HT2A selective antagonists such as ketanserinand sarpogrelate Figures 1 and 2 respectively clearlyshow that at equilibrium ketanserin did not completelydisplace [3H]AT-1015 and that AT-1015 did not com-pletely displace [3H]ketanserin Thus AT-1015 is a non-competitive antagonist that allosterically modulates thebinding of ketanserin However further studies arerequired to clarify the exact mechanism of this phenom-enon

In conclusion this study has demonstrated that AT-1015 a newly synthesized 5-HT2 receptor antagonistdissociates slowly from 5-HT2 receptor sites and thisresult provides further evidence of its previously reportedslower dissociation from rabbit cerebral cortex mem-branes These reg ndings have also revealed possible expla-nations for the insurmountable antagonism of AT-1015on vasoconstriction in the rat artery (Kihara et al 2000)and porcine coronary artery (Gong et al 2000)

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Zifa E Fillon G (1992) Hydroxy tryptamine receptors Pharmacol

Rev 44 401plusmn 458


dissociated from bound 5-HT2 receptors after washoutunlike ketanserin and sarpogrelate Recently Kihara et al(2001) reported that AT-1015 was a potent and long actingoral antithrombic agent with a low risk of bleeding-timeprolongation in a photochemically induced arterial throm-bosis model in the rat femoral artery The duration of theantithrombic eŒect of AT-1015 was much longer than thatof sarpogrelate The author also suggested that this eŒectof AT-1015 might be elicited by its potent and long-actinginhibition of vasoconstriction through the 5-HT2A receptor

The purpose of this study is to investigate the as-sociation and dissociation kinetics of the binding of[3H]AT-1015 a radioligand of AT-1015 to 5-HT2 recep-tors in rabbit cerebral cortex membranes and make acomparison with those of [3H]ketanserin binding Theinvestigation may provide further evidence of its slowerdissociation that has been reported previously in wash-out experiments using the method of centrifugationresuspension and radioligand binding assay The studywas also performed to compare the dissociation of[3H]AT-1015 caused by addition of excess unlabelledAT-1015 with that induced by the addition of excessunlabelled other 5-HT2A selective antagonists such asketanserin and sarpogrelate

Materials and Methods

Materials

[3H]Ketanserin (633 Ci mmolshy 1) was purchased fromNEN Life Science Products Inc (Boston MA) [3H]AT-1015 (181 TBq mmolshy 1) was obtained from ADMETOX Research Institute Daiichi Pure Chemicals CoLtd Ibaraki Japan AT-1015 (N-[2-[4-(5H-dibenzo[ad]cyclohepten - 5 -ylidene) - piperidino]ethyl] - 1 - formyl -4-piperidinecarboxamide monohydrochloride monohy-drate) used in the study was synthesized and donated byAjinomoto Co Ltd Tokyo Japan Ketanserin wasobtained from RBI (Research Biochemical Incorpor-ated Natick MA) and sarpogrelate was obtained fromMitsubishi Chemical Corporation Tokyo Japan

Preparation of membrane-enriched fraction

Membrane-enriched fraction of rabbit cerebral cortexwas prepared by the method described previously(Hosohata et al 1995) In brief the brain was removedfrom a male rabbit (Japanese white 3plusmn 4 kg) after therabbit had been anaesthetized with sodium pentobar-bital (25 mg kgshy 1 iv) and exsanguinated The cerebralcortex was then separated from the whole brain andminced in 10 volumes of sucrose buŒer (10 mm Tris-HCl 025 m sucrose pH 74) and was homogenized byusing a glass homogenizer The homogenate was thencentrifuged at 40000 g for 30 min at 4 deg C The super-natant was discarded and the resultant pellet was resus-pended in the incubation medium containing 120 mm

Tris-HCl and 40 mm MgCl2 (pH 74) The membrane-enriched fraction was then immediately frozen in liquidnitrogen and stored at reg 80 deg C until used Membraneprotein concentrations were determined by the methodof Bradford (1976) with bovine serum albumin used asstandard

Radioligand binding studies

5-HT

receptor binding assays

The radioligand binding assay for 5-HT2 receptor sub-types was performed according to the method describedpreviously (Nagatomo et al 1988) Experiments wereperformed at a reg nal volume of 200 L of assay buŒer(60 mm Tris-HCl 20 m m MgCl2 01 ascorbic acidand 10 m pargylline pH 74) with 50 L of [3H]AT-1015 100 L of membrane preparation (01 mg of pro-tein) and 50 L of vehicle or test solution containingvarious concentrations of the test compounds Theconcentration of [3H]AT-1015 ranged from 003 n m to60 n m for the saturation binding experiments After60 min incubation at 23deg C the reaction mixture wasterminated by the addition of 20 mL of ice-cold buŒer(50 mm Tris-HCl pH 74) and rapidly reg ltered undervacuum through a Whatman GFC glass reg ber reg lterpre-soaked in 01 polyethyleneimine (PEI) The reg lterswere washed reg ve times with 20 mL of ice-cold buŒer(50 mm Tris-HCl pH 74) The retained radioactivity inthe vial that contained the reg lter with 2 mL of toluene-trinitron based scintillation macr uid was determined using aliquid scintillation counter (Packard 2200 Tri-CarbScintillation Analyzer Packard Instrument Co IncMariden CT) The specireg c binding was determined bysubtracting the remaining nonspecireg c binding in thepresence of 10 m unlabelled AT-1015 from the totalbinding

Kinetic studies of [$H]AT-1015 and [$H]ketanserinbinding to 5-HT

receptors

Kinetic studies of [3H]AT-1015 and [3H]ketanserinbinding to 5-HT2 receptors were carried out accordingto the method of Ojima et al (1997) with slight modireg ca-tion In association experiments the amount of specireg cbinding of [3H]AT-1015 (10 n m ) and [3H]ketanserin(125 n m ) to the rabbit cerebral cortex membranes weredetermined at a reg nal volume of 02 mL of assay buŒer(60 mm Tris-HCl buŒer 20 m m MgCl2 01 ascorbicacid and 10 m pargylline pH 74) with 50 L of[3H]AT-1015 and [3H]ketanserin 100 L of membranepreparation (01 mg of protein) and 50 L of vehicle ornonspecireg c agent after various time intervals between 1and 90 min and 1 and 60 min respectively at 23deg CAfter incubation at the various time intervals at 23deg Cthe reaction mixture was terminated by the addition of20 mL ice-cold buŒer (50 m m Tris-HCl pH 74) andrapidly reg ltered under vacuum through a WhatmanGFC glass reg ber reg lter pre-soaked in 01 PEI Thereg lters were washed reg ve times with 20 mL of ice-cold




Data analysis





2macr







Results

































membrane


(kobs) (minshy 1)



[3H]AT-1015 01418sup3 00128 01229sup3 00128




U-test





















Addition of

unlabelled ligand


2) (min)
















Discussion












References






92 267plusmn 277















18 1066plusmn 1071


































189plusmn 194






1188plusmn 1190


















Data analysis





2macr







Results

































membrane


(kobs) (minshy 1)



[3H]AT-1015 01418sup3 00128 01229sup3 00128




U-test





















Addition of

unlabelled ligand


2) (min)
















Discussion












References






92 267plusmn 277















18 1066plusmn 1071


































189plusmn 194






1188plusmn 1190



































membrane


(kobs) (minshy 1)



[3H]AT-1015 01418sup3 00128 01229sup3 00128




U-test





















Addition of

unlabelled ligand


2) (min)
















Discussion












References






92 267plusmn 277















18 1066plusmn 1071


































189plusmn 194






1188plusmn 1190


















Addition of

unlabelled ligand


2) (min)
















Discussion












References






92 267plusmn 277















18 1066plusmn 1071


































189plusmn 194






1188plusmn 1190





















References






92 267plusmn 277















18 1066plusmn 1071


































189plusmn 194






1188plusmn 1190















at-1015, a newly synthesized 5-ht2 receptor antagonist, dissociates slowly from the 5-ht2 receptor...

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