Functions of 5HT 2A receptor and its antagonists in the cardiovascular system

www.elsevier.com/locate/pharmthera

Pharmacology & Therapeu

Associate editor: I. Kimura

Functions of 5-HT2A receptor and its antagonists in

the cardiovascular system

Takafumi Nagatomoa,*, Mamunur Rashidb, Habib Abul Muntasira, Tadazumi Komiyamac

aDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences,

5-13-2 Kamishinei-cho, Niigata 950-2081, JapanbDepartment of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh

cDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan

Abstract

The serotonin (5-hydroxytryptamine, 5-HT) receptors have conventionally been divided into seven subfamilies, most of which have

several subtypes. Among them, 5-HT2A receptor is associated with the contraction of vascular smooth muscle, platelet aggregation and

thrombus formation and coronary artery spasms. Accordingly, selective 5-HT2A antagonists may have potential in the treatment of

cardiovascular diseases. Sarpogrelate, a selective 5-HT2A antagonist, has been introduced clinically as a therapeutic agent for the treatment of

ischemic diseases associated with thrombosis. Molecular modeling studies also suggest that sarpogrelate is a 5-HT2A selective antagonist and

is likely to have pharmacological effects beneficial in the treatment of cardiovascular diseases. This review describes the above findings as

well as the signaling linkages of the 5-HT2A receptors and the mode of agonist binding to 5-HT2A receptor using data derived from molecular

modeling and site-directed mutagenesis.

D 2004 Elsevier Inc. All rights reserved.

Keywords: 5-HT; 5-HT2A; 5-HT2A antagonists; Sarpogrelate; Cardiovascular diseases

Abbreviations: AA, arachidonic acid; AC, adenylyl cyclase; ACE-I, angiotensin converting enzyme inhibitor; AR-A000002, (R)-N-[5-methyl-8-(4-

methylpiperazin-1-yl)-1,2,3,4-tetrahydro-2-naphthyl]-4-morp holinobenzamide; ARF, ADP-ribosylation factor; B-20991, 2[[4-(o-methoxyphenyl)piperazin-1-

yl]-methyl]-1.3-dioxoperhydroimidazo[1.5-a]pyridine; BMY 7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione dihydro-

chloride; BRL 15572, 1-(3-chlorophenyl)-4-[3,3-diphenyl(2-(S,R) hydroxypropanyl)piperazine]hydrochloride; cAMP, cyclic AMP; CaM, Calmodulin; CP-135

807, 3-(N-methylpyrrolidin-2R-ylmethyl)-5-(3-nitropyrid-2-yl)amino-1H-indole; CP 93129, 3-1 2 4 6 tetrahydropyrid-4-ylpyrrolo-3 2-B-pyrid-5-one; cPLA2,

cytosolic phospholipase A2; DOCA, deoxycorticosterone; DP-5-CT, N,N-di-n-propyl-5-carboxamidotryptamine; ERK, extracellular signal-regulated kinase; 5-

F-8-OH-DPAT, 5-fluoro-8-hydroxy-2-N,N-dipropylaminotetraline; FRTL-5, Fischer rat thyroid cell line; GPCR, G-protein coupled receptor; Jak, janus kinase; L-

694 247, 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl ]- 1H-indole-3-yl]ethylamine; LY 334370, 5-(4-fluorobenzoyl)amino-3-(1-methyl-

piperidin-4-yl)-1H-indole fumarate; LY 344864, R-(+)N-(3-dimethylamino-1,2,3,4-tetrahydro-9H-carbazol-6-yl)-4-fluorobenzamide; MAO-A, monoamine

oxidase-A; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MCT, monocrotaline; MDL 72222, 8-methyl-8-azabicyclo[3.2.1]oct-3yl

3,5-dichlorobenzoate; MDL 73005, 8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl)methylamino]-8-azaspiro[4,5] decan-7,9-dione methyl sulphonate; MEK, mitogen

and extracellular signal-regulated kinase; MI, myocardial infarction; NAN 190, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine; NAS 181, R-(+)-

2-[[[3-(Morpholinomethyl)-2H-chromen-8-yl]oxy]methyl] morpholine methane sulfonate); 8-OH-DPAT, 8-hydroxy-2-N,N-dipropylaminotetraline; PI, phos-

phoinositide; PKA, protein kinase A; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PNU-109, 291, (S)-(-)-1-

[2-[4-(4-methoxyphenyl)-1-piperazinyl]ethyl]-N-methyl-isochroman-6-carboxamide; R 102444, (2R,4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]

phenoxy]ethyl-1-methylpyrrolidine hydrochloride; Ro 04-6790, 4-amino-N(2,6bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide; Ro 63-0563, 4-

amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide; RS 39604, 1-[4-Amino-5-chloro-2-(3,5-dimethoxyphenyl)methyloxy]-3-[1-[2-methylsul-

phonylamino]ethyl]piperidin-4-yl]propan-1-one; SB-258719, (R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl) propyl]benzenesulfonamide; SB

269970-A, (R)-3-(2-(2-(4-methyl-piperidin-1-yl)-propylidine-1-sulfonyl)-phenol; SB-271046, 5-Chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-

benzothiophenesulfon-amide; SB-656104-A, 6-((R)-2-[2-[4-(4-Chloro-phenoxy)-piperidin-1-yl]-ethyl]-pyrrolidine-1-sulphonyl)-1H-indole hyrochloride; SHR,

spontaneously hypertensive rat; SL 65.0472, 7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide; STAT,

signal transducers and activators of transcription; TGF, transforming growth factor; TMD, transmembrane domain; TMH, transmembrane helix; TS-951,N-[endo-

8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl]-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide; WAY 100635, N-[2-[4-(2-ethoxyphenyl)-1-pipera-

0163-7258/$ - s

doi:10.1016/j.ph

* Correspon

E-mail addr

tics 104 (2004) 59–81

ee front matter D 2004 Elsevier Inc. All rights reserved.

armthera.2004.08.005

ding author. Tel.: +81 25 268 1185; fax: +81 25 268 1280.

ess: [email protected] (T. Nagatomo).

T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8160

zinyl]ethyl]-N-(2-pyridinyl)cyclohexane-carboxamide trihydrochloride; WKY, WistarKyoto normotensive rat; YM-060, (-)-(R)-5-[(1-methyl-1H-indol-

3-yl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole monohydrochloride; YM-31636, 2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

2. Serotonin receptor subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.1. 5-Hydroxytryptamine1 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.1.1. 5-Hydroxytryptamine1A subtype . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.1.2. 5-Hydroxytryptamine1B subtype. . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.1.3. 5-Hydroxytryptamine1D receptor . . . . . . . . . . . . . . . . . . . . . . . . . . 62

2.1.4. 5-Hydroxytryptamine1E and 5-hydroxytryptamine1F receptors . . . . . . . . . . . 62

2.1.5. 5-Hydroxytryptamine1-like receptors . . . . . . . . . . . . . . . . . . . . . . . . 62







3. Serotonin receptors in the cardiovascular system . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4. 5-Hydroxytryptamine2A receptor and its antagonists. . . . . . . . . . . . . . . . . . . . . . . . . 65

5. Signaling pathway of 5-hydroxytryptamine2A receptor . . . . . . . . . . . . . . . . . . . . . . . 66

5.1. The 5-hydroxytryptamine2A receptor activates phospholipase C . . . . . . . . . . . . . . . 66

5.2. The 5-hydroxytryptamine2A receptor activates phospholipase A2 . . . . . . . . . . . . . . 66

5.3. The 5-hydroxytryptamine2A receptor activates the Janus kinase/signal transducers and

activators of transcription (STAT) pathway. . . . . . . . . . . . . . . . . . . . . . . . . . 66

5.4. The 5-hydroxytryptamine2A receptor activates phospholipase D . . . . . . . . . . . . . . . 67

5.5. The 5-hydroxytryptamine2A receptor can regulate cyclic adenosine monophosphate

accumulation in certain cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

5.6. The 5-hydroxytryptamine2A receptor activates the extracellular signal-regulated

mitogen-activated protein kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

5.7. The 5-hydroxytryptamine2A receptor regulates calmodulin . . . . . . . . . . . . . . . . . . 67

5.8. The 5-hydroxytryptamine2A receptor regulates channels . . . . . . . . . . . . . . . . . . . 67

6. Molecular aspects of 5-hydroxytryptamine2A receptors . . . . . . . . . . . . . . . . . . . . . . . 67

7. Pharmacological and molecular aspects of sarpogrelate . . . . . . . . . . . . . . . . . . . . . . . 69

7.1. Chemistry of sarpogrelate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7.2. Radioligand binding studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7.3. Inhibitory effects on platelet aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7.4. Effects on vasoconstriction response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

7.5. Effects on vasodilatation response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

7.6. Hemodynamic effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

7.7. Relationship between binding affinities and functional potency . . . . . . . . . . . . . . . 71

7.8. Binding sites of 5-hydroxytryptamine2R family with sarpogrelate assessed by molecular

modeling study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

8. Clinical significance of 5-hydroxytryptamine2A receptor and its antagonists . . . . . . . . . . . . 73

9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

1. Introduction

5-Hydroxytryptamine (5-HT, serotonin) is an indole-

amine neurotransmitter and was identified in 1948. Seroto-

nin was given as the name of the vasoconstrictor substance

that appears in serum after blood has clotted, and enteramine

as that of the smooth muscle-contracting substance present

in enterochromaffin cells of the gut mucosa. The synthesis

of 5-HT in 1951 permitted the identification of serotonin

and enteramine as the same metabolite of 5-hydroxytrypto-

phan. 5-HT has been identified for almost 55 years as an

effector for various types of smooth muscle and subse-

quently, as an agent that enhances platelet aggregation and

as a neurotransmitter in the central nervous system (CNS;

Sanders-Bush & Mayer, 1996). 5-HT is found in both the

central nervous system and the peripheral nervous system,

and is important for a variety of physiological functions,

including platelet aggregation, smooth muscle contraction,

appetite, cognition, perception, mood, and other CNS

functions (Hoyer et al., 1994; Roth, 1994). These diverse

Table 1

Functional responses of 5-HT receptors

5-HT receptors Functional responses

5-HT1A Anxiety, depression, hypo tension

5-HT1B Effect on locomotion, penile erection, hypophagia,

vasoconstriction (rat caudal artery)

5-HT1D Migraine, vasoconstriction (bovine and human

cerebral arteries), relaxation (pig coronary artery

with endothelium), inhibition of plasma extravasations

(guinea pig)

5-HT1E Not known

5-HT1F Not known

5-HT1-like Smooth muscle contraction, relaxation

(endothelium-dependent), depression

5-HT2A Vasoconstriction, platelet aggregation and thrombus

formation, coronary artery spasm, bronchoconstriction,

increase of vascular permeability and body

temperature; central effects include serotonin-induced

wet-dog shake behavior and behavioral excitation

5-HT2B Rat stomach fundus muscle contraction, relaxation of

T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 61

physiological functions are mediated by large number of 5-

HT receptor subtypes that are encoded by distinct genes. It

now appears that there are at least 15 receptor subtypes that

belong to four classes of receptors: 5-HT1/5, 5-HT2 (A, B, C),

5-HT3, and 5-HT4/6/7 (Hoyer et al., 1994). 5-HT2A receptors

are expressed in the CNS and periphery. The 5-HT2A

receptor mediates 5-HT-induced platelet aggregation, vas-

cular and nonvascular smooth muscle contraction, percep-

tion, and emotion (Roth et al., 1998). It has been implicated

in the pathogenesis of a wide variety of ischemic heart

diseases.

This review article will emphasize the following topics:

receptor classification, 5-HT receptors in the cardiovascular

diseases, several 5-HT2A antagonists, their pharmacological

actions and molecular aspects, the signaling pathway of the

5-HT2A receptor, and the clinical significance of 5-HT2A

receptor and its antagonists.

vascular smooth muscle

5-HT2C Locomotion, feeding, anorexia nervosa, CSF

formation, adrenocortrophic hormone release,

obsessive-compulsive disorders, and anxiety

5-HT3 Inhibition or stimulation of heart by a combination

of local and reflex effects, vasodilatation (human

forearm), chemotherapy-induced emesis, anxiety,

depression, schizophrenia,

migraine, irritable bowel syndrome

5-HT4 Contraction (guinea pig ileum and colon; human

colon), tachycardia, neuronal excitability, relaxation

(sheep pulmonary vein, rat ileum, and rat esophagus)

5-HT5A

and 5-HT5B

Unknown

5-HT6 May be related to behavioral disorder

5-HT7 May be related to stress states and depression,

relaxation (smooth muscle)

2. Serotonin receptor subtypes

The structural, operational, and transductional character-

istics of 5-HT receptors are the main three criteria to classify

these molecules in a comprehensive manner (Hoyer &

Martin, 1996). The IUPHAR classification of receptors for

5-HT proposed at the 3rd Serotonin Satellite Meeting in

Chicago and discussed in detail by Hoyer et al. (1994) is

summarized in Table 1. 5-HT is divided into seven major

classes, 5-HT1–7, most of which have several subtypes.

2.1. 5-Hydroxytryptamine1 receptors

5-HT1 receptors are found in the brain. The subtypes 5-

HT1A, 5-HT1B, and 5-HT1D are distinguished on the basis of

their regional distribution and their pharmacological activity

(Bruinvels et al., 1991). They respond mainly by the

inhibition of neurotransmitter release and are linked to

inhibition of adenylate cyclase.

2.1.1. 5-Hydroxytryptamine1A subtype

5-HT1A subtype is localized within the CNS, particularly

in the dorsal raphe, hippocampus, and cortex. The activation

of the central 5-HT1A receptors induces a behavioral

syndrome (Tricklebank, 1985), anxiety (Traber & Glaser,

1987), depression (Cervo et al., 1988), and hypotension

(Dreteler et al., 1990). Recent studies have revealed that the

activation of 5-HT1A receptors lowers cutaneous vaso-

constriction and fever associated with acute inflammatory

response (Ootsuka & Blessing, 2003; Blessing, 2004) and

causes hypothermia (Blier et al., 2002). Agonists selective

for 5-HT1A receptors are 8-OH-DPAT, DP-5-CT, 5-CT, 5-

methyl-urapidil (Richardson & Hoyer, 1990), tandospirone

(Kannari et al., 2002), B-20991 (Caicoya et al., 2001), and

Flesinoxan (Bantick et al., 2004). The most selective

antagonists are NAN 190 (Glennon et al., 1988), MDL

73005 (Hibert & Moser, 1990), 5-F-8-OH-DPAT (Hillver et

al., 1990), BMY 7378 (Yocca et al., 1987), and WAY

100635 (Barros et al., 2003).

2.1.2. 5-Hydroxytryptamine1B subtype

The 5-HT1B binding sites are pharmacologically distinct

from the 5-HT1A binding sites. 5-HT1B receptor binding

sites are species specific (Hoyer & Middlemis, 1989). 5-

HT1B receptors are found in rodent brains, particularly in

the substantia nigra and basal ganglia, and are apparently

absent in other mammalian species, including pig, calf, and

man (Hoyer et al., 1986). 5-HT1B receptors control the

activity of basal ganglia that are not linked to the

dopaminergic innervations. They also control the release

of other neurotransmitters such as acetylcholine and

glutamate (Limberger et al., 1991). Some central behavioral

effects are mediated by 5-HT1B receptors (Lucki, 1992).

There are few selective 5-HT1B agonists, and recently it was

shown that CP 93129 appears to be 5-HT1B selective

(Marcor et al., 1990). Selective antagonists for 5-HT1B

receptors are NAS 181 (de Groote et al., 2003) and AR-

A000002 (Hudzik et al., 2003).


2.1.3. 5-Hydroxytryptamine1D receptor

5-HT1D receptors have been identified in the brains of

mammalian species including guinea pig, rabbit, dog, pig,

calf, and human (Heuring et al., 1987; Herrick-Davies &

Titeler, 1988; Hoyer & Schoeffter, 1988; Waeber et al.,

1988; Beer et al., 1992; Maura et al., 1993). Molecular

biological studies have revealed that there are two human 5-

HT1D receptor subtypes, 5-HT1Da and 5-HT1Dh. It has been

found that 5-HT1D receptors are distinct and separate from

the 5-HT1B receptors, but that these two types of receptors

have very close structural homology in rodents (Hartig et al.,

1992). Selective 5-HT1D agonists are PNU-109,291 (Cutrer

et al., 1999), CP-135, 807 (Mansbach et al., 1996), and L-

694,247 (De Castro-e-Silva et al., 1997). The most selective

5-HT1D antagonist is BRL15572 (De Vries et al., 1998).

Both 5-HT1B and 5-HT1D receptors have potential roles

in the pathogenesis of migraine headaches. These receptors

are thought to mediate the effects of triptan-like drugs in the

treatment of migraine. The triptans such as sumatriptan,

naratriptan, rizatriptan, zolmitriptan, almotriptan, eletriptan,

and frovatriptan, which are potent 5-HT1B/1D receptor

agonists, are effective for the treatment of acute migraine

(Goadsby, 2003).

2.1.4. 5-Hydroxytryptamine1Eand 5-hydroxytryptamine1F receptors

Little is known about the distribution and function of

these newly identified receptors. However, homogenate-

binding studies and in situ hybridization studies have

indicated that these receptors are present in the CNS (Adam

et al., 1993). These two receptors appear to be negatively

coupled to adenylyl cyclase (McAllister et al., 1992). 5-

HT1F receptors are involved in blocking migraine pain

transmission through the trigeminal ganglion and nucleus

caudalis, and selective 5-HT1F receptor agonists

(LY334370; LY344864) inhibit dural inflammation in the

neurogenic plasma protein extravasation (Filla et al., 2003;

Ramadan et al., 2003). Thus, these agonists are effective for

the treatment of migraine, causing no side effects of

vasoconstriction (Goldstein et al., 2001). N-[3-(2-Dimethy-

laminoethyl)-2-methyl-1H-indol-5-yl]-4-fluorobenzamide, a

potent, selective, and orally active 5-HT1F receptor agonist,

is also potentially useful for migraine therapy (Xu et al.,

2001). No other agonists or antagonists for 5-HT1E and 5-

HT1F receptors have been reported yet.

2.1.5. 5-Hydroxytryptamine1-like receptors

It has been suggested that 5-HT-like receptors are a group

of related receptors that can be considered as unclassifiable or

orphan receptors identified in the CNS and blood vessels

(Hoyer, 1989). 5-HT receptor-mediated endothelium-

dependent relaxation in pig coronary artery has been

classified as 5-HT1-like (Molderings et al., 1989). 5-HT1-

like receptors are involved in mediating vasoconstriction in

the coronary artery (MacLean et al., 1996). Villalon et al.

(1998) suggested that the inhibition of sympathetically

induced vasopressor responses produced by 5-HT is medi-

ated by 5-HT1-like receptors. Only sumitriptan is a selective

5-HT1-like receptor agonist (Connor et al., 1992) and GR

127935 is a potent antagonist for 5-HT1-like receptors

(Terron, 1996). Since the functional responses once attributed

to 5-HT1-like receptors have been shown to bemediated by 5-

HT1B, 5-HT1D, and 5-HT7 receptors, the 5-HT1-like recep-

tors are now considered redundant (Saxena et al., 1998).


Among the 5-HT receptors, 5-HT2 receptors are of

significant clinical interest because of their potential

involvement in mediating many of the central and peripheral

physiological functions of serotonin. There are three

receptor subtypes within the 5-HT2 classes: 5-HT2A, 5-

HT2B, and 5-HT2C. 5-HT2A subtype is functionally the most

important, the others having a much more limited distribu-

tion and functional role.

The cDNAs for 5-HT2A receptor in rat (Prichett et al.,

1988), hamster (Chambard et al., 1990), mouse (Yang et al.,

1992), and human (Saltzman et al., 1991) have been cloned.

The human 5-HT2A receptor gene is located on chromosome

13q14–q21 (Sparkes et al., 1991), consists of three exons

separated by two introns, and spans over 20 kb (Chen et al.,

1992). 5-HT2A receptor subtypes have been identified in both

the CNS and the periphery. 5-HT2A receptors have been

found in many parts of the CNS including the cerebral cortex,

basal ganglia, hippocampus, thalamus, cerebellum, and

hypothalamus. However, it is most highly enriched in the

cerebral cortex (Roth et al., 1987). In the periphery, 5-HT2A

receptors are located in platelets (De Chaffoy et al., 1985),

vascular smooth muscle (Cohen et al., 1981), and uterine

smooth muscle (Wilcox et al., 1992). 5-HT2A receptor has

been implicated in various processes such as vascular smooth

muscle contraction (De Chaffoy et al., 1985), extravascular

smooth muscle contraction (including uterine contraction)

(Ichida et al., 1983) and platelet aggregation (Leysen et al.,

1984), and is related to such disorders as migraine headaches

(Humphery et al., 1990), anxiety (Taylor, 1990), and mental

depression (Meltzer & Lowy, 1987; Table 1).

The 5-HT2B receptor was cloned from rat and mouse in

1992 (Foguet et al., 1992a, 1992b) and from humans in 1994

(Kursar et al., 1994). The human 5-HT2B receptor gene is

localized to chromosome 2q36.3–2q37.1 and has two introns,

and thus the gene structure is similar to that of the 5-HT2A and

5-HT2C receptors. mRNA encoding the 5-HT2B receptor is

expressed most abundantly in the human liver and kidney.

Lower levels of expression have been detected in the

pancreas and spleen (Bonhaus et al., 1995), but the presence

of mRNA for the 5-HT2B receptor in the brain appears to be

relatively rare (Kursar et al., 1994). 5-HT2B receptors were

functionally characterized in the rat stomach fundus and

porcine pulmonary artery, where they trigger muscle con-

traction (Cohen & Fludzinski, 1987) and relaxation (Glusa &

Pertz, 2000), respectively.


The 5-HT2C receptor was cloned from mouse (Yu et al.,

1991), rat (Julius et al., 1988), and human (Saltzman et al.,

1991). The receptor gene is located on chromosome Xq24,

and contains three introns (Xie et al., 1996). 5-HT2C receptors

are expressed nearly exclusively throughout the brain

(Mengod et al., 1990). High levels of 5-HT2C receptor

expressions have been detected in the choroid plexus, the

cortex, the nucleus accumbens, the amygdala, the hippo-

campus caudate nucleus, and the substantia nigra (Pazos et

al., 1985; Mengod et al., 1990; Palacois et al., 1990;

Abramowski et al., 1995). This receptor type has been

reported to increase grooming, penile erection, oxytocin

secretion (Bagdy et al., 1992), and transferron levels in the

choroid plexus (Esterle & Sanders-Bush, 1992). The 5-HT2C

receptor plays an important role in the serotonergic regulation

of body weight and food intake (Heisler et al., 1998).

Activation of this receptor decreases appetite and body

weight in obese subjects (Sargent et al., 1997). Thus,

selective 5-HT2C agonists have the potential to be effective

as anti-obesity agents (Bickerdike, 2003).


5-HT3 receptors are found mainly in the peripheral

nervous system, particularly on pre- and post-ganglionic

autonomic neurons and on neurons of the sensory and enteric

nervous system, on which 5-HT exerts a strong excitatory

effect. 5-HT itself evokes pain when injected locally

(Richardson et al., 1985), and when given intravenously

elicits a fine display of autonomic reflexes, which results

from excitation of many types of vascular (Blauw et al.,

1988), pulmonary (Mcqueen & Mir, 1989), and cardiac

sensory nerve fibers (Saxena & Villalon, 1991).

5-HT3 receptor activation elicits a rapidly desensitizing

depolarization mediated by the gating of cations (Peters et

al., 1991). 5-HT3 receptors also occur in the brain,

particularly in the area prostrema, a region of the medulla

involved in the vomiting reflex, and selective 5-HT3

antagonists are used as antiemetic drugs (Rang et al., 1999).

With respect to agonists, m-chlorophenylbiguanide is

appreciably more potent than either phenylbiguanide or 2-

methyl-5-HT. Pyrroloquinoxaline derivatives have high

affinity and selectivity for 5-HT3 receptors (Campiani et

al., 1999). Ito et al. (2000) suggested that YM-31636 is a

potent and selective 5-HT3 agonist. With respect to

antagonists, compounds that show high potency and

selectively for 5-HT3 receptors are MDL 72222, tropisetron

or ondansetron, granisetron (Hoyer et al., 1994), ramose-

tron, and (R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetra-

hydro-1H-benzimidazol hydrochloride (YM060; Miyata et

al., 1991).


5-HT4 receptors have recently been identified as a

subclass distinct from 5-HT3 receptors. They occur in the

brain, as well as in peripheral organs, such as the gastro-

intestinal tract, bladder, and heart. Their main physiological

role appears to be in the gastrointestinal tract, where they

produce neuronal excitation, and mediate the effect of 5-HT

in stimulating peristalsis (Rang et al., 1999). Tegaserod, a 5-

HT4 agonist, may offer new treatment options that normalize

GI motor and sensory functions in patients with irritable

bowel syndrome (Rivkin, 2003; Mach, 2004). Cisapride,

another 5-HT4 agonist, affects oesophageal motility and

lower sphincter function involved in the control of gastro-

oesophageal reflux (Finizia et al., 2002). Cartier et al. (2003)

and Mannelli et al. (2003) reported that cisapride might

stimulate cortisol secretion in patients with Cushing’s

syndrome. Other reported 5-HT4 agonists are TS-951 (Kajita

et al. 2001) and mosapride (Ruth et al. 2003). RS39604 is a

selective 5-HT4 antagonist (Orsetti et al., 2003).


A group of researchers reported the cloning of the genes

for two putative mouse and rat receptors that are called the

recombinant receptors 5-HT5A and 5-HT5B. At present, the

functional correlation between these receptors and their

transductional characteristics is unknown. As such they

cannot be fully characterized and therefore can only be

provisionally classified (Hoyer et al., 1994).


5-HT6 receptors, like 5-HT4 and 5-HT7 receptors,

stimulate adenylate cyclase activity. The prominent local-

ization of 5-HT6 mRNA in the striatum, nucleus accumbens,

olfactory tubercle, and substantia nigra, together with its

high affinity for both typical and atypical neuroleptics, has

led to speculation that this receptor might be one of the

target sites for the action for antipsychotic agents (Murphy

et al., 1999). Recent studies have revealed that 5-HT6

receptor appears to regulate glutamatergic and cholinergic

neurotransmission, suggesting that it may be involved in the

regulation of cognition and feeding (Woolley et al., 2004).

Recently, some potent and selective antagonists have been

developed, including Ro 04-6790, Ro 63-0563 (Sleight et

al., 1998), SB-271046 (Bromidge et al., 1999), and 4-(2-

bromo-6-pyrrolidin-1-ylpyridine-4-sulfonyl)phenylamine

(Riemer et al., 2003).


5-HT7 receptors genes have been identified in rodent and

human brains and, like 5-HT6 receptors, they have high

binding affinity for antidepressant and antipsychotic drugs

(Murphy et al., 1999). 5-HT7 receptors are thought to mediate

canine external carotid vasodilatation (Villalon et al., 2001)

and smooth muscle relaxation produced by 5-HT in canine

cerebral arteries (Terron & Falcon-Neri, 1999). Ishine et al.

(2000) suggested that 5-HT7 receptors partly mediate 5-HT-


induced inhibition of rhythmic contractions. The most

selective 5-HT7 antagonists are SB-258719 (Guscott et al.,

2003), SB-269970-A, and SB-656104-A (Thomas & Hagan,

2004). No selective 5-HT7 agonist has been reported yet.

However, 8-OH-DPAT has agonistic activity for 5-HT7

receptors (Sprouse et al., 2004).

3. Serotonin receptors in the cardiovascular system

The effects of serotonin in the cardiovascular system are

complex. It is involved in both central and peripheral

mechanisms, acting through numerous receptor subtypes. Its

cardiovascular effects have been associated with bradycar-

dia or tachycardia, hypotension or hypertension, and vaso-

dilatation or vasoconstriction. 5-HT also acts as an ideal

neurotransmitter candidate for many aspects of cardiovas-

cular regulation. Recent pharmacological studies have

suggested that compounds acting on 5-HT receptors could

be of therapeutic use in the treatment of migraine, hyper-

tension, and heart and vascular diseases (Villalon & Saxena,

1997; Frishmann & Grewall, 2000).

Among the 5-HT receptors, 5-HT1A (sympathoinhibitory

and vagal bradycardia) and 5-HT2 (sympathoexcitatory)

receptor subtypes predominantly execute the central regu-

lation of the cardiovascular system (McCall & Clement,

1994). Stimulation of 5-HT1A receptors produces a vaso-

pressor and bradycardiac effect, which results from a

combination of central sympathetic inhibition and central

stimulation of the vagus nerve. On the other hand,

stimulation of central 5-HT2 receptors results in vaso-

constriction and bradycardia due to increased sympathetic

discharge.

Peripherally, numerous serotonergic receptors and recep-

tor subtypes modulate a range of cardiovascular functions,

including vasoconstriction, vasodilatation, platelet aggrega-

tion, and positive inotropic and chronotropic effects (Yusuf

et al., 2003). Activation of both 5-HT1B and 5-HT1D

receptor subtypes induces vasoconstriction in the human

coronary artery, which leads to the development of

myocardial ischemia in patients with stable angina, vaso-

spastic angina, and acute coronary syndrome. The potent

and selective 5-HT (1B/1D) receptor agonists, domitriptan

and sumitriptan, have significant effects on vasoconstriction

(Akin & Gurdal, 2002; Van den Broek et al., 2002). Ishida

et al. (2001) reported that atherosclerotic rabbit coronary

arteries exhibited enhancement of contraction and Ca2+

mobilization in response to serotonin. They suggested that

5-HT1B receptor, which is up-regulated by atherosclerosis,

most likely mediated the augmented vasoconstriction

effects of serotonin. 5-HT2A receptors are of significant

clinical interest because of their potential involvement in

mediating many cardiovascular diseases. The 5-HT2A

receptor mediates several important pathophysiological

effects in both the peripheral nervous system and CNS. It

has been associated with the contraction of vascular smooth

muscle, platelet aggregation, and thrombus formation and

coronary artery spasm. All of these processes play an

important role in the pathogenesis of a wide variety of

ischemic heart diseases. 5-HT2A receptors are also involved

in migration and cell proliferation (Tamura et al., 1997;

Sharma et al., 1999). 5-HT2B receptor is expressed in

embryonic (Choi et al., 1997) and adult (Choi &

Maroteaux, 1996) cardiovascular tissues, gut, and brain

from the rat, mouse, and human species. Several lines of

evidence suggest that 5-HT regulates cardiovascular func-

tions through 5-HT2BR during embryogenesis and child-

hood. Genetic ablation of 5-HT2BR in mice leads to partial

embryonic and neonatal death as a result of the following

heart defects: (1) 5-HT2BR mutant embryos exhibit a lack

of trabeculae in the heart, leading to mid-gestation lethality

(Nebigil et al., 2000). (2) In newborn mice, contractility

and structural deficits at cellular junctions in 5-HT2BR

mutant cardiomyocytes lead to cardiac dilation. (3) In the

adult 5-HT2BR mutant mice, echocardiography and electro-

cardiography both confirm the presence of left ventricular

dilation and decreased systolic function typical of dilated

cardiomyopathy (Nebigil et al., 2001). These results

provide a basis for understanding how 5-HT, via 5-HT2BR,

can regulate the differentiation and proliferation of the

developing heart and the structure and function of the adult

heart and neonatal cardiomyocytes. Identification of factors

controlling myocardial differentiation and proliferation is

very important for understanding the pathogenesis of

congenital heart disease. Nebigil et al. (2003) reported that

overexpression of 5-HT2BR leads to hypertrophic cardio-

myopathy and is associated with altered mitochondrial

function. These findings promise to have important

implications for the understanding of congenital heart

disease and the development of potential therapeutic

interventions for cardiovascular disease Nebigil & Mar-

oteaux, 2003. 5-HT2B receptor also mediates 5-HT-induced

arterial contraction in deoxycorticosterone (DOCA)-salt

hypertension (Watts, 1998b). Thus, an increase in 5-HT2B

receptor activation plays a role in maintaining high blood

pressure in DOCA-salt hypertension (Banes & Watts,

2003). 5-HT3 receptors are present on human afferent

vagal nerve endings and are responsible for the Von

Benzold-Jarisch reflex in humans (Richardson et al.,

1985). Stimulation of 5-HT3 receptors on afferent cardiac

vagal nerve endings produces an initial hypotensive

response to 5-HT, an effect that results from an abrupt

and transient bradycardia (and the consequent decrease in

cardiac output). Fu and Longhurst (2002) reported that

during myocardial ischemia the activated platelets stimulate

cardiac sympathetic afferents, at least in part through a 5-

HT3 receptor mechanism. 5-HT4 receptors are present in

human atrial cells, and 5-HT increases human heart rate and

atrial contractile force and hastens atrial relaxation through

5-HT4 receptors. Moreover, 5-HT may be arrhythmogenic

and give rise to atrial fibrillation through the mediation of

the 5-HT4 receptor in the human heart (Bach et al., 2001).


Salle et al. (2001) reported that 5-HT4 receptor is

physiologically important since it could be a target for

auto antibodies in mothers at risk of giving birth to children

with neonatal atri-ventricular block.

4. 5-Hydroxytryptamine2A receptor and its antagonists

Serotonin is known to participate in the regulation of the

cardiovascular system and is therefore linked to both

vascular and cardiac events (Viekenes et al., 1999). Among

the 5-HT receptors, 5-HT2A receptor subtype mediates

several important pathophysiological effects in both the

peripheral nervous system and CNS. After vascular injury,

the released 5-HT induces vasoconstriction, platelet aggre-

gation, increase of vascular permeability and cell prolifer-

ation. Moreover, factors such as age, atherosclerosis, and

hypertension are known to augment 5-HT-induced vaso-

constriction. 5-HT2A receptor has been implicated in the

contraction of vascular smooth muscle, contraction of

uterine smooth muscle, platelet aggregation, and thrombus

formation and coronary artery spasm. All of these processes

play an important role in the pathogenesis of a wide variety

of ischemic heart diseases. Therefore, 5-HT2A antagonists

have been used to treat cardiovascular diseases.

In 1981, Leysen et al. discovered ketanserin, which

selectively binds to 5-HT2 receptor and has no significant

effect on the 5-HT3, 5-HT4, or 5-HT1 receptor families. The

5-HT2A receptor antagonist ketanserin has been suggested to

have therapeutic potential in hypertension as well as in

peripheral vascular disease (Vanhoutte et al., 1988; Brogden

& Sorkin, 1990) and to exert a cardioprotective effect in the

ischemic myocardium (Grover et al., 1993). Long-term

treatment with ketanserin significantly decreased blood

pressure variability, ameliorated impaired arterial baroreflex

function, and significantly prevented the target organs of

spontaneously hypertensive rats (SHR) from being damaged

(Du et al., 2003; Miao et al., 2003). But despite its

effectiveness at lowering blood pressure it was withdrawn

due to tendency to proarrhythmia. Moreover, ketanserin’s

effect of lowering blood pressure due to its effects on the 5-

HT2 receptor has always been controversial. Ketanserin has

also high affinity for a1-adrenergic and histamine H1

receptors (Janssen, 1983), central sympathoinhibitory prop-

erties and direct vasodilatory properties (Saxena & Villalon,

1990).

Ritanserin is a more selective 5-HT2A-receptor antagonist

than ketanserin with low affinity for a1-adrenergic recep-

tors. It lowers portal pressure without causing systemic

hemodynamic changes in portal hypertensive rats (Fernan-

dez et al., 1993). It increases cerebral blood flow in focal

cerebral ischemia in rats (Back et al., 1998).

Cyproheptadine has prominent 5-HT blocking activity on

smooth muscle by virtue of its binding to 5-HT2A receptors,

although it is an effective H1-receptor antagonist. It does not

effectively lower the blood pressure. Xin et al. (1994)

suggested that cyproheptadine might be useful for the

treatment of cerebral ischemic damage.

Methysergide is a 5-HT2A/2C receptor antagonist,

although it acts as a partial agonist of 5-HT1 receptors. It

has been used for the prophylactic treatment of migraine. It

inhibits the vasoconstrictor and pressor effects of 5-HT as

well as the actions of 5-HT on various types of extravascular

smooth muscle. Fujiwara and Chiba (1995) reported that

methysergide markedly inhibited the 5-HT2 mediated vaso-

constrictions in atheresclerotic rabbit common carotid

arteries.

AT-1015, a potent 5-HT2A receptor antagonist selectively

inhibited 5-HT2A receptor-mediated platelet aggregation and

5-HT induced vasoconstriction with insurmountable antag-

onism and also ameliorated laurate-induced peripheral

vascular lesions in rats (Kihara et al., 2000). It is a potent

and long-lasting antithrombotic agent and has a low risk of

prolongation of bleeding time (Kihara et al., 2001).

Komiyama et al. (2004) reported that AT-1015 improves

oxygen resaturation of ischemic calf muscle after exercise in

hypercholesterolemic rabbits.

Sarpogrelate is a novel, selective 5-HT2A receptor

antagonist that has been introduced as a therapeutic agent

for the treatment of ischemic diseases associated with

thrombosis (Ito & Notsu, 1991). It is a new type of

compound for 5-HT2A receptor subtype, which is structur-

ally different from ketanserin and other 5-HT2 receptor

antagonists. These chemical differences are likely to

account for the different characteristics of sarpogrelate.

Hara et al. (1991b) investigated the antithrombotic effect

of sarpogrelate in three different experimental thrombosis

models in mice. Simultaneous injection of serotonin and

collagen into the tail vein in mice induced acute

pulmonary thromboembolic death. Coronary artery spasm

plays an important role in the pathogenesis of a wide

variety of ischemic heart diseases (Fuster et al., 1992).

Miyata et al. (2000) showed that sarpogrelate dose-

dependently inhibits the serotonin-induced coronary artery

spasm in a porcine model. 5-HT-induced vasoconstriction

promotes hemostasis and vascular occlusion. Gong et al.

(2000) demonstrated that sarpogrelate inhibits 5-HT-

induced contraction of coronary artery in the porcine

model. Cardiac hypertrophy is a major problem in cardiac

diseases. Ikeda et al. (2000) designed a study to elucidate

the effects of sarpogrelate on cardiac hypertrophy in rat.

Their results indicated that sarpogrelate might have

antihypertrophic effects and could be a useful aid for

cardiovascular disease. Neointimal hyperproliferation and

platelet activation/aggregation are two major cardiovascu-

lar abnormalities commonly observed in blood vessels

after cellular injury, mechanical or physiological stress, or

overload due to peripheral resistance (Schwartz et al.,

1986; Schwartz & Reidy, 1987). Sharma et al. (1999)

demonstrated the antiproliferative behavior of sarpogrelate

in cultured rat aortic smooth muscle cells and reported

that sarpogrelate operates as a specific inhibitor of 5-HT-


mediated cell proliferation and is a good candidate for

preventing serotonin-induced neointimal hyperplasia.

Obata et al. (2000) investigated the antinociceptive effect

of sarpogrelate in rats. Their results implied that the

antinociceptive effect of sarpogrelate results mainly from

its action at peripheral sites. Temsah et al. (2001) reported

that sarpogrelate causes a significant improvement in

cardiac performance and high energy stores as well as a

decrease in ultrastructural changes in ischemic-reperfused

hearts. Brasil et al. (2002) showed that pretreatment or

post-treatment of myocardial infarction (MI) rats with

sarpogrelate reduces electrocardiographic changes and

infarct size, and improves cardiac function. Satomura et

al. (2002) studied baseline and maximal coronary blood

flow of patients with various cardiovascular diseases after

the administration of sarpogrelate and found that both

baseline and maximal coronary blood flow increased

significantly without a change of the systemic hemody-

namics. These findings support the view that in coronary

artery disease, sarpogrelate improves microcirculation by

antagonizing the vasoconstrictor products of aggregating

platelets. Sarpogrelate inhibits monocrotaline (MCT)-

induced pulmonary hypertension and prolongs survival

in rats (Hironaka et al., 2003). Umrani et al. (2003)

suggested that sarpogrelate prevents streptozotocin-

induced down-regulation of cardiac 5-HT2A receptors

and increases platelet aggregation in diabetic rats. It

retards the progression of atherosclerosis in rabbits (Hay-

ashi et al., 2003).

Recently, Ogawa et al. (2002) found that R-102444, a

novel and selective 5-HT2A receptor antagonist, inhibited

platelet aggregation in rabbits and rats and was more

potent than sarpogrelate. SL 65.0472, a 5-HT1B/5-HT2A

receptor antagonist, inhibited 5-HT-induced vasoconstric-

tion in a canine model of hindlimb ischemia (Barbe et al.,

2003).

5. Signaling pathway of 5-hydroxytryptamine2A receptor

A total of 15 serotonin receptor subtypes have been

reported to date, and they may be further subdivided into

seven receptor classes. These subfamilies have been

characterized according to overlapping pharmacological

properties, amino acid sequences, gene organization, and

second messenger coupling pathways (Hoyer et al., 1994).

The 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7

receptors couple to G-proteins, whereas the 5-HT3 receptors

are 5-HT-gated ion channels. Recent studies have revealed a

rich diversity of coupling mechanisms for each 5-HT

receptor subtype. The multiplicity of coupling pathways

for each of the receptors suggests that each individual 5-HT

receptor subtype can regulate a broad array of potential

signals that can be affected by variables such as cell type,

receptor number, numbers and types of G-proteins

expressed in the target cells, and the specific agonist

through which the receptor is activated. In this review, we

will focus only on the signaling linkages of the G-protein

coupled 5-HT2A receptors.

5.1. The 5-hydroxytryptamine2Areceptor activates phospholipase C

5-HT2A receptor couples to Gq/11 proteins and activates

PLC-h in most tissues and cells in which it is expressed, to

increase inositol triphosphate and increase intracellular Ca2+

(Briddon et al., 1988; Grotewiel & Sanders-Bush, 1999).

These effects can result in activation of l-type Ca2+

channels (Mckune & Watts, 2001) and stimulation of PKC

(Takuwa et al., 1989).

5.2. The 5-hydroxytryptamine2Areceptor activates phospholipase A2

The 5-HT2A receptor can mediate stimulation of phos-

pholipase A2 (PLA2); thereby generating the second mes-

senger arachidonic acid (AA; Tournois et al., 1998).

Kurrasch-Orbaugh et al. (2003a) reported that the 5-HT2A

receptor can couple to PLA2 activation through two parallel

signaling cascades: (1) activation of the pertussis toxin-

sensitive G protein, namely Gai/o, causes the release of Ghg,which is free to initiate activation of the Ras-Raf-MEK-ERK

signaling cascade, ultimately leading to ERK-mediated

phosphorylation of cPLA2; (2) activation of receptor-coupled

pertussis toxin-insensitive Ga12/13, which functions to

activate Rho, and ultimately results in p-38 mediated

phosphorylation of cPLA2. In addition, because inhibition

of either pathway caused a nearly identical reduction in AA

release, it seems likely that the two pathways share a common

final enzyme known as mitogen activated protein kinase

(MAPK) prior to PLA2 activation. They also showed that

none of the inhibitors, toxins, or constructs employed (alone

or in combination) was able to completely abolish 5-HT-

induced AA release. Only the 5-HT2A receptor antagonist

ketanserin and the PLA2 inhibitor mepacrine were able to

inhibit all 5-HT-induced AA release in NIH3T3-5-HT2A

cells. Kurrasch-Orbaugh et al. (2003b) suggested that the 5-

HT2A receptor could differentially regulate the PLA2 and

PLC signaling pathways in NIH3T3-5-HT2A cells, and that a

larger receptor reserve exists for 5-HT-induced PLA2

activation than for 5-HT-induced PLC activation.

5.3. The 5-hydroxytryptamine2A receptor

activates the Janus Kinase/signal transducers

(Jak) and activators of transcription (STAT) pathway

In fetal myoblasts, serotonin binding to the 5-HT2A

receptor results in the stimulation of the Janus kinase (Jak)/

signal transducers and activators of transcription (STAT)

pathway. 5-HT2A triggers a rapid and transient tyrosine

phosphorylation of Jak2 kinase in response to serotonin. This

serotonin-induced association of Jak2 with the carboxy


terminal tail (ct) of the 5-HT2A receptor allows the recruit-

ment of STAT3 to the receptor complex and its subsequent

tyrosine phosphorylation by the phosphorylated Jak2 kinase,

and leads to STAT3 nuclear translocation (Guillet-Deniau et

al., 1997). Guillet-Deniau et al. (1997) also reported that the

5-HT2A and STAT3 co-precipitate with Jak2, indicating that

they are physically associated (Table 2).

5.4. The 5-hydroxytryptamine2Areceptor activates phospholipase D

The 5-HT2A receptor can also signal through the

activation of phospholipase D (PLD), an enzyme that can

be controlled by the small G proteins ADP-ribosylation

factor (ARF; Mitchell et al., 1998). Both coimmunopreci-

pitation experiments and the effects of negative mutant ARF

constructs on 5-HT2AR-induced PLD activation have

suggested that ARF1 may play a greater role than ARF6

in the function of this receptor. The association of ARF1

with the ct domain of the receptor is stronger than its

interaction with the third intracellular loop (i3), or the

interactions of ARF6 with either construct. Therefore, a

negative mutant construct of ARF1, but not ARF6, inhibits

the activation of PLD by 5-HT2AR (Robertson et al., 2003).

5.5. The 5-hydroxytryptamine2Areceptor can regulate cyclic adenosine

monophosphate accumulation in certain cells

The 5-HT2A receptor can both stimulate and diminish

cyclic adenosine monophosphate (cAMP) accumulation in

specific cell types. It increases cAMP in A1A1 cells by the

intermediate actions of PKC-a and/or PKC-y and Ca2+/CaM(Berg et al., 1994), and in FRTL-5 thyroid cells through a

pertussis toxin-sensitive mechanism (Tamir et al., 1992). It

can inhibit forskolin-stimulated cAMP accumulation and

AC activity in rat renal mesangial cells (Garnovskaya et al.,

1995).

5.6. The 5-hydroxytryptamine2Areceptor activates the extracellular

signal-regulated mitogen-activated protein kinase

The 5-HT2A receptor activates ERK MAP kinases in

cells with contractile phenotypes, vascular smooth muscle

Table 2

Signaling characteristics of human 5-HT2A receptors

Receptor Common signaling

pathways

Other signaling

linkages

G-protein

coupling

5-HT2A Activates PLC Inhibits AC Gqa and

G11azGia

Activates PKC Activates Jak2/STAT3

Stimulates ERK Activates Ca2+ channels

Activates PLA2,

Activates PLD

cells, requiring inputs from PLC, l-type Ca2+ channels, and

MEK1 (Florian & Watts, 1998; Watts, 1998a) and in renal

mesangial cells, involving stimulation of PKC, activation of

an NAD(P)H oxidase-like enzyme, and production of

reactive oxygen species (H2O2 and/or superoxide; Grewal

et al., 1999; Greene et al., 2000). Xu et al. (2002) reported

that in sheep aortic valve interstitial cells (SAVIC), 5-HT

also mediates strong extracellular signal-regulated kinase

(Erk1/2) signaling via the MAP-kinase pathway, only in part

mediated through 5-HT2AR activity. Both PKC and Src/Src-

like tyrosine kinase are involved in mediating the stimula-

tory effects of serotonin on Erk1/2 activity. They also

reported that 5-HT2A receptors are the most functionally

active of the 5-HT2R in this cell type, and are involved in

both up-regulation of transforming growth factor (TGF)-h1expression and activity, which may contribute to the

progression of 5-HT-related heart valve disease.

5.7. The 5-hydroxytryptamine2Areceptor regulates calmodulin

There is some evidence that the 5-HT2A receptor signals

through calmodulin (CaM). Chen et al. (1995) showed that

agonist-mediated up-regulation of the 5-HT2A receptor

depends upon CaM and Ca2+/CaM-dependent kinase 2.

Inhibition of CaM-dependent kinase 2 or calcineurin (a

CaM-dependent phosphatase) inhibits 5-HT-induced cyclo-

oxygenase 2 mRNA expression in renal mesangial cells

(Goppelt-Struebe et al., 1999).

5.8. The 5-hydroxytryptamine2A receptor regulates channels

The 5-HT2A receptor increases intracellular Ca2+ levels

by liberating intracellular stores of Ca2+ and/or by

activating Ca2+ channels, depending upon the cell of

interest. It may activate l-type Ca2+ channels in some

cell types (Watts, 1998a). The Ca2+ channels coupled to

the 5-HT2A receptor have been characterized as both

voltage-dependent and voltage-independent (Eberle-Wang

et al., 1994; Hagberg et al., 1998). The increases in Ca2+

levels evoked by the 5-HT2A receptor have been linked

to subsequent opening of Ca2+-activated K+ channels in

C6 glial cells (Bartrup & Newberry, 1994) and to an

inward current mediated through Ca2+-activated Cl-

channels in Xenopus oocytes (Montiel et al., 1997). In

rat cortical astrocytes, the 5-HT2A receptor activates both

an l-type Ca2+ channel and an apamin-sensitive Ca2+-

activated small conductance K+ channel (Jalonen et al.,

1997).

6. Molecular aspects of

5-hydroxytryptamine2A receptors

The 5-HT2A receptor is a member of the G protein-

coupled receptor superfamily, for which structure-activity


studies have identified key interactions in the ligand-

receptor complexes. One notable group of ligands for this

receptor are the serotonergic hallucinogens, such as lysergic

acid diethylamide (LSD) and N,N-dimethyl 5-HT (bufote-

nin), which have high affinity for the 5-HT2A receptor.

Studying the binding pocket of the receptor and identifying

the molecular mechanisms that determine ligand affinity,

specificity, and coupling efficiency may help elucidate the

basis for the specific biological effects of these chemicals.

An important approach to investigate structure-function

relations of the 5-HT2A receptor is to introduce structural

perturbations via site-directed mutagenesis and evaluate the

resulting receptor phenotype in binding and signal trans-

duction assays. Molecular modeling has facilitated the

integration of experimental observations and biophysical

data into a mechanistic scheme for receptor structure and

function. The availability of the crystal structure of

rhodopsin has improved the accuracy of computational

modeling of homologous receptors (Palczewski et al.,

2000). This approach has been used to determine the

binding site of the 5-HT2A receptor and the pattern of

interaction of 5-HT2A agonists with the specific trans-

membrane helices (TMH).

The 5-HT2A receptor has an aspartate residue at a

homologous location in the third TMH domain. Site-

directed mutagenesis of these receptors has indicated that,

for most ligands, an interaction between the basic nitrogen

of the ligand and the carboxyl side chain of the conserved

TMH3 aspartate (Asp3.32) stabilizes ligand binding. The

same charged amino group of 5-HT that interacts with the

TMH3 aspartate was predicted to form a hydrogen bond

with the side chain of a second TMH3 serine. In the

molecular model of the receptor, this serine residue is

positioned on the same face of the helix as the aspartate.

Mutation of the serine site that interacts specifically with the

free amino group of 5-HT allows the ligands to fit similarly

in the binding pocket and to activate the receptor to a similar

degree (Almaula et al., 1996). Manivet et al. (2002) reported

that several amino acids stabilize the interaction of 5-HT and

Asp3.32 via hydrogen bonding, these amino acids are Ser3.36,

Ser4.57, Ser5.43, Asp2.50, Asn7.49, and Glu7.36.

The highly conserved amino acids in the TMH regions

have a key functional role in the agonist-induced rearrange-

ment of the TMH that constitutes the mechanism of receptor

activation. Hibert et al. (1991) reported that the two highly

conserved aromatic residues Trp6.48 and Phe6.52 are directly

involved in the binding of several neurotransmitters (5-HT,

dopamine, and adrenaline) to their corresponding receptors.

They also speculated that the side chain conformations of

these hydrophobic residues probably changed during the

binding process. Such changes could directly affect the

conformation of the adjacent helices, in particular in the

vicinity of proline residues, and of other helices by

propagation along the backbone of interacting conserved

aromatic residues. Site-directed mutagenesis experiments

also supported the crucial role of these two aromatic

residues (Roth et al., 1997). In all 5-HT G-protein coupled

receptors (GPCR), six conserved aromatic residues (Trp3.28,

Phe3.35, Trp4.50, Phe6.44, Trp6.48, and Trp7.40) are involved in

hydrophobic interactions. In addition, Trp3.28, Phe3.35,

Trp6.52, and Phe7.38 define an aromatic box that surrounds

5-HT. This box maintains the 5-HT indole ring in a

favorable orientation to interact with 5-HT in the 5-HT2A

receptor (Manivet et al., 2002). Mutations of the highly

conserved aromatic residues, including W200[2.50],

W336[6.48], W367[7.40], F340[6.52], and Y370[7.43],

located in the neighboring helices markedly reduced agonist

affinity and efficacy at 5-HT2A receptors (Choudhary et al.,

1995; Roth et al., 1997), whereas mutations of other

aromatic residues (e.g., F365[7.38]), predicted to be near

the binding pocket, had no or little effect on agonist affinity,

although they diminished agonist efficacy (Roth et al.,

1997). Egan et al. (1998) mutated amino acid 322 to lysine

(C322K), glutamate (C322E), or arginine (C322R), and

showed that the mutant 5-HT2A receptor exhibited an

increase in agonist affinity and potency, and an increase in

basal inositol phosphate production. Shapiro et al. (2000)

examined the effects of four single-point mutations in

TMH5 (S239A, F240A, F243A, and F244A) of the rat 5-

HT2A receptor on ligand binding and agonist-stimulated

phosphoinositide (PI) hydrolysis. The F243A mutation

decreased the binding of 5-HT2A antagonists (e.g.,

ketanserin, ritanserin) and had no effect on the binding

of 5-HT. The F240A mutant had no effect on the binding

of any of the ligands, whereas F244A caused an agonist-

specific decrease in binding affinity. The S239A mutation

reduced the binding affinity of tryptamine. F243A and

F244A reduced PI hydrolysis, whereas S239A and F240A

had no effect. They proposed that the interaction of 5-

HT2A agonists with TMH6 (H6) via aromatic residues

facilitates H6 motion and subsequent receptor activation.

They also predicted that agonist binding to residues in H6

leads to the disruption of a strong ionic interaction

between H3 and H6. Shapiro et al. (2002) tested this

bH3–H6 interaction modelQ and suggested that disruption

of the strong ionic interaction between Arg-173(3.50) in

TMH3 and Glu-318(6.30) in TMH6 of the 5-HT2A

receptor by an E318(6.30)R mutation would lead to a

highly constitutively active receptor with enhanced affinity

for agonist. Serotonin forms hydrogen bonds with Ser3.36

in TMH3 and Ser5.46 in helix 5 of the 5-HT2A receptor.

Disruption of these bonds by methyl-substitution of the

cationic primary amine or of the backbone N1 amine,

respectively, reduces the agonist efficacy (Ebersole et al.,

2003). Eborsole et al. (2003) also proposed that ligands

with free, unsubstituted primary amines, which interact

with Ser3.36, and ligands with substitution of the N1-

amine, which interact with Ser5.46, increase the agonist

efficacy toward the Ser3.36Ala and the Ser5.46Ala mutant

5-HT2A receptor.

In conclusion, these studies give us an understanding of

the nature and consequences of ligand-receptor interactions


at a molecular level, and provide a basis for proposing

mechanisms for the pharmacological actions of the 5-HT2A

receptor.

7. Pharmacological and

molecular aspects of sarpogrelate

7.1. Chemistry of sarpogrelate

Chemically, sarpogrelate is (F)-1-[2-[2-(3-methoxyphe-

nyl)ethyl]phenoxy]-3-(dimethyl amino)-2-propyl hydrogen

succinate hydrochloride (Fig. 1). In rats, dogs, monkeys, and

man, oral sarpogrelate is first hydrolyzed to (F)-1-[2-[2-(3-

methoxyphenyl)ethyl]phenoxy]-3-(dimethyl amino)-2-prop-

anol (M-1; Komatsu et al., 1992). M-1 is a major metabolite,

formed by displacement of the succinate ester portion from

sarpogrelate. M-1 exhibited a more potent antiserotonergic

effect on in vitro platelet aggregation and smooth muscle

constriction assay than sarpogrelate, while the antithrom-

botic effects of oral treatment with M-1 in thrombosis

models were weaker than those of sarpogrelate. This

discrepancy between the in vitro and in vivo activities of

M-1 is explained by its low absorbability in oral admin-

istration (Hara et al., 1991a, 1991b). Sarpogrelate lacks the

basic chemical structure of serotonin. However, it contains

an ethylene chain and an amine group. It contains two

methyl groups in the amine position and a large hydrophobic

group on the h-carbon of the ethylene chain (Fig. 1). On the

other hand, other 5-HT2A antagonists, e.g., ketanserin,

ritanserin, and AT-1015 do not contain methyl groups in

the amine position or large groups on the h-carbon of

ethylene chain. They contain N-ethyl piperidine. Cyprohep-

tadine has a different type of chemical structure from

sarpogrelate, ketanserin, and ritanserin, and has no similarity

to the basic chemical structure of serotonin (Fig. 2). For this

reason, the pharmacological properties of sarpogrelate may

be different from those of ketanserin and other 5-HT2

selective antagonists.

Fig. 1. Chemical structure of sarpogr

7.2. Radioligand binding studies

Radioligand binding assays showed (Rashid et al.,

2001a, 2001b, 2002a) that sarpogrelate had high displace-

ment potencies for [3H]ketanserin binding to 5-HT2A

subtype in rabbit cerebral cortex, rabbit platelet, and rat

frontal cortex membrane fractions compared with other 5-

HT2 antagonists such as ketanserin, ritanserin, cyprohep-

tadine, miancerin, and methysergide. In washout experi-

ments, we demonstrated (Rashid et al., 2001a) that both

sarpogrelate and ketanserin rapidly dissociated from 5-HT2

receptor sites in rabbit cerebral cortex membranes com-

pared with a potent and long-acting 5-HT2 antagonist,

ritanserin (Leysen et al., 1985). Previous studies based on

radioligand binding assay and functional studies have

shown that sarpogrelate exhibits specificity toward 5-HT2

receptors, since it lacks significant 5-HT1, 5-HT3, 5-HT4,

a1-, a2-, and h-adrenoreceptors, histamine H1, H2, and

muscarinic M3 antagonistic activity (Maruyama et al.,

1991; Tsuchihashi et al., 1991; Pertz & Elz, 1995; Nishio

et al., 1996). Studies in rat cortical membranes using

different radioligands have shown that M-1, the active

metabolite of sarpogrelate, has high affinity for 5-HT2A

receptors, with very low affinity for 5-HT1B and 5-HT4

receptors (Nishio et al., 1996). Both ketanserin and

ritanserin also exhibit specificity toward 5-HT2 receptors.

However, ketanserin has high affinity for a-adrenergic

receptors and histamine H1 receptors, whereas ritanserin

has low affinity for a1-adrenergic receptors.

7.3. Inhibitory effects on platelet aggregation

Kikumoto et al. (1990) first synthesized a series of [2-

[(omega-aminoalkoxy)phenyl]ethyl] benzene derivatives

and evaluated for their ability to inhibit collagen-induced

platelet aggregation in vitro and to protect against exper-

imental thrombosis in mice. They observed that sarpogrelate

((F)-1-[o-[2-(m-methoxyphenyl)ethyl]phenoxy]-3-(dime-

thylamino)-2-propyl hydrogen succinate hydrochloride,

elate and its metabolite (M-1).

Fig. 2. Chemical structure of serotonin and several 5-HT2A antagonists.


MCI-9042) and other derivatives inhibited collagen-induced

platelet aggregation in vitro and thrombosis formation in

mice. Nakamura et al. (1999) suggest that extracellular

release of serotonin and P-selectin from platelets was caused

by induction of aggregation, and that these responses were

suppressed by sarpogrelate. The platelet anti-aggregatory

effect of sarpogrelate has been explained by its 5-HT2A-

receptor blocking properties. Furthermore, sarpogrelate

inhibits the 5-HT release accompanied by collagen-induced

platelet aggregation in human, rabbit, and rat platelet-rich

plasma and also the secondary wave of aggregation induced

by ADP and adrenaline (Hara et al., 1991a). The effective-

ness of sarpogrelate in thromboembolic therapy might

depend on the extent of vascular damage (Yamashita et

al., 2000). Ketanserin (5-HT2 antagonist and a1-adrenergic

antagonist; Van Nueten et al., 1981) and cyproheptadine (5-

HT2 antagonist and H1-histaminergic antagonist; Remy et

al., 1977) caused more potent inhibition of serotonin-plus

collagen-induced platelet aggregation than sarpogrelate.

However, M-1 (active metabolite of sarpogrelate) showed

almost equal inhibitory potency to ketanserin with respect to

platelet aggregation.

7.4. Effects on vasoconstriction response

It has been demonstrated that 5-HT-induced vaso-

constriction of arteries is mainly mediated by a 5-HT2A

receptor subtype and is also mediated by 5-HT1-like

receptor (Connor et al., 1989; Toda & Okamura, 1990).

Several studies have dealt with the inhibitory effect of

sarpogrelate on the contraction response induced by 5-HT.

Hara et al. (1991a) reported that sarpogrelate potently

inhibited the 5-HT2A receptor-mediated contraction of the

rat caudal artery by 5-HT in a competitive manner, while

5-HT1 or adrenergic receptor-mediated vasoconstriction

was inhibited more weakly. Pertz and Elz (1995) also

reported that sarpogrelate produced concentration-depend-

ent antagonism of the contraction response induced by 5-

HT in the rat tail artery, causing parallel dextral shift of 5-

HT concentration-effect curves with no or little effect on

the maximum response. Our studies (Gong et al., 2000)

showed that both sarpogrelate and ketanserin inhibited

contraction responses induced by both 5-HT and a-Me-5-

HT in the porcine coronary artery without endothelium,

while only sarpogrelate caused a 55% increase of

maximum contraction at a higher concentration of 5-HT.

The results suggested that the 55% increase of maximum

contraction induced by sarpogrelate treatment might have

been due to 5-HT1-like or as yet uncharacterized receptors.

The results also indicated the presence of two 5-HT

receptor subtypes involved in the contraction of coronary

arteries. Sarpogrelate inhibited only the 5-HT2A subtype

response, while ketanserin affected both the 5-HT2A and

5-HT1-like receptor-mediated contraction responses, sug-

gesting that the former was more selective for 5-HT2A

receptor than the latter. The antagonistic activity of

sarpogrelate was lower than that of ketanserin for

contraction responses induced by 5-HT and a-Me-5-HT,

respectively.

7.5. Effects on vasodilatation response

5-HT causes both endothelium-dependent and endothe-

lium-independent relaxation of a number of isolated blood


vessels in a variety of animals. In porcine coronary and

pulmonary arteries, 5-HT causes endothelium-dependent

relaxation responses. So far, it has been reported that

endothelium-dependent relaxant effects of 5-HT in pig

coronary and pulmonary arteries are mediated by 5-HT1-

like and/or 5-HT2B receptors (Scoeffter & Hoyer, 1989;

Glusa & Pertz, 2000). Our investigation (Rashid et al.,

2002b) showed that sarpogrelate (10�7–10�5 M) had a very

weak antagonistic effect on 5-HT-induced endothelium-

dependent relaxation in the porcine coronary artery

mediated by 5-HT1-like receptors and that its effect was

weaker than those of ritanserin (10�9–10�7 M) and

cyproheptadine (10�8–10�6 M). Sarpogrelate had no

inhibitory effect on the bradykinin-induced relaxation

response. The rank order of the calculated ratio of

concentration of pA2 versus K i was: sarpogrela-

teNritanserinNcyproheptadine, and the results suggested

that sarpogrelate had the highest selectivity towards 5-

HT2A receptor and it might also be the safer with respect to

its clinical effects in comparison with ritanserin and

cyproheptadine.

7.6. Hemodynamic effects

Many reports on the hemodynamic actions of sarpog-

relate have been published. Brasil et al. (2002) examined

the effect of sarpogrelate in preventing cardiac dysfunc-

tions due to MI in rats. They observed that sarpogrelate

attenuates cardiac dysfunction, infarct size, and changes

in the electrocardiogram due to MI. Satomura et al.

(2002) reported that sarpogrelate increased both baseline

and maximal coronary blood flow without changing the

systemic hemodynamics. Setoguchi et al. (2002) inves-

tigated the effects of long-term sarpogrelate administra-

tion on the systolic blood pressure of Wistar-Kyoto

normotensive rats (WKY) and SHR, and compared these

effects with those of quinapril (ACE-I). The results

showed that quinapril induced a dose-dependent decrease

in systolic blood pressure in WKY and SHR, while

sarpogrelate had no effect on systolic blood pressure.

Therefore, we suggested that the 5-HT2A antagonist

sarpogrelate might not be useful for controlling systolic

blood pressure. On the other hand, ketanserin signifi-

cantly reduces the systemic and pulmonary arterial

pressure and total systemic and pulmonary vascular

resistance (Domenighetti et al., 1997; Hood et al.,

1998). Bolte et al. (1998) compared the hemodynamic

efficacy of ketanserin with that of dihydralazine in the

management of severe early-onset hypertension in preg-

nancy. They found that dihydralazine significantly

increased cardiac output and decreased systemic vascular

resistance, while ketanserin induced a minor change in

cardiac output and a moderate decrease in systemic

vascular resistance. Ritanserin decreases portal pressure

without causing systemic hemodynamic changes in portal

hypertensive rats (Fernandez et al., 1993).

7.7. Relationship between

binding affinities and functional potency

In our previous study (Rashid et al., 2002a), a relation-

ship was found between binding affinity and functional

potency of sarpogrelate as well as other 5-HT2 antagonists

such as ketanserin, ritanserin, cyproheptadine, miancerin,

and methysergide. The correlation study showed that the

binding affinities of all 5-HT2 antagonists in the rabbit

cerebral cortex and rat frontal cortex membranes had a good

relationship with their inhibitory potency against the

contraction response in vascular smooth muscle. In contrast,

the binding affinities of 5-HT2 antagonists in rabbit platelets

do not correlate with their vascular functional potency. It

was suggested that the variations of these relationships in

rabbit platelets might be due to the selectivity of 5-HT2

antagonists for 5-HT2 receptor subtypes, and the character-

istics of 5-HT2 receptors in the membranes of rabbit cerebral

cortex, rat frontal cortex, rabbit platelets, and vascular

smooth muscle. In addition, these variations might also be

due to the nonspecific binding sites and chemical structures

of the antagonists.

7.8. Binding sites of 5-hydroxytryptamine2R family

with sarpogrelate assessed by molecular modeling study

5-HT receptors belong to the gene super family of

GPCR, with the exception of the 5-HT3 receptor class,

which is a ligand-gated ion channel (Maricq et al., 1991).

GPCR are characterized by seven transmembrane segments,

an extracellular amino-terminus and a cytoplasmic carboxy-

terminus (Ballesteros & Weinstein, 1995). 5-HT2R family

members share similar intron-exon distribution, high pri-

mary sequence homology (68–79% in the transmembrane

segments), and pharmacological properties, and all stimulate

phospholipase C activity, leading to increased phosphoino-

sitide hydrolysis in the cell (Baxter et al., 1995). Using

molecular modeling techniques, we clearly demonstrated

the specificity of the binding sites and selectivity of

sarpogrelate for human 5-HT2A versus 5-HT2B and 5-

HT2C receptor subtypes.

Our recent molecular modeling (Rashid et al., 2003)

investigation revealed that molecular dynamics (MD)

simulations predict the strongest interaction for the 5-

HT2AR/sarpogrelate complex. Upon binding, sarpogrelate

constrained an aromatic residues’ network (Trp3.28, Phe5.47,

Trp6.48, Phe6.51, Phe6.52 in 5-HT2AR; Phe3.35, Phe6.51, Trp7.40

in 5-HT2BR; Trp3.28, Phe3.35, Phe5.47, Trp6.48, Phe6.51,

Phe6.52 in 5-HT2CR) in a stacked configuration, preventing

activation of the receptor (Figs. 3 and 4). The model

suggested that the structural basis of the selectivity of

sarpogrelate for 5-HT2AR versus both 5-HT2BR and 5-

HT2CR was based on the following: (1) In the 5-HT2AR, the

number of electrostatic interactions established with sarpog-

relate was higher than in the two other subtypes (5-HT2BR

and 5-HT2CR). A tight interaction was formed between the

Fig. 3. Stereo view of the binding site of sarpogrelate to 5-HT2AR (a), 5-HT2BR (b), and 5-HT2CR (c) receptor; view orthogonal to the membrane plane from

the extracellular side of the cell.


antagonist and the transmembrane domain (TMD) 3. Asp3.32

neutralized the cationic head of sarpogrelate, which allowed

the antagonist molecule to prevent the fixation and the action

of agonist molecules, and interacted simultaneously with the

carboxylic group hydrogen of sarpogrelate. Additional

contacts were made with Trp3.28 and Trp6.48. (2) Due to

steric hindrance, Ser5.46 (vs. Ala5.46 in 5HT2B and 5HT2C),

prevented sarpogrelate from entering deeply inside the

hydrophobic core of the helix bundle and interacting with

Pro5.50. (3) The side chain of Ile4.56 (vs. Ile4.56 in 5HT2BR

and Val4.56 in 5HT2CR) constrained sarpogrelate to adjust its

position by moving toward the strongly attractive Asp3.32.

In 5-HT2BR and 5-HT2CR, sarpogrelate interacted with

the acidic moiety of Asp3.32 (Figs. 3 and 4). Moreover, in

5-HT2CR, the carboxylate group oxygen O1 of sarpogrelate

was H-bonded with the Ser7.46 OH group. In 5-HT2AR and

5-HT2BR, interactions with Ser7.45 and Ser7.46 were not

possible due to the axial position of sarpogrelate. In

5HT2CR, sarpogrelate was deeply embedded in the helix

bundle and the methoxy group of the aromatic ring could

Fig. 4. Two-dimensional views of sarpogrelate docked to three-dimensional models of human 5-HT2A (a), 5-HT2B (b) and 5-HT2C (c) receptors. Amino acids in

ball-and-stick models are those that make electrostatic interactions with sarpogrelate. Small red lines around interacting atoms represent Van der Waals

interactions. H bonds are depicted as green lines. The numbers of amino acids indicate helix numbers and the positions of amino acids.


interact with Pro5.50. Additional contacts were made with

Ala5.46 and Phe5.47. In 5HT2BR, sarpogrelate was less

embedded than in 5-HT2CR. Interaction with Pro5.50 was

not possible since the movement of sarpogrelate was

blocked by the presence of the Thr5.49 side-chain (vs.

Ile5.49 in 5HT2AR and 5HT2CR). On the other hand, it was

observed that sarpogrelate displayed higher binding affinity

for human cloned 5-HT2A subtype than 5-HT2B and 5-

HT2C subtypes. The modeling results were in good

agreement with the binding affinities of sarpogrelate for

human 5-HT2 receptor family members expressed in

transfected cells.

In conclusion, sarpogrelate, a novel 5-HT2A antagonist,

has several unique properties. It has high binding affinity for

5-HT2A receptor with lack of inhibition against 5-HT1, 5-

HT3, 5-HT4, and other receptors, and rapidly dissociates

from 5-HT2 receptor sites. It inhibits platelet aggregation

and thrombus formation, inhibits vasoconstriction and

vasospasm, while it has a very weak effect on vaso-

relaxation and it may not be useful for controlling systolic

blood pressure. Recent advances in molecular biology and

molecular modeling have allowed elucidation of the

structure, binding sites, and pharmacological functions of

5-HT2 receptors. We identified possible interaction sites

between sarpogrelate and 5-HT2 receptor subtypes, and also

evaluated the precise selectivity of sarpogrelate for 5-HT2A

receptor subtype compared with 5-HT2B and 5-HT2C

subtypes. The data reviewed here suggest that sarpogrelate

is a 5-HT2A-selective antagonist and is likely to have

pharmacological effects beneficial in the treatment of

cardiovascular diseases.

8. Clinical significance

of 5-hydroxytryptamine2A receptor and its antagonists

5-HT has important effects on cardiovascular function. It

stimulates platelet aggregation and has a prothrombic effect.

It potentiates platelet aggregation in the presence of other

agonists, such as collagen, ADP, epinephrine, and thrombin

(De Clerk et al., 1982; Holmsen, 1985). 5-HT is also known

to be a strong vasoconstrictor at sites of endothelial injury

(Van Nueten et al., 1981). Therefore, during thrombus

formation, platelet-derived 5-HT plays an important role in

creating a positive feedback cycle on further platelet

aggregation followed by release of vasoconstrictors, such

as thromboxane A2. Increased plasma levels can also be

observed during thrombus formation (Wester et al., 1992).

Moreover, elevated plasma 5-HT is associated with coro-

nary artery disease and cardiac events (Golino et al., 1994;


Viekenes et al., 1999). All of these effects are mediated by

5-HT2A receptors on platelet and vascular smooth muscle

(Roth et al., 1998). 5-HT2A receptors are also involved in

migration and cell proliferation (Tamura et al., 1997;

Sharma et al., 1999). A drug that can suppress these

pathological processes would therefore be of therapeutic

value.

Ketanserin is a selective 5-HT2A antagonist with vaso-

dilator properties in the systemic and pulmonary circulation.

It decreases systolic and diastolic blood pressure in patients

with acute and chronic hypertension. Oral ketanserin 40 mg

twice daily is effective for the treatment of hypertension.

Dizziness, tiredness, edema, dry mouth, and weight gain are

the most commonly reported side effects (Brogden &

Sorkin, 1990). Ketanserin also effectively lowers the blood

pressure in the treatment of severe hypertension during

pregnancy both intravenously (Steyn & Odendaal, 2000)

and orally (Steyn & Odendaal, 2001), but the optimal

dosage has not been established. Intracoronary administra-

tion of ketanserin increases coronary collateral flow and

decreases myocardial ischemia during balloon angioplasty.

This could be clinically useful in the management of acute

ischemic syndromes (Kyriakides et al., 1999). However,

despite its effectiveness in the treatment of severe hyper-

tension, it has been withdrawn due to its tendency to cause

proarrythmia.

Sarpogrelate has been introduced clinically as a ther-

apeutic agent for the treatment of ischemic diseases

associated with thrombosis (Ito & Notsu, 1991). Sarpogre-

late is used to improve ischemic symptoms including ulcer,

pain, and the cold sensation resulting from chronic arterial

occlusion (Moro et al., 1997). Several reports have

supported the therapeutic usefulness of sarpogrelate. Kinu-

gawa et al. (2002) investigated the effectiveness of 2-week

treatment with sarpogrelate administered orally (100 mg 3

times a day) on anginal symptoms and exercise capacity in

anginal patients. Their findings indicated the therapeutic

effectiveness of sarpogrelate for anginal patients, especially

for patients with well-developed collaterals. Tanaka et al.

(1998) investigated whether orally administered sarpogre-

late (200 mg daily) improved exercise capacity as a result of

augmented collateral circulation in patients with effort

angina. They reported that sarpogrelate augmented flow

reserve of the collateral circulation and improved exercise

capacity in anginal patients with well-developed collaterals.

Their findings indicated that sarpogrelate is useful not only

as an antiplatelet drug, but also as an antianginal drug. Usui

et al. (2000) evaluated the clinical efficacy of sarpogrelate to

reduce the intravascular hemolysis problems suffered

frequently by patients implanted with heart valve pros-

theses. Sarpogrelate was given daily, 100 mg orally for the

first 6 months and 200 mg thereafter. They reported that

sarpogrelate is a useful drug for patients with implanted

heart valve prostheses and resultant high serum lactate

dehydrogenase because it works as an antiplatelet drug and

reduces mechanical hemolysis. Kato et al. (2000) examined

the effect of 12 months of 300 mg oral sarpogrelate

hydrochloride once daily on the symptoms of Raynaud’s

phenomenon, respiratory failure, and cardiac function in

seven patients with systemic sclerosis. After 2 and 12

months of sarpogrelate administration, a significant decrease

was found in the frequency and duration of Raynaud’s

phenomenon, as well as the coldness, numbness, and pain of

Raynaud’s phenomenon, respiratory failure, and right

ventricular failure.

9. Conclusion

This review describes the potential involvement of 5-

HT2A receptors in mediating many cardiovascular processes

and diseases, including the contraction of vascular smooth

muscle, platelet aggregation, thrombus formation, and

coronary artery spasm. Sarpogrelate is a more selective

and specific 5-HT2A receptor antagonist than other 5-HT2A

antagonists and is an excellent drug for the treatment of

peripheral vascular disease. Molecular modeling studies

have provided the first structural hypothesis about the 5-

HT2A receptor selectivity of sarpogrelate, which may be

further examined by site-directed mutagenesis experiments

and radioligand binding assays in the future. Important

determinants for selective binding appear to involve mostly

Trp3.28, Asp3.32, Ile4.56, and Ser5.46. These results will be

useful for research into improving 5-HT2AR versus 5-

HT2BR/5-HT2CR selectivity by modifying the chemical

structure of sarpogrelate. This review has also highlighted

some fascinating new insights into 5-HT2A receptor signal-

ing and molecular findings that have resulted from several

lines of investigation over the last few years.

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Functions of 5HT 2A receptor and its antagonists in the cardiovascular system

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