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
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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
2.2. 5-Hydroxytryptamine2 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.3. 5-Hydroxytryptamine3 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.4. 5-Hydroxytryptamine4 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.5. 5-Hydroxytryptamine5 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.6. 5-Hydroxytryptamine6 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.7. 5-Hydroxytryptamine7 receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
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).
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8162
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).
2.2. 5-Hydroxytryptamine2 receptors
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.
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 63
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).
2.3. 5-Hydroxytryptamine3 receptors
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).
2.4. 5-Hydroxytryptamine4 receptors
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).
2.5. 5-Hydroxytryptamine5 receptors
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).
2.6. 5-Hydroxytryptamine6 receptors
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).
2.7. 5-Hydroxytryptamine7 receptors
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-
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8164
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).
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 65
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-
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8166
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
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 67
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
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8168
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
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 69
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.
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8170
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
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 71
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.
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8172
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.
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–81 73
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;
T. Nagatomo et al. / Pharmacology & Therapeutics 104 (2004) 59–8174
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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