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Cellular and Molecular Neurobiology, Vol. 15, No. 5, 1995
Molecular Neurobiology and Pharmacology of the Vasopressin/Oxytocin Receptor Family
J. Peter H. Burbach, t'6 Roger A. H. Adan, 1 Stephen J. Lolait, 2 Fred W. van Leeuwen, 3 Eva Mezey, 4 Miklos Palkovits, 2 and Claude Barberis s
Received March t, 1995; accepted April 1, 1995
KEY WORDS: vasopressin; oxytocin: G-protein coupled receptors.
SUMMARY
1. VP and OT mediate their wealth of effects via 4 receptor subtypes V~a, V~b, V2, and OT receptors.
2. We here review recent insights in the pharmacological properties, structure activity relationships, species differences in ligand specificity, expression patterns, and signal transduction of VP/OT receptor.
3. Furthermore, the existence of additional VP/OT receptor subtypes is discussed.
INTRODUCTION
Physiological research at the beginning of this century had indicated the existence of substances in the neurohypophysis causing vasoconstriction and "rapid birth" in animal models. On the guidance of these biological activities two nonapeptides
~Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
2 Laboratory of Cell Biology National Institute of Mental Health, Bethesda, MD 20892. 3 Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. 4 Clinical Neuroscience Branch, National Institute of Neurological Diseases and Stroke, Bethesda,
MD 20892. 5 Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier
Cedoc 5, France. 6 To whom correspondence should be addressed.
573 0272.434019511000.0573507.5010 ~ 1995 Plenum Publishing Corporation
574 Burbach et aL
were the first peptide hormones to be isolated and chemically identified and termed vasopressin and oxytocin (d~K~'~ = rapid, rdKo~ = birth). Nowadays we know that vasopressin (VP) and oxytocin (OT) are the major peptide hormones produced in the hypothalamo-neurohypophysial system. Released in the blood they serve hormonal functions in peripheral organs. VP is the antidiuretic hormone acting on the kidney. VP also acts on the vascular system, has ACTH-releasing activity on pituitary corticotrophs and has mitogenic and metabolic activities. OT is primarily involved in reproductive functions in females. It has uterotonic and milk-ejecting activities and it stimulates the release and synthesis of prolactin in the anterior pituitary. Despite the structural relatedness VP and OT generally serve separate hormonal functions in the periphery. Four receptor subtypes have been identified that mediate these biological effects. There are three receptors selectively binding VP divided in the V~-type receptors, coupling to the protein kinase C pathway and the V2 receptor coupling to the protein kinase A pathway (Jard et al., 1988). The V~a receptor is responsible for the classical vasopressor activity: it is present on vascular epithelium and also in the liver. The V~b receptor has originally been found on pituitary corticotrophs, mediating the effect of VP on ACTH release. The V2 receptor mediates the antidiuretic action of VP on the kidney. The OT receptor is expressed in the uterus, mammary gland, and pituitary lactotrophs. The OT receptor can also bind VP with appreciable affinity and may therefore be considered as a nonselective VP receptor.
Neuronal fiber systems originating from hypothalamic cell groups innervate the nervous system and deliver VP and OT in several brain regions. VP and OT have many actions in the CNS. They play a role in the control of adaptive, social, and sexual behaviors and regulative body temperature and blood pressure (De Wied et al., 1993; Kov~ics and De Wied, 1994). In the CNS several of the known VP/OT receptors are expressed, but the distinction of biological activities of VP and OT is less clear in the brain than in the periphery. A number of central functions can be exerted by both peptides, sometimes resulting in opposite effects. Furthermore, in the CNS VP and OT are metabolized to hexa- and pentapeptides with more selective and potent effects than the nonapeptides. This chapter addresses the molecular biology and pharmacology of the known VP/OT receptors and discusses the existence of addition subtypes in the CNS.
THE VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY: ARCHITECTURE AND SIGNAL TRANSDUCTION
In the last few years, receptors of most neuropeptides have been identified by molecular cloning. Almost all of them belong to the superfamily of G-protein coupled receptors, showing the typical architecture of severe transmembrane (TM) domains. Within this superfamily, the four known VP/OT receptors
Vasopressin/Oxytocin Receptor Family 575
extracellular
cytoplasmic ~tial itoylation
Fig. 1. Schematic diagram of the rat Via AVP receptor showing it is similarity to the rat Vlh and V 2 receptors, and the rat OT receptor. Shaded circles represent amino acids that are identical between the four receptors; open circles represent amino acids that are not identical. The seven putative transmembrane (TM) domains are marked with Roman numerals I-VII, and are based on the hydropathy profiles and sequence comparison with other G protein-coupled receptors. Note that most amino acid identify is in the TM domains (particularly TMs II and VI) and the second and third extracellular domains between TMs II and 1II, and TMs VI and V, respectively. The NH2- and COOH-terminals, as well as the cytoplasmic domains are quite divergent. The AVP and OT receptors probably have a disulphide bridge between cysteine residues present in the second and third extracellular domains (Cys TM and Cys 1'~5 in the rat V1~ receptor). Potential palmitoylation sites (Cys 37° and Cys 371 in the rat V~a receptor) which may anchor the receptors in the plasma membrane (Dohlman et aL, 1991) are conserved among all the AVP/OT receptors. The sequences were aligned using the Bestfit and Gap programs (Genetics Computer Groups (GCG) software (University of Wisconsin); maximum alignment with the Via receptor was facilitated by introducing gaps in the sequences.
constitute a subclass (Figs. 1 and 2). Thus far, four members of the VP/OT receptor family have been cloned. These are the OT (Kimura et at., 1992), the V~ (Morel et al., 1992; Thibonnier et al., 1994), the V~b (Sugimoto et al., 1994a,b; de Keyzer et al., 1994; Lolait et al., 1995), and the V2 receptors (Birnbaumer et al., 1992; Lolait et al., 1992). The primary structures of several V~, V~b, V2, and OT receptors of several other species including lower vertebrates as fish and snail are now available. Their relatedness resides predominantly in the TMs and the extracellular loops 1 and 2 (Fig. 2).
VP and OT receptors have been classified on the basis of (i) the second messenger system coupled to the receptors, and (ii) the affinity of a series of VP and OT analogues with enhanced selectivity for a certain receptor type. The V2 receptor couples to the cAMP signal transduction pathway (Birnbaumer et al., 1992; Lolait et al., 1992). The Vta, the Vlb, and the OT receptors all couple to the PI/Ca ÷÷ signal transduction pathway (Thibonnier et al., 1994; de Keyzer et al.,
576 Burbach et aL
A V 2 human
I V 2 rat
V 2 pig V~o human
_ ~ ..... [ Vlo rat v-r fish OT human
t OT pig OT rat Vlb human CP-1 snail CP-2 snail
Fig, 2, Structural relations within the VP/OT re- ceptor family. Panel A shows the alignment of primary structures of several cloned VP/OT recep- tors in the central portion that spans transmembrane domains 1-7. Panel B shows the degree of similarity in a dendogram. Alignments were produced using the Pileup programs of the GCG software package (see also the legend to Fig. 1); sequences were from Morel et al. 1992; Lolait et aL, 1992, t995; Kimura et at., 1992; Rozen et aL, 1995; Gorbulev et al., 1993; Thibonnier et al., 1994; Mahlmann et al., 1994).
1994; Kimura et al., 1992). Activation of additional signal transduction pathways has been observed. Stimulation of VI~ receptors in Avr5 cells stimulated multiple signal transduction pathways: phospholipases A2, C and D were activated (Thibonnier et al., 1991). Furthermore, there is evidence that activation of V1, receptors rapidly stimulates tyrosine phosphorylation. In Swiss 3T3 cells VP stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in a Ca ++ and PKC independent manner (Zachary et al., 1993a,b). In cultured rat smooth muscle cells VP stimulated the MAP kinase signal transduction pathway which was only partially mediated in a Gi and PKC dependent manner (Granot et al., 1993). The signal transduction pathways activated by the Vlb and OT receptors have not been studied as extensively as those of the VI~ receptor. As for other G-protein-coupled receptor families, homologies within the VP/OT recep- tor family do not provide clues to the type of G-proteins that couple to the different members.
PHARMACOLOGY OF VASOPRESSIN AND OXYTOCIN RECEPTORS
Radiolabeled ligands have been so far the best tools for identification, pharmacological characterization, and localization of VP and OT receptors, in particular in the CNS. With respect to the localizational additional new tools have been developed recently. They include probes for in situ hybridization of transcripts and antisera for immunocytochemistry of the receptor proteins (see below).
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578 Burbach et al.
Tritiated oxytocin [3H]OT, arginine vasopressin [3H]VP and lysine vasopres- sin [3H]LVP have allowed the characterization of VP and OT receptors in a great number of tissues (Jard et aL, 1988). It must be pointed out that [3H]VP, [3H]LVP and [3H]OT are not as highly selective ligands for VP and OT receptors respectively as commonly assumed. Thus, [3H]VP has an affinity for V~ receptors in the rat which is only 2- to 5-fotd higher than its affinity for the OT receptor in the same species (see Table I). In the rat, [3H]LVP binds to the OT receptor with a higher affinity than to VP receptors.
More specific analogues have been tritiated, like the OT receptor agonist [4-threonine, 7-glycine]-oxytocin ([Thr4-GlyT]OT) (Elands et al., 1988a), the V2 receptor agonist [1-deamino, 8-D-arginine]vasopressin (dDVP) (Marchingo et al., 1988), and the antagonists d(CH2)5[Tyr(Me2)]VP (Via receptor selective) (Van Leeuwen et al., 1987; Dorsa et al., 1988; Cornett and Cate, 1989), desGly-NH~ d(CH2)5 [D-Ile 2, Ile4]VP (Vz receptor selective) (Trinder et al., 1991). The new potent and selective nonpeptide vasopressin V~ antagonist, Sr 49059 was also tritiated (Serradeil-Le Gal et al., 1994; see Table I).
There is a general agreement that radioiodinated VP and OT are not useful as labeled ligands for VP and OT receptors; but several iodonated antagonists are useful tools. The OT receptor antagonist [125I]-d(CHz)5[Tyr(Me)2,Thr4,Tyr - NHg]OVT (Elands et al., 1988b) has a high affinity and selectivity for OT receptors. It is a convenient tool for autoradiographic localization of OT receptors in the brain and kidney (Tribollet, 1992). Several V3a antagonists have also been radioiodinated such as d(CH2)5[SarT]Vp (Kelly et al., 1989; Gersteber- ger and Fahrenholz, 1989), Phaa-D-Tyr(Me)Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (Linear VP antagonist) (Schmidt et al., 1991), and HO-Phaa-D-Tyr(Me)-Phe-Gln- Asn-Arg-Pro-Arg-NH2 (HO-Linear VP antagonist) (Barberis et al., 1995). This last ligand has the highest affinity and selectivity for Via receptor reported to date, rendering it a highly sensitive tool for the localization and characterization studies of V~ receptors present in the brain, liver, and blood vessel walls (Barberis et al., 1995).
A huge amount of pharmacological data on the biological actions of VP and OT and on their receptors have been published. The very large spectrum of responses elicited by VP and OT and the widespread distribution of their receptors explain at least partly this profusion of pharmacological data. A more obvious reason to explain this profusion is that hundreds of VP and OT structural analogues have been designed and produced by several active groups of organic chemists and pharmacologists. Among these, Drs. M. Mannning and W. H. Sawyer's groups deserve a special mention. By generously distributing VP and OT analogues to interested laboratories all over the world, Manning and Sawyer have a most eminent role in the development of our knowledge on the pharmacology of VP and OT receptors and actions.
In the rat, the pharmacological characterization of VP receptors revealed striking similarities in the ligand setectivities of V~ receptors from all tissues tested except the adenohypophysis. Both the observed rank order of potencies and absolute values of the affinity constants determined on acellular preparations were similar. In particular, combined pharmacological and autoradiographical
Tab
le I
. R
adio
labe
lled
Pro
bes
for
Vas
opre
ssin
and
Oxy
toci
n R
ecep
tors
< O ,q
Dis
soci
atia
tion
C
onst
ants
(n
M)
for
Vt,
(li
ver)
re
cept
ors
V,,
fr
om V
2
(hyp
ophy
sis)
(k
idn
ey)
Bin
ding
to
OT
(u
teru
s)
Ago
nist
(A
) an
tago
nist
(A
N)
©
-i
[3H
I-L
VP
8"
["
HIA
VP
0.
6-3'
[:
~HI-
OT
78
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aHl.
['rh
r',G
ly7l
OT
>8
000
~ [!
H]-
dD
AV
P
t00'
H
]-d(
CH
.)s-
[Tyr
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P
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ecep
tor
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uman
ute
rus
OT
rec
epto
r ex
pres
sed
in C
os7
celt
s.
h Ja
rd e
t al
., 19
86.
'" B
arbe
ris
et a
l. (i
n pr
ess)
, a
Can
tau
et a
l.,
1980
. "
Man
ning
and
Saw
yer,
199
3,
[ E
land
s et
aL,
19
88.
Gai
llar
d et
al.
, 19
84.
a A
udig
ier
and
Bar
beri
s, 1
985.
'
But
len
et a
L,
1978
. t
Raj
eris
on e
t al
..
1974
.
~' S
tass
cn e
t al
., 19
85.
t S
chw
artz
et
al..
1991
. "K
iral
y et
al.
, 19
86.
" W
illi
ams
et a
l.,
1994
. "
Sug
imot
o et
al.
, 19
94.
P S
erra
deiI
-Le
Gal
et
al.,
1993
. '~
Yam
amur
a et
al.
. 19
92.
• Y
amam
ura
et a
l.,
1991
. '
Ant
oni,
198
4.
, S
chlo
sscr
et
aL,
1994
.
ol
,.,.
I
580 Burbach et al.
studies on all brain areas where specific vasopressin binding sites could be detected failed to reveal regional differences in the ligand selectivity of central receptors (Tribotlet et al., 1988). All potent antagonists of the vasopressor response (except dP[Tyr(Me)2]Vp) which bind with high affinity to V~ receptors in a large number of tissues, exhibit lower affinity for adenohypophysial receptors while a good correspondence is found for peptides with agonistic properties (Table II). This was the basis for the conclusion that adenohypophysial receptors are different from other VI receptors and to the proposal that adenohypophysial receptors should be named V~b receptors as opposed to an apparently common type of receptor (V1~ receptors) expressed in other tissues (Jard et al., 1986). Recently the V~b receptor has also been denoted as V3 (De Keyzer et al., 1994), but this nomenclature will not be used here. It is of interest to note that for peptide hormone and neuropeptide receptors it was the first example in which subtypes should be distinguished by the use of specific antagonists as it is the general case for receptors for neurotransmitters.
The presence of V2 receptor seems to be confided to the kidney. The only extrarenal tissue in which V2 vasopressin receptors were identified, through the presence of a vasopressin-sensitive adenylate cyclase is the porcine seminal vesicle (Maggi et al., 1986). Molecular studies underscore the restricted expression of the V2 receptor (Lolait et al., 1995). The possible existence of several subtypes of V2 receptors in mammals is still an open question. In particular, a V2 receptor subtype has been suggested to exist in limbic brain areas (see below.)
STRUCTURE-ACTIVITY RELATIONSHIPS
Below the structure-activity relationships for binding of VP or OT analogues to VP/OT receptors is outlined and the structural analogues exhibiting the highest selectivity for the different subtypes of VP and OT receptors present on kidney membranes (V2 receptors), liver membranes (VI~ receptors), hypophysial membranes (Vlb receptors), and either uterine or mammary gland membranes (OT receptors) are described.
As apparent from Table II, the most selective VIa/V2 analogues tested so far are peptides with V1 antagonistic/V2 agonistic properties such as desGly 9- d(CH2)sVP. The HO-Linear VP antagonist is also very potent and selective for V1~ receptor. The nonpeptide antagonist OPC 21268 developed by Yamamura et al. (1991) is orally active and is much more selective for the vascular V1~ receptor than for the V2 receptor. Another nonpeptide Via antagonist SR 49059, developed by Serradeil-Le Gal et aL (1993) is also very potent, selective, and orally active. No highly selective, V~JV2 agonist is presently available.
Conversely, the most V2/V~ selective ligands are the highly potent anti- duiretic peptides of the DVP and VVP series. Several peptide V1a/V2 antagonists also discriminate between these two receptor subtypes although less efficiently than agonists of the DVP and W P series. By modifying the structure of the nonpeptide V~ vascular antagonist OPC 21268 a nonpeptide V2 antagonist was
Ta
ble
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Me)
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dP
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e)2
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8"
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31i
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9059
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,000
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-3P
AL
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2.
0"
220"
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Gly
%d
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2)5
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lD(T
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00"
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N
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25
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AN
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CH
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NH
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2)s
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6.
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pA
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00 q
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70
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AN
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,q 3.
° R
at l
iver
AV
P
rece
pto
r o
r h
um
an
ute
rus
OT
re
cep
tor
exp
ress
ed i
n C
os
7 ce
lls.
b
Jard
et
al.,
1986
. c
Bar
ber
is e
t aL
, (i
n pr
ess)
. d
Can
tau
et
al.,
1980
. e
Man
nin
g a
nd
Saw
yer
, 19
93.
[ E
lan
ds
et a
l.,
1988
. R
Gai
llar
d e
t aL
, 19
84.
* A
ud
igie
r an
d
Bar
ber
is,
1985
. B
utl
en e
t al
., 19
78.
i R
ajer
iso
n e
t aL
, 19
74.
k S
tass
en e
t al
.,
1985
. t
Sch
war
tz e
t al
., 19
91.
"Kir
aly
et
al.
, 19
86.
" W
illi
ams
et a
l.,
1994
. "
Su
gim
oto
et
al.,
1994
. P
Ser
rad
eiI-
Le
Gal
et
aL,
1993
. q
Yam
amu
ra
et a
l.,
1992
. •
Yam
amu
ra
et a
l.,
1991
. ~
An
ton
i,
1984
. '
Sch
loss
cr e
t aL
, 19
94.
582 Burbach et aL
obtained: OPC 31260, which is potent, selective, and orally active (Yamamura et al., 1992). This compound has no partial agonistic action in the Brattleboro rat (Yamamura et aL, 1993), as it is usually the case for the peptide antagonists in human (Kinter et al., 1991).
Highly selective ligands for Vjb receptors have not been found so far, except for 1-deamino[D-3-(3' pyridyl)Ala2]Vp (Schwartz et al., 1991). This compound seems to be a full agonist specific for the Vlb receptor, while it is a weak antagonist for the V~a vascular vasopressin receptor and a weak agonist for the Vz receptor. Thus, it may be a unique analogue for studies on the V~b receptor. Several antagonists, in particular V~a/V2 antagonists, discriminate very efficiently the V~b receptors, for which they have a low affinity, from either V~a or V2 receptors for which they have a high affinity. The most potent V~b antagonists are the peptide antagonists dP[Tyr(Me)2]VP (Antoni et al., 1984; River et aL, 1984), and HO-Linear VP antagonist (Barberis et aL, 1995). They are also potent V~a antagonists (Bankowski et aL, 1978; Manning et aL, 1992).
As for as OT receptors are concerned, OT itself can be considered as a fairly selective ligand (Table II). It must be noticed that several VP antagonists do interact with high affinity with OT receptors. Among the analogues tested [Thr4-Gly7]OT and desGly-NH9-d(CH2)5[Tyr(Me)Z-Thr4]OVT are the most OT receptor-selective ligands which have been identified so far. Intravenous ad- ministration of the OT receptor antagonist 1-deamino [D-Tyr(Et)2,Thr4,OrnS]OT (AtosibanR) provides significantly beneficial treatment of primary dysmenor- rhoea and effectively stops uterine contractions in pre-term labor (Melin, 1993). A new class of cyclic peptide OT antagonists derived from a bacterial source has also been reported (Pettibone et al., 1989) and nonpeptide antagonists of the oxytocin receptor have been developed (Williams et al., 1994).
SPECIES DIFFERENCES IN THE LIGAND SPECIFICITY
There are marked differences among mammalian species in the ligand selectivity and distribution of VP and OT receptors (see for instance Pettibone et aL, 1992). Transposition of pharmacological data obtained in the rat to other mammalian species may be hazardous and lead to erroneous interpretations. This has been exemplified by the characterization of LVP receptors present on porcine seminal vesicle membranes (Maggi et al., 1986). Based on pharmacological studies with selective analogues for rat V1 and V2 receptors, the authors concluded that LVP receptors in pig seminal vesicles might represent a new type of vasopressin receptor. However examination of these pharmacological data indicates that the observed ligand selectivity of VP receptors in pig seminal vesicles is not markedly different from that of renal adenylate cyclase-coupled Vz receptor from the same species.
There are marked differences in the ligand selectivities of the renal V2 receptors from the three mammalian species which are the most extensively studied so far: human, rat, and pig. For most of the vasopressin analogues tested the human V2 receptor has a lower affinity than the rat V2 receptor. In particular,
Vasopressin/Oxytocin Receptor Family 583
this is the case for dDVP, a highly potent agonist in the rat and for desGlygd(CH2)5[D-Tyr(Et)2,vala]Vp, one of the most potent anti-antidiuretic peptides in the rat. It should be noted that all the peptide V2 antagonists which exhibit anti-antidiuretic activity in the rat and exhibit vasopressin-induced activation of human kidney adenylate cyclase do not behave as an aquaretic agent in humans. On the contrary, the nonpeptide antagonist OPC 31260 is a pure antagonist and, when administered intravenously, it increases the excretion of hypotonic urine in humans (Yamamura et aL, 1993; Ohnishi et al., 1995).
The comparison of the ligand selectivities of V1 receptors from different tissues has not been carefully explored in other mammalian species than the rat. The pharmacological profiles of V1 receptors in human platelets and uterus are similar but not identical. Differences might also exist in the pharmacological profiles of human and rat V~ receptors. In particular, two V~ antagonists, d(CH2)sVP and its 9-des-glycyl derivative bind to rat V~a receptors with a much higher affinity than to human uterus VI~ receptors. The V~a nonpeptide antagonist OPC 21268 and the OT nonpeptide antagonists L369243 and L369255 also bind to rat VI~ receptors fvith a higher affinity. Conversely, several V2 agonists (dVDVP, (OH)VDVP) and the OT agonist [Thr4,GlyT]OT have a low affinity for rat VI~ receptors and a more than ten-fold higher affinity for human V~ receptors. The OT antagonist 1-deamino[D-Tyr(Et)2,Thr4]OVT binds with 200-fold higher affinity to human than to rat V~a receptors.
As yet there is no direct evidence for the existence of marked differences in the ligand selectivity of OT receptors in different tissues, thus for the existence of OT isoreceptors. No clear differences in the ligand selectivity of oxytocin receptors in the human, rat and sow uterus were evidenced. In particular, OT receptors in the rat hypothalamus do not appear to be different from peripheral receptors in the rat uterus, mammary gland, oviduct, and fat cells (Tribollet, 1992). It must be emphasized that a common characteristic of OT receptors is to bind VP with high affinity (Table II). Hence, the OT receptor can be considered as a "nonselective" VP receptor. The OT receptor has equal affinity for VP and OT, whereas the V~a receptor has a 30-fold higher affinity for VP than for OT. However, high doses of OT were able to stimulate contraction of rat vascular smooth muscle cells via VI, receptors (Briner et al., 1992). Vice versa occupation of OT receptors by VP may influence OT receptor activity: VP and OT have opposite effects in the extinction of a passive avoidance task in rats (De Wied et al., 1991). Since both these effects were blocked by an OT receptor antagonist, it was suggested that VP may act as an inverse agonist on the OT receptor. These data suggest that for OT receptors OT and VP both are endogenous ligands may influence OT receptor activity.
MOLECULAR BASIS OF THE PHARMACOLOGICAL PROPERTIES OF VP AND OT RECEPTORS
A new, unexplored area is the structure-function relationships of VP and OT receptors. The recent cloning of all four classical VP/OT receptors has opened
584 Burbach et al,
this field of research. For small ligand's such as catecholamines, the binding site is fully located within the plane of the membrane involving several residues of different transmembrane domains of the G-protein-coupled receptors. For larger peptide agonists, the binding pocket may not be restricted to the transmembrane regions. For example, in receptors of the neurokinin- or the angiotensin-receptor families, the extracellular domains are of crucial importance in the binding of either agonist or antagonist analogues (Schwartz, 1994). Using synthetic peptides that mimic the extracellular domains of neurohypophysial hormone receptors, Howl et al., (1995) have identified binding site determinants in the extracellular domains of the rat Via receptor which selectively recognize neurohypophysial hormones as well as peptide and nonpeptide antagonists. These binding sites are mainly located in the N-terminal domain and the first and second extracellular loops. Insight into the binding domain of VP receptors has recently come from the purification of the renal V2 receptor after labeling with a photoreactive VP agonist bearing an azido group on the lateral chain of Lys ~ (Kojro et al., 1993). This analogue reacted with two amino acids located in the first extraceltular loop of the bovine V2 receptor. The authors suggested that an interaction might occur between the side chain of residue 8 in the natural hormone and one of the three Asp residues present in the first extracellular loop of the V 2 receptor. Indeed, by changing the Asp 1°3 residue in the first extracellular loop of the bovine V2 receptor by a Tyr residue, present in the porcine V2 receptor, the same group identified this Asp 1°3 residue as being responsible for the high affinity binding of dDAVP for the renal V2 receptor (Ufer et al., 1995). This is in agreement with the three dimensional model of the VP receptor developed by Mouillac et aL (1995). In this model the side chain of Arg 8 in the hormone points toward the outisde of the transmembrane core of the receptor and might interact with receptor residues situated in the first extracellular loop. In particular, it might form a hydrogen bond with a Tyr residue at position 115 in the first extracellular loop of the V~ receptor. Using site-directed mutagenesis, Chini et aL (1995) have shown that this Tyr 115 is crucial not only for agonist high affinity binding but also for receptor selectivity. Thus, when this Tyr ~5 is replaced in the VI~ receptor by Asp or Phe (the residues occurring in the V 2 and the OT subtypes), the agonist selectivity of the VI~ receptor switches accordingly (Fig. 3). The three- dimensional model of the vasopressin V~a receptor developed by Mouillac et al. (1995) was further verified and refined by site-directed mutagenesis.
Substitution of the residues shown in Fig. 3 led to a dramatic impairment in VP binding, suggesting that VP could be completely buried into a 15-20 & deep cleft defined by the transmembrane helices of the receptor and interacting with amino acids such as G l n 214'218'311"413'620 and Lys 3°8, located within this region (see Fig. 3 for the numbering of the residues). The agonist binding cleft of the Vta receptor is therefore situated in a position equivalent to that described for the cationic neurotransmitters. Moreover, it has been proposed that the same agonist binding site is shared by all members of the VP/OT receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to VP, is not affected by the mutations. This indicates a different mode of
Vasopressin/Oxytocin Receptor Family 585
8 O
O00000000000000000000g Fig. 3. A two-dimensional diagram of the neurohypophysial hormone recep- tors. Residues suggested to be involved in the binding of the natural peptide agonists are shown in white on black (Mouillac et al., 1995). These residues are numbered in the text according to their position in the putative transmembrane regions: the left digit indicates the number of the transmembrane a-helix, the next two digits indicate the rank of the residue in this transmembrane region (e.g., Lys 308 is the 8th residue of helix 3). Tyrosine 115, shown in the black square in the first extracellular loop, has also been suggested to be involved in the receptor selectivity (Chini et al., 1995).
binding for agonists and antagonists in the VP receptor, as already shown for other peptide receptors such as the NK-1 receptor (Rosenkilde et al., 1994).
EXPRESSION OF VASOPRESSIN/OXYTOCIN RECEPTOR IN THE BRAIN
The classification and localization of VP/OT receptors in the brain is based on four lines of studies. First, studies focused on biological effects using pharmacological, behavioral, (electro)physiological neurochemical, and neuroch- emical parameters in combination with selective agonists and antagonists have
586 Burbach et al.
pointed to the existence of various subtypes of this receptor family in the rodent brain. With respect to the classical VP/OT receptors these studies generally agree on the presence of the VI~ and OT receptor subtypes in the CNS, Furthermore, several studies along these lines suggest addition subtypes (see below). Second, in vitro autoradiographic binding studies with labeled agonists and antagonists support the presence of the Via and OT receptor subtypes in the brain. Such binding studies failed to demonstrate expression of the V2 receptor. Third, the recent cloning of all four classical receptor subtypes has allowed detailed studies on the localization of receptor transcripts in the brain by in situ hybridization, and, fourth, immunocytochemistry for the receptor proteins has been developed. These studies localized Via receptor mRNA and OT receptor mRNA in the rat brain, but failed to show expression of the V2 receptor in neural tissues (Lolait et aL, 1995). Both the Via and OT receptor genes are expressed in adult rat brain, albeit at much lower levels than in peripheral tissues such as liver and uterus. The distribution of mRNAs encoding the receptors is widespread, and in many instances overlapping, e.g., the septal-hippocampal area, cortex, cerebellum, certain hypothalamic nuclei, olfactory tubercle, and nuclei and nucleus of the solitary tract (Via) (Ostrowski et al., 1992, 1994; Szot et al., 1994; Young et al., 1993; Yoshimura et aL, 1993). Binding of VP and OT in many of these brain regions correlates with effects on memory, reproductive, social, and sexual behavior, temperature regulation, and cardiovascular function (De Wied et al., 1993). However, it cannot be determined from these studies whether single neurons in the brain express both receptor genes. In situ hybridization for the V~b receptor showed expression in the majority of pituitary corticotrophes (Lolait et aL, submitted) unlike the V~ receptor gene which is expressed in a small number of cells bordering the neural lobe and in scattered cells of the anterior pituitary (Ostrowski et al., 1992). A surprising finding is the wide and abundant expression of the Vlb receptor in the adult rat brain (Fig. 4). If the V~b receptor protein is expressed in these tissues, it may point to previously undescribed functions for VP or suggest that some functions of VP ascribed to V~JOT receptors (e.g., see Meisenberg et al., 1984) may indeed be due to activation of the Vlb receptor.
Recently, antisera have been raised against synthetic peptides derived from the third intracellular loop of the rat OT receptor (Adan et al., 1995). Immunocytochemical analyses of brain and peripheral tissues showed expression of the OT receptor protein in several brain areas, expected to express the receptor, e.g., the ventromedial nucleus (Fig. 5), the bed nucleus of the stria terminalis, the ventral pallidium, and the paraventricular and supra-optic nuclei. In the latter two nuclei, the OT receptor protein likely serves as autoreceptor on oxytocinergic magnocellular neurons and is involved in the synchronized bursting activity of these cells. OT receptor protein was also found in the neural lobe of the pituitary gland. No OT receptor immunoreactivity, however, was detected in two areas with outspoken density of OT binding: the ventral hippocampus- subiculum area and the central nucleus of the amygdala. This observation may be explained by postulating an OT binding site different from the cloned rat OT receptor.
A • Z •
"~ .~ : ~1: ,
/ NTS
B
~ J . ° ,
,. -:.'.,'
CB
. -
J .
7 Chp
C CA1
..': : .. .~: . - : - ~:..: ~ : : , . .
• . ] ,
Fig. 4. Localization of V~ b receptor mRNA in 12/zM coronal sections of brain from an adult male Sprague-Dawley rat by in situ hybridization. (A, C) Dark-field photomicrographs (silver grains appear white in the negative image). (B) The brightfield image of A. In A, signal is prominent in the granule (but not Purkinje) cell layer of the cerebellum (CB) with lower amounts in the nucleus of the solitary tract (NTS) and chloroid plexus (Chp) (×25). In C, strong labeling is found in the hippocampal formation (e.g., CA1 region) and dentate gyrus (DG) (×50). Considerable variability in the brain (and pituitary) Vlb receptor mRNA levels between individual, untreated rats has been noted--the underlying basis for this has not been determined. Vlb receptor mRNA was detected using a 3sS-labeled antisense RNA probe (a 618-base pair fragment of the Vlb receptor cDNA, extending from TMIII to TMVII). Exposure against Hyperfilm/3max (Amersham) was 21 days at room temperature. The sense RNA probe showed negligible, background labeling.
588 Burbach et aL
I I I "
Fig. 5. Immunocytochemical staining of OT receptors in a 50-~m thick transversal vibratome section of a lactating rat containing the ventromedial hypothalamic nucleus (VMH; staining with OT-Rec, an antibody raised against the oc- tapeptide, GRAALARV, present in the third intracellular loop of the rat OT receptor). Note that the VMH and the immediate surroundings are clearly stained, whereas the remaining hypothalamic structures are negative, tli = third ventricle. Bar = 200 ~m.
RECEPTORS FOR METABOLITES OF VP AND OT
A number of pharmacological observations have suggested the existence of VP/OT receptor subtypes beyond the four classical members. Most of these observations concern effects of agonists and antagonists that deviate in phar- macological profile from the ones of the classical VI~, VID, V2, and OT receptors. Perhaps the most intriguing and less understood findings concern the biologically active metabolites of VP and OT: [pGlu4,Cyt6]Vp 4-9 (~hort VP 4-9) and [pGtu4,Cyrr]OT 4-9 (short OT 4-9). These peptides were characterized as major metabolites of VP and Ot incubated with brain membranes in vitro (Burbach et al., 1983a). VP 4-9 also exists as endogenous VP metabolite at low levels in brain
Vasopressin/Oxytocin Receptor Family 589
tissue and is rapidly formed after local or ICV injection of VP in the brain (Burbach et aL, 1984; Stark et al., 1989). VP 4-9 and its derivative [pGlua,Cyr6]Vp 4-8 (short VP 4-8) share effects with VP, but are more restricted in activity. Like VP, VP 4-9 facilitates avoidance behaviors but is active in doses that are 10- to 1000-fold smaller than VP (Burbach et al., 1983b; De Wied et al., 1984, 1987). VP metabolites (VP 4-9 or VP 4-8) display central activities at other levels as well. Electrophysiological studies showed that VP 4-9/4-8 potentiates synaptic trans- mission. In femtomolar concentration VP 4-9/4-8 caused a long-lasting enhance- ment of glutamate-mediated transmission in the lateral septum and ventral hippocampus, and it decreased the threshold of hippocampus and lateral septum neurons to synaptic excitatory input (Rong et al., 1993; Urban and Killian, 1990). Several cellular responses can be elicited by VP 4-9/4/8. VP metabolites enhanced IP turnover in hippocampal slices and primary cultures (Gu and Du, 1993) and could stimulate the accumulation of cAMP in hippocampal cultures (Diaz-Brinton et al., 1993). Cellular effects also include induction of immediate- early gene expression after in vitro treatment. C-los and c-src were induced in distinct hippocampal neurons after icv administration of VP 4-9/4-8 (Giri et al., 1990; Gu and Du, 1991). VP 4-8 also affected the phosphorytation of the neural phosphoprotein B50/GAP43 in infant rats (Chen et al., 1993). The activities of VP and OT metabolites are more restricted than that of the nonapeptides themselves. VP 4-9 lacks several central activities of VP, like the hypothermic effects. VP 4-9 does not seem to interact with the classical receptors. It is not able to displace labeled VP from the Via and V2 receptors and does not display a potent intrinsic activity on the Via, V~b, and V2 receptors expressed in cell lines (Adan, in press; Lolait et al., in press). These properties are in agreement with the lack of any vasopressor, antidiuretic, uterotonic and ACTH-releasing ac- tivities of VP 4-9 in bioassays (Burbach, 1989). Furthermore, autoradiographic binding studies on brain slices have revealed binding sites for 35S-labeled VP 4-9 that were anatomically distinct from the classical VP/OT receptors (Brinton et al., 1986; De Kloet et aL, 1985; Jurzak et aL, 1994). VP 4-9 binding was pre- dominantly associated with circumventricular organs. Moreover, sites with a similar localization were stained with an anti-idiotypic VP-antibody, suggesting that it could recognize the VP 4-9 binding sites (Jurzak et al., 1994). As yet, there is no evidence that these binding sites confer a biological response of VP 4-9, hence that they are true receptors. These results seem to disagree with a recent report that indicates binding of [355]VP 4-8 to pyramidal and granule cells of the hippocampus ( D u e t al., 1994). Recently, in vitro binding of VP 4-8 to brain synaptosomal membranes has been described ( D u e t al., 1994). Binding was observed to preparations of all investigated brain regions. The Ka was in the order of 3 nM and binding was independent of the divalent cations Ni :÷ or Mg 2÷. The highest specific binding was found in the amygdala and hypothalamus and the lowest in the cerebellum. VP was not able to displace the 35S-labeled tracer. It remains to be clarified if these binding sites relate to the biological effects of metabolites of VP.
The OT metabolite OT 4-9 has been studied less, but from the limited data available it is apparent that it is very similar to VP 4-9 with respect to biological
590 Burbach et al.
properties. OT 4-9 is active in avoidance paradigms and more potent than OT. It lacks classical endocrine activities (Burbach et al., 1983c). The behavioral activities of OT 4-9, however, are opposite to those of VP 4-9, but both the effects of OT 4-9 and VP-4-9 can be antagonized most efficiently by the OT receptor antagonist d(CH2).~[Tyr(Me)2,Thr4,Tyr-NH9]OVT and less efficiently by V~a and V~ antagonists (De Wied et at., 1990).
Taken together, the available binding data on VP metabolites indicate that binding sites exists for these peptides that are not recognized by VP or OT and conversely, that VP metabolites do not bind to classical VP/OT receptors. Also the anatomical distribution displays distinction, although data are not yet conclusive at this point. Binding of VP 4-8 has indeed been observed in the hippocampus in one study (Du et al., 1994). Moreover, the recently cloned V~b receptor is expressed widely in the brain and its expression pattern overlaps all VP 4-9/4-8 binding sites. The neurochemical data showing changes in 2nd messenger pathways indicate that the VP 4-9 receptor belongs to the superfamily of G-protein-coupled receptors. Furthermore, behavioral pharmacological data suggest a structural relationship between the VP 4-9 receptor and the known members of the VP/OT receptor family. In particular, the antagonism by an OT receptor antagonist and N~-acetyl-VP points to this relationship. It remains to be determined if the demonstrated binding sites represent true biological receptors. The cloning of the VP 4-9 receptor will eventually solve urgent questions on the receptor identitv and function.
Additional VP/OT receptor subtypes have been suggested to exist in the brain. Effects of VP analogues (on cAMP accumulation) in hippocampal cultures have been interpreted as evidence for a cAMP-coupled VP receptor with a Vl-like pharmacological profile, termed "V2b" (Diaz-Brinton et al., 1993). A novel receptor subtype has been proposed from an electrophysiological study involving vasotocin (Ingrahar et al., 1994). Behavioral studies have also suggested a "vasotocin-like" receptor in the brain, that is responsible for the effects of VP and OT on memory-related processes (De Wied et al., 1991). The latter was based on the most efficient blockade of behavioral effects of VP and OT by the OT receptor antagonist. This could be explained by the pharmacology of the OT receptor that binds VP almost equally well as OT. Only one OT receptor has been demonstrated as yet by molecular cloning. However, the expression of this receptor protein could not be demonstrated in all brain areas in particular the subiculum and amygdala that display abundant binding of OT ligands. Thus, an additional form of the OT receptor may still exist, that perhaps is related to the behavioral effects of OT and VP in these limbic brain areas.
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