I. INTRODUCTION

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The neurohypophysial hormone oxytocin (OT) was the first peptide hormone to have its structure determined and the first to be chemically synthesized in biologically active form (143). It is named after the "quick birth" ( = quick; oo = birth) which it causes due to its uterotonic activity (124). OT was also found to be responsible for the milk-ejecting activity of the posterior pituitary gland (428). The structure of the OT gene was elucidated in 1984 (270), and the sequence of the OT receptor was reported in 1992 (299).

OT is a very abundant neuropeptide. This became obvious in a study where the most prevalent hypothalamic-specific mRNAs were analyzed. OT was found to be the most abundant of 43 transcripts identified (202). Today, we recognize that OT exerts a wide spectrum of central and peripheral effects. The actions of OT range from the modulation of neuroendocrine reflexes to the establishment of complex social and bonding behaviors related to the reproduction and care of the offspring. Overall, the cyclic nonapeptide OT and its structurally related peptides facilitate the reproduction in all vertebrates at several levels.

In this review, we summarize the present knowledge of the OT receptor system gained in the different fields of research, thereby focusing mainly on the work over the past decade. For more details to the different topics, the reader is referred to many excellent reviews that have been recently published (1, 42, 43, 155, 160, 161, 261, 265, 266, 295, 298, 335, 369, 389, 413, 443, 572, 573, 621). In the following two sections, we describe the structural features of the OT receptor system on the molecular level. OT has long been considered to be restricted to stimulation of uterine contractions during labor and milk ejection during lactation. These classical functions of the OT system are treated in the first parts of section IV. The fact that OT is found in equivalent concentrations in the neurohypophysis and plasma of both sexes suggests that OT has further physiological functions. The expression of OT and its receptor has now been identified in a variety of peripheral tissues. For example, evidence was provided that OT acts in concert with atrial natriuretic peptide (ANP) in the control of body fluid and in cardiovascular homeostasis in the rat. It is important to note that most of our knowledge about functions of OT derives from studies with rats. However, localization and expression patterns of OT receptors show marked species differences, suggesting that some of the described OT activities may be species specific. Over the past decade, particularly the central actions of OT have been intensively studied revealing a profound regulation by steroids. The regulation by steroids is in fact a common theme throughout the review and is probably the most remarkable feature of the OT receptor system. Finally, in the last section, we summarize the contributions that have been reported on behavioral effects mediated or modulated by OT. Ironically, the classical "oxytocic" function of OT is again open for discussion due to the results with OT-deficient mice.

	II. OXYTOCIN AND OXYTOCIN-LIKE PEPTIDES

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All neurohypophysial hormones are nonapeptides with a disulfide bridge between Cys residues 1 and 6. This results in a peptide constituted of a six-amino acid cyclic part and a COOH-terminal -amidated three-residue tail. Based on the amino acid at position 8, these peptides are classified into vasopressin and OT families: the vasopressin family contains a basic amino acid (Lys, Arg), and the OT family contains a neutral amino acid at this position (Table 1). Isoleucine in position 3 is essential for stimulating OT receptors and Arg or Lys in position 8 for acting on vasopressin receptors. The difference in the polarity of these amino acid residues is believed to enable the vasopressin and OT peptides to interact with the respective receptors (42).

Virtually all vertebrate species possess an OT-like and a vasopressin-like peptide. Bony fishes (Osteichthyes), predecessors of the land vertebrates, possess isotocin and vasotocin. Thus two evolutionary molecular lineages have been proposed: an isotocin-mesotocin-OT line, associated with reproductive functions, and a vasotocin-vasopressin line involved in water homeostasis. Because vasotocin has been found in the most primitive cyclostomes, the OT and vasopressin genes may have arose by duplication of a common ancestral gene after the radiation of cyclostomes. Based on calculations from the nucleotide level of OT and vasopressin gene, the ancestral gene encoding the precursor protein should be more than 500 million years old. The exceptional structural stability of the nonapeptides during evolution suggests a strong selective pressure, e.g., by coevolution with the corresponding receptors and/or with specific processing enzymes. A conspicuous diversity of OT-like peptides is found in cartilaginous fishes (Chondrichthyes). These marine fishes use urea rather than salts for osmoregulation. It was hypothesized that the OT-like hormones gained their high diversity in Chondrichthyes as they have been relieved from the control of ionic homeostasis (1). Notably, OT, the typical hormone of placental mammals, has been identified in the Pacific ratfish, a Chondrichthyes species.

Mesotocin is the OT-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all nonmammalian tetrapods (amphibians, reptiles, and birds). Only two South American marsupials express OT exclusively, whereas all other marsupials have mesotocin. In the Northern brown bandicoot (Isoodon macrourus) (488) and the North American opossum (Didelphis virginiana) (102), OT is present together with mesotocin. Overall, mesotocin has the largest distribution in vertebrates after vasotocin found in all nonmammalian vertebrates and isotocin identified in bony fishes. Despite this invariability, no clear physiological role has been ascribed to this peptide so far. It is unknown whether the marsupial species that are endowed with both OT and mesotocin have two distinct receptors. The earthworm Eisenia foetida is the most primitive species from which an OT-related peptide (annetocin) has been isolated (429). Injection of annetocin in the earthworm or in leechs results in induction of egg-laying behavior (430).

In all species, OT and vasopressin genes are on the same chromosomal locus but are transcribed in opposite directions (Fig. 1). The intergenic distance between these genes range from 3 to 12 kb in mouse (224), human (500), and rat (391). This type of genomic arrangement could result from the duplication of a common ancestral gene, which was followed by inversion of one of the genes. The human gene for OT-neurophysin I encoding the OT prepropeptide is mapped to chromosome 20p13 (472) and consists of three exons: the first exon encodes a translocator signal, the nonapeptide hormone, the tripeptide processing signal (GKR), and the first nine residues of neurophysin; the second exon encodes the central part of neurophysin (residues 10-76); and the third exon encodes the COOH-terminal region of neurophysin (residues 77-93/95).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Organization of the oxytocin (OT) and vasopressin (VP) gene structure including schematic depiction of the putative cell-specific enhancers (open circle, enhancer of OT gene; shaded circle, enhancer of VP gene). [Modified from Gainer et al. (200).] A: details of the approximately 160-bp region (composite hormone response element) of the upstream OT gene promoter conserved across five species including the sequences of the response elements estrogen response element (ERE), chicken ovalbumin upstream promoter transcription factor I (COUP-TF), and steroidogenic factor-1 (SF-1) are indicated. [Modified from Ivell et al. (273a).] B: domain organization of preprooxytocin including the processing sites. The precursor is split into the indicated fragments by enzymatic cleavages, one involving a glycyl-lysyl-arginine (GKR) sequence and leaving a carboxamide group at the COOH-terminal end of OT. Signal, signal peptide.

The high homology of the OT-like precursor polypeptides is well documented in the sequence of preproannetocin from Eisenia foetida, a primitive invertebrate. It consists of a signal peptide, annetocin (flanked by a Gly COOH-terminal amidation signal and a Lys-Arg dibasic endoproteolytic sequence), and a neurophysin domain. Notably, 14 cysteine residues that play a crucial role in constructing the correct tertiary structure of a neurophysin are completely conserved in the Eisenia neurophysin domain (499).

The OT prepropeptide is subject to cleavage and other modifications as it is transported down the axon to terminals located in the posterior pituitary (74). The mature peptide products, OT and its carrier molecule neurophysin, are stored in the axon terminals until neural inputs elicit their release (475). The main function of neurophysin, a small (93-95 residues) disulfide-rich protein, appears to be related to the proper targeting, packaging, and storage of OT within the granula before release into the bloodstream. OT is found in high concentrations (>0.1 M) in the neurosecretory granules of the posterior pituitary complexed in a 1:1 ratio with neurophysin. In such complexes, OT-neurophysin dimers are the basic functional units as suggested by the crystal structure of the neurophysin-OT complex (486). Cys-1 and Tyr-2 in the OT molecule are the principal neurophysin binding residues. In particular, the protonated -amino group (Cys-1) in OT forms an essential contact site to neurophysin via electrostatic and multiple hydrogen bonding interactions. Due to its dependence on amino group protonation (pK_a ~6.4), the binding strength between OT and neurophysin is much higher in an acidic compartment like the neurosecretory granules (pH ~5.5). Conversely, the dissociation of the complex is facilitated as the complex is released from the neurosecretory granules and enters the plasma (pH 7.4).

Due to the lack of an appropriate cell culture system, the regulation of OT gene expression was studied in heterologous systems and in transgenic mice. The expression patterns found with bovine OT transgenes in mice suggested very complex mechanisms for the cell type-specific expression of OT genes. A bovine OT transgene consisting of the OT structural gene flanked by 600 bp of upstream and 1,900 bp of downstream sequences contained sufficient information to direct expression to murine oxytocinergic magnocellular neurons, within which it was subject to physiological regulation (240). However, enlargement of OT transgene constructs by addition of 700 bp of contiguous downstream sequences repressed the hypothalamic expression. Analysis of various gene constructs in transgenic mice led to the proposal that cell-specific enhancers for OT and vasopressin gene expression are not located on the 5'-upstream regions of these genes, but are present in the intergenic region 0.5-3 kb downstream of the vasopressin gene (Fig. 1). So, constructs containing genomic DNA from 0.5 to 9 kb 5'-upstream of the OT and vasopressin genes but with no endogeneous 3'-downstream sequences did not show significant expression in the hypothalamic magnocellular neurons (200).

The OT mRNA in the rat shows an increase in poly(A) tail length in response to the activation of the hypothalamoneurohypophysial system, e.g., during pregnancy, lactation, and dehydration. This could augment mRNA stability and may be an additional level of OT gene control (95, 623). Hexanucleotide AGGTCA motifs and variations thereof are present in the proximal 5'-flanking region of cloned OT genes. This motif is part of binding sites for all members of the nuclear receptor superfamily, except the glucocorticoid, mineralocorticoid, progesterone, and androgen receptor. Various combinations of this motif exist, ranging from single hexanucleotides, direct or inverted repeats with spacing varying from one to at least six nucleotides (436). Thus potentially several members of the nuclear receptor family including many orphan receptors could interact with the OT gene and regulate its expression. The human and rat OT promoters could be stimulated by the ligand-activated estrogen receptors ER and ER, the thyroid hormone receptor THR, and the retinoic acid receptors RAR and RAR in a variety of cells (3, 477, 478). However, it is important to note that these results were obtained from cotransfection experiments in cell lines, i.e., under nonphysiological circumstances.

A highly conserved DNA element exists at ~160 nucleotides upstream from the transcriptional initiation site (Fig. 1). Deleting the region between 172 and 148 resulted in complete loss of thyroid hormone responsiveness and most of the responsiveness to estrogen and retinoic acid (4). This special "composite" hormone response element is composed of three TGACC motifs. Two of them form an inverted repeat with a spacing of three nucleotides that differs in one nucleotide from the palindromic, canonical estrogen response element (ERE) (79). The rat and human OT promoter shows a good homology with the classic palindromic ERE. Accordingly, in a heterologous transfection system, the rat and human but not the bovine OT gene promoter could be stimulated by estradiol (477). In the appropriate cellular context, the strongest activators were ER and ER. The composite hormone response element was suggested to synergize with proximal elements for the estrogen responsiveness and was found to be essential for the positive regulation by retinoic acid (341, 478). However, estrogen receptor expression was not detected in oxytocinergic cells of the rat hypothalamus (37). Thus direct estrogen-dependent activation may not regulate the OT gene expression in magnocellular neurons in vivo. An estrogen responsiveness was reported for a parvocellular OT-expressing cell group that contains ER (112). Although THRs have been localized in supraoptic nucleus (SON) and paraventricular nucleus (PVN), only a small influence of thyroid hormones on OT gene expression was found in rats in vivo (4).

The rat uterus displays a marked upregulation of OT gene expression before delivery. The main site of steroid-induced uterine OT gene expression was the endometrial epithelium. A strong increase in OT mRNA (~150-fold) preceded the increase in uterine OT binding sites that occurs very shortly before the onset of labor (326, 526). The estrogen-induced rise in uterine OT mRNA was probably mediated via the common hormone response element in the OT gene promoter. The palindromic structure at the composite hormone response element at approximately 160 bp was identified as necessary and sufficient for estrogen induction of the OT gene promoter. However, the high level of uterine OT mRNA at term was not achieved by any of the steroid treatment regimens tested so far (624).

Several investigations have focused on the role of nuclear orphan receptors in the regulation of the OT gene. Unlike in the hypothalamus, the bovine OT gene could be tissue-specifically expressed in the gonads by a minimal functional promoter contained within 600 bp of the transcription start site (15, 586). As mentioned above, the bovine OT gene is unresponsive to estradiol. Nuclear orphan receptors of corpus luteum granule cells have been identified to interact with the common hormone response element (~160 bp, Fig. 1): chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) and steroidogenic factor-1 (SF-1). The levels of these factors could be responsible for the regulation of the endogeneous OT gene in this tissue. SF-1 is a factor with constitutive activating properties on the OT gene (586). The orphan COUP-TFI repressed the activation of the rat OT gene induced by retinoic acid, thyroid hormone, and estrogens through competitive binding to the composite hormone response element (79). Another orphan receptor identified in the hypothalamus, testis receptor 4 (TR4), interacts, unlike all other nuclear receptors, at a region further downstream (112/77 bp) of the common response element in the OT gene (80).

The 5'-flanked region of the rat OT gene also contains binding sites for class III POU homeodomain proteins. Brn-2, a member of this family, is involved in the regulation of OT genes in magnocellular neurons. Brn-2 null mice lack magnocellular vasopressin- and OT-expressing neurons in SON and PVN (225). Brn-2 is required for a specific step in the developmental fate of magnocellular neurons. However, POU class III proteins did not display a significant regulatory activity on the OT gene in heterologous expression systems (80).

Taken together, OT gene regulation in vivo appears to be governed by multiple enhancers and repressors interacting in a complex yet ill-defined fashion.

	III. OXYTOCIN RECEPTORS

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Kimura et al. (299) first isolated and identified a cDNA encoding the human OT receptor using an expression cloning strategy. The encoded receptor is a 389-amino acid polypeptide with 7 transmembrane domains and belongs to the class I G protein-coupled receptor (GPCR) family. To date, the OT receptor encoding sequences from pig (215), rat (489), sheep (480), bovine (44), mouse (315), and rhesus monkey (492) have also been identified.

The human OT receptor mRNAs were found to be of two sizes, 3.6 kb in breast and 4.4 kb in ovary, endometrium, and myometrium. The OT receptor gene is present in single copy in the human genome and was mapped to the gene locus 3p25-3p26.2 (254, 386, 519). The gene spans 17 kb and contains 3 introns and 4 exons. Exons 1 and 2 correspond to the 5'-prime noncoding region. Exons 3 and 4 encode the amino acids of the OT receptor. Intron 3, which is the largest at 12 kb, separates the coding region immediately after the putative transmembrane domain 6. Exon 4 contains the sequence encoding the seventh transmembrane domain, the COOH terminus, and the entire 3'-noncoding region, inluding the polyadenylation signals (Fig. 2). Although many GPCRs have an intronless gene structure, the genes for some other members of the GPCR family including the human vasopressin V2 receptor (511) contain an intron at the same location after transmembrane domain 6. The transcription start sites lie 618 and 621 bp upstream of the initiation codon as demonstrated by primer extension analysis. Nearby, a TATA-like motif and a potential SP-1 binding site is found in the human OT receptor gene. The 5'-flanking region also contains invert GATA-1 motifs, one c-Myb binding site, one AP-2 site, two AP-1 sites, but no complete ERE. Instead, there were two half-palindromic 5'-GGTCA-3' motifs and one half-palindromic 5'-TGACC-3' motif of ERE. Moreover, there were two nucleofactor interleukin-6 (NF IL-6) binding consensus sequences and two binding site sequences for an acute phase reactant-responsive element at the 5'-flanking region (254).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Organization of the human OT receptor gene including the localization of consensus sequences for transcription factors. The human OT receptor gene consists of four exons. Exons 3 and 4 encode the amino acid sequence for the OT receptor. The start (ATG) and stop (TGA) codons of the receptor cDNA are indicated. The DNA sequences encoding for transmembrane regions I-VII are indicated by black areas. [Modified from Inoue et al. (254).]

In the OT receptor gene of the mouse, the promoter region lacks an apparent TATA box but contains multiple putative interleukin-response elements, several half-palindromic motifs, and a classical ERE (315).

In case of the rat OT receptor gene expression at parturition, three transcripts (2.9, 4.8, and 6.7 kb) were identified that differ in the length of their 3'-untranslated regions (489). The promoter region of the rat OT receptor gene also contains multiple putative interleukin-response elements, NF IL-6, and acute-phase response elements (APRE) (489). Further sequence analysis of 4 kb of the 5'-flanking DNA of the rat OT receptor gene revealed the presence of a cAMP response element (CRE) as well as several other potential regulatory elements, including AP-1, AP-2, AP-3, AP-4 sites, an ERE, and a half-steroid response element. A palindromic ERE was identified ~4 kb 5' of the translational start site. An OT receptor reporter construct of this promoter in the human cancer breast cell line MCF-7 demonstrated pronounced induction by both forskolin and phorbol ester, but contrary to in vivo findings, only a weak transcriptional response to estradiol (39). Constructs of the CRE and half-steroid response elements from the promoter of the rat OT receptor gene function as active enhancers. This suggests a potential role for protein kinase A and C pathways in OT receptor gene regulation. The protein kinase A pathway, for example, may be activated when forskolin treatment promotes upregulation of OT receptors in cultured rabbit amnion cells (235, 236). Protein kinase C may act to increase fos/jun activity at AP-1 sites in response to phorbol ester treatment (41). In a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OT receptors, a DNA region containing an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif was required for both basal and serum-induced OT receptor gene expression. The ets factor GABP/ slightly induced OT receptor gene expression in this human breast cell line. The gene expression was markedly potentiated following cotransfection with c-fos/c-jun (242).

APREs are typically found in genes for acute-phase proteins such as ₂-macroglobulin or T kininogen, which are induced by infection or inflammation. The presence of these elements in the promoter region of the human and rat OT receptor gene suggests that the acute induction of OT receptor expression could be a phenomenon similar to the induction of acute-phase reponse genes. The decidua has "macrophage-like" properties and functions. Possibly, inflammatory cytokines are able to induce labor and, thereby, take usage of the transcriptional activation of the OT receptor gene.

However, the lack of classical EREs in a promoter does not exclude a potential direct effect of estrogens on gene expression, since the present half-palindromic ERE motifs can also act synergistically to mediate estrogen activation as shown in the ovalbumin gene (284). Gonadal steroids have an important influence on the uterine OT receptor mRNA accumulation in vivo. Estrogens administered to ovariectomized rats increased OT receptor binding sites and increased OT receptor mRNA accumulation severalfold. Although progesterone leads to a marked decline of OT receptor binding sites, the mRNA levels of OT receptor were nearly unchanged (622). This and several other findings (528) imply the involvement of nongenomic effects of progesterone (see sect. IIIF).

The OT receptor gene is differentially expressed in various tissues. In uterus or hypothalamus, the OT receptor regulation correlates with the pattern of sex steroids, in particular estradiol. As shown with knock-out mice, ER is not necessary for basal OT receptor synthesis but is absolutely necessary for the induction of OT receptor binding in the brain by estrogen (612). However, it is unclear whether OT receptor gene transcription is predominantly regulated by estrogen. The continuous presence of receptors in certain brain regions after gonadectomy suggests the existence of alternate mechanisms of regulation. In this context, a study of the tammar wallaby, an Australian marsupial, is interesting. This species has a twin uterus attached to a double cervix so that each uterus forms an independent environment. During pregnancy, only one uterus becomes gravid, the other remains empty and can thus be regarded as a natural control for uterine changes occurring during pregnancy. It was shown that mesotocin receptor concentrations and the responsiveness to mesotocin differed between the gravid and nongravid myometrium during pregnancy. This indicates that the stimulating agent for the mesotocin receptor is unlikely a circulating factor but rather a local factor, possibly of fetal or placental origin (438). Another well-studied system is the OT receptor gene expression in bovine endometrial cells. In vivo, bovine endometrial OT receptors are upregulated in a cycle-dependent fashion. This regulation appears to be completely at the transcriptional level. Even if the receptors are downregulated in vivo, they show upregulation when explanted and cultured in vitro (516). This indicates that the OT receptor regulation is partly due to gene suppression in vivo. Despite the presence of steroid receptors in bovine endometrial cells, the level of OT receptor mRNA could neither be affected by progesterone or estradiol nor by a progesterone withdrawal protocol. The only factor that affected the OT receptor mRNA level was interferon-. As in vivo, this cytokine suppressed the OT receptor mRNA production (267).

Nuclear protein binding and transfection experiments suggested that constitutive upregulation is a feature of the OT receptor promoter (267). So, specific gene suppression is likely to play an important role for physiological control of the OT receptor expression. Of interest, a genomic element within the third intron of the human OT receptor gene was found to be associated with transcriptional gene suppression. The intronic region was hypermethylated in nonexpressing tissues, but relatively hypomethylated in the myometrium of the cycle and at term, when the OT receptor gene is upregulated (390). Taken together, it was concluded that sex steroids have an indirect effect on both the OT and OT receptor genes, possibly involving intermediate transcription factors or cofactors (273).

The transcriptional regulation of OT receptor shows species-specific differences. The brain OT receptor varies across species in its distribution as well as in its regional regulation by gonadal steroids (261, 262). For the OT receptor as for many other genes, the DNA sequences located in the 5'-flanking region upstream from the coding region are primarily responsible for conferring tissue-specific expression. Transgenic mice carrying 5 kb of the 5'-flanking region of the prairie vole OT receptor gene showed the typical expression pattern of prairie vole OT receptors in mice (616). However, the regulatory elements that confer the specific expression patterns for the OT receptor gene in vivo are yet unknown.

One of the fundamental questions concerns the possible existence of OT receptor subtypes (443, 572). Such subtypes have been suggested to be present, e.g., in the rat uterus (101, 103), kidney (34), or brain (5, 132), to explain differential pharmacological profiles or immunoreactivity patterns. Application of polymerase chain reaction methods and Southern analysis in several tissues known to possess OT binding activity failed to identify a gene encoding a further OT receptor subtype. However, the applied techniques only screen for genes with high homology to the uterine-type OT receptor, and therefore, a putative further OT receptor with low homology to the uterine-type OT receptor would have been kept undetected (43, 572).

The OT receptor is a typical member of the rhodopsin-type (class I) GPCR family. The seven transmembrane -helices are most highly conserved among the GPCR family members. Conserved residues among the GPCRs (outlined in black in Figs. 3 and 4) may be involved in a common mechanism for activation and signal transduction to the G protein. On the basis of studies with model GPCRs, it is assumed that the switching from the inactive to the active conformation is associated with a change in the relative orientation of transmembrane domains 3 and 6, which then unmasks G protein binding sites. In the class I GPCR family, an Asp in transmembrane domain 2 (Asp-85 in human OT receptor, see Figs. 3-5) and a tripeptide (E/D RY) at the interface of transmembrane 2 and the first intracellular loop are believed to be important for receptor activation (57). With respect to Asp-85, this was confirmed for the human OT receptor. When Asp-85 is exchanged by the residues Asn, Gln, or Ala, agonist binding and signal transduction of the receptor becomes impaired (166, 587). Mutations at the conserved tripeptide motif DRY (DRC in case of the OT receptor) result in an either inactive or a constitutively active OT receptor (see below) (166) (for an overview of the published mutagenesis studies see Fig. 5 and Table 2).

View larger version (91K): [in this window] [in a new window]   Fig. 3. Primary sequence alignments of the human OT receptor (OTR), the human vasopressin 2 receptor (V2R), the human vasopressin 1A receptor (V1aR), and the human vasopressin 1b receptor (V1bR). The putative transmembrane helices 1-7 are underlined (asterisks). The residues conservative within the subfamily (~25% of the whole sequence) are outlined in gray, while those conservative for the whole G protein-coupled receptor superfamily are outlined in black.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Schematic structure of the human OT receptor with amino acid residues shown in one-letter code. The residues are marked in the same manner as in Fig. 3, i.e., residues conservative within the OT/vasopressin receptor subfamily are outlined in gray, and residues conservative for the whole G protein-coupled receptor superfamily are outlined in black. The putative N-glycosylation ("Y") and palmitoylation (at C346/C347) sites are marked.

View larger version (76K): [in this window] [in a new window]   Fig. 5. Schematic model of the human OT receptor indicating amino acid residues that are putatively involved in ligand-binding and associated signal transduction events (described in sect. III, B-E). The amino acid residues in gray solid circles are conserved (identical) between the OT receptors from different mammalian species (human, rhesus monkey, pig, bovine, sheep, rat, and mouse). Residues in open circles show interspecies variation. At these positions, an amino acid substitution may be tolerated through mammalian evolution without influencing the functional properties of the receptor. Residues in black solid circles have been subjected to mutagenesis (see Table 2 and sect. III, B-E, for more details). The glutamine and lysine residues highly conserved within the vasopressin/OT receptor family may partly define an agonist-binding pocket that is common to all the different subtypes of this receptor family (42, 398). According to a molecular modeling approach (166), an OT docking site has been proposed (corresponding residues are marked by arrows). In the inactive receptor conformation, the highly conserved arginine (R137) may be constrained in a pocket that is formed by polar residues (indicated by asterisks). After agonist binding, this arginine side chain may be shifted out of the "polar pocket," thereby unmasking a G protein binding site. Receptor domains putatively interacting with OT, a peptide OT antagonist and Gq are marked by lines (for details see sect. III, C and D).

The cysteine residues in the first and second extracellular loops are highly conserved within the GPCR family and are probably connected by a disulfide bridge. Two other well-conserved Cys residues reside within the COOH-terminal domain. Most likely, they are palmitoylated as demonstrated for the V2 receptor (491) and other GPCRs and anchor the cytoplasmic tail in the lipid bilayer. However, for the V2 receptor as well as for the rat OT receptor, elimination of palmitoylation sites by mutagenesis failed to produce significant alterations in receptor function (505).

The OT receptor has two (mouse, rat) or three (human, pig, sheep, rhesus monkey, bovine) potential N-glycosylation sites (N-X-S/T consensus motif) in its extracellular NH₂-terminal domain. For the "core" OT receptor, a molecular mass of ~40-45 kDa can be calculated on the basis of the amino acid sequence derived from the known cDNA sequences of several species. In photoaffinity labeling experiments using myometrial membranes obtained from guinea pig during late pregnancy, a 68- to 80-kDa protein was specifically labeled by a photoreactive OT antagonist (306) developed by Elands et al. (151). Deglycosylation of the photolabeled receptor with endoglycosidase F gave rise to protein with 38-40 kDa (306). Similarly, in the same tissue, a 78-kDa protein was labeled by a photoreactive vasopressin analog (164). In contrast, in membranes from rat mammary gland and rabbit amnion cells, photoreactive OT analogs specifically incorporated into a 65-kDa binding protein (237, 399). It is possible that the different molecular masses for the myometrial versus the mammary gland and amnion OT receptor are due to differential glycosylation patterns. With the assumption of a mass of ~10 kDa for a typical glycosylation core, all of the potential glycosylation sites could be occupied by glycosylation moieties. Recombinant deglycosylation mutants of the human OT receptor have been created by site-directed mutagenesis by exchanging Asp for Asn in positions 8, 15, and 26. The deglycosylated receptors were highly expressed in HeLa cells and showed unaltered receptor binding characteristics (296). Thus the receptor glycosylation appears not to be necessary for proper expression and has no effect on the functional properties of the receptor. Similar findings have been reported for other GPCRs, e.g., the vasopressin V2 receptor (251).

The high homology of the nonapeptides of the evolutionary line isotocin-mesotocin-OT (see sect. IIA and Table 1) is also reflected in the high homology of the corresponding receptors. Accordingly, the mammalian OT receptors share the highest degree of sequence similarity with the toad mesotocin receptor (70%) (6) and the isotocin receptor of teleost fish (66%) (229), whereas the sequence homologies with the vasopressin V1 (nearly 50%) and V2 receptors (40%) are significantly lower. About 100 amino acids (~ 25%) are invariant among the 370-420 amino acids in the human receptors for vasopressin V2, V1a, V1b, and OT (see Figs. 3 and 4). The highest homology between the vasopressin/OT receptor types is found in the extracellular loops and the transmembrane helices. The NH₂ terminus and the COOH terminus have lower similarities, and the intracellular loops are the least of all conserved. Structural common features of the OT/vasopressin receptor family could play an important role in ligand/receptor recognition, e.g., the sequences FQVLPQ at the end of transmembrane domain 2, the sequences GPD (APD in mesotocin receptor) in the first extracellular loop, and DCWA (DCRA and DCWG in mesotocin and isotocin receptor) and PWG in the second extracellular loop.

For small molecules like catecholamines, the ligands bind in a cavity between the -helical segments formed by transmembrane domains 3-6. Peptide ligands, on the other side, bind more superficially and also interact with extracellular loops and/or the NH₂-terminal domain. For the binding of the peptides OT and arginine vasopressin (AVP), residues located in the transmembrane domains as well as residues within extracellular domains are involved in ligand binding. Because the OT/vasopressin peptides as well as their receptors are well conserved, the ligand binding interaction should consist of both common and selective contact sites. As derived from molecular modeling in combination with mutagenesis studies, the agonist binding site for the vasopressin/OT peptides was proposed to be located in a narrow cleft delimited by the ringlike arrangement of the transmembrane domains. An equivalent position was described for the binding of cationic neurotransmitters. In the rat V1a receptor, the conserved Gln residues in the transmembrane domains 2, 3, 4, and 6 and a Lys residue localized in transmembrane domain 3 were replaced by Ala residues. All the receptor mutants had a decreased affinity for the agonists vasopressin, OT, and vasotocin (see Table 2 and Fig. 5). Because the corresponding Gln and Lys residues are highly conserved, it was proposed that the agonist-binding pocket is common to all the different subtypes of this receptor family (42, 398).

Photoaffinity labeling of the first extracellular loop of the bovine vasopressin V2 receptor using a photoreactive lysine vasopressin analog provided direct evidence for the involvement of the first extracellular loop in agonist binding (305). In the first extracellular loop, the homologous residues F103, Y115, and D115 in the human OT, V1a/V1b, and V2 receptor were found to be crucial for the determination of the ligand selectivity (105, 562). For example, the mutation in the equivalent position (Y115F) of the rat V1a receptor led to a 19-fold increase of OT binding compared with the native receptor (105). Molecular modeling of ligand binding interaction to the V1a receptor supported the view that the side chain of Arg-8 in AVP projects outside the transmembrane core of the receptor and could interact with Tyr-115 located in the first extracellular loop. Arg-8 in AVP is known to be necessary for its high-affinity binding to the V1a receptor. When Tyr-115 in the V1a receptor is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the V1a receptor switches accordingly (Table 2) (105). The corresponding residue also determined the agonist specificity of the bovine and pig V2 receptor (562). Thus this residue certainly contributes to agonist selectivity. Additionally, by a peptide mimetic approach it was found that a synthetic dodecapeptide, which is homologous to the first extracellular loop of the human OT receptor, inhibits the binding of tritiated AVP to the human OT receptor (245). The second extracellular loop is also thought to be important for hormone binding of the human OT receptor, since it is conserved only within the nonapeptide receptor family (Fig. 5).

The OT receptor has a weak ligand selectivity profile: hormones with the same cyclic part and either Arg-8 (in arginine vasotocin) or Leu-8 (in OT) are bound with the same affinity, whereby Ile-3 (in OT) in the cyclic hormone part contributes more to affinity than Phe-3 (in oxypressin). This indicates that the cyclic part of OT is more important in conferring binding selectivity for the OT receptor compared with the linear tripeptidic part of the hormone. Using chimeric "gain in function" V2/OT receptor constructs, Postina et al. (463) demonstrated that the NH₂ terminus and the first and second extracellular loops were necessary for agonist binding and selectivity. In particular, the exchange of the NH₂ terminus of the V2 receptor for the corresponding first extracellular domain of the OT receptor resulted in a sixfold increase in binding affinity for OT. Presumably, the NH₂ terminus of the OT receptor takes part in hormone binding and probably interacts with the hydrophobic leucyl residue in position 8 of the ligands. The NH₂-terminal domain and the first extracellular loop of the OT receptor are proposed to interact with the linear COOH-terminal tripeptidic part of OT, whereas the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part (463) (Fig. 5).

Concerning the binding for OT and AVP, the OT receptor is relatively unselective with only about 10-fold higher affinity of the receptor for OT (297, 463). AVP acts as a partial agonist on the OT receptor. To elicit the same response as induced by OT, ~100-fold higher concentrations of AVP are necessary (106, 297). However, AVP becomes a full agonist when two aromatic residues of the OT receptor (Y209 and F284) are replaced by the residues F and Y present at equivalent positions in the vasopressin receptor subtypes (Table 2). These two residues are therefore crucial for the response of the OT receptor to the partial agonist AVP (106).

Chimeric constructs encoding parts of the white sucker fish [Arg⁸]vasotocin receptor and parts of the isotocin receptor have shown that the NH₂ terminus and a region spanning the second extracellular loop and its flanking transmembrane segments contribute to the affinity of the [Arg⁸]vasotocin receptor (230). For the isotocin receptor from teleost fish, it has been shown that the sixth transmembrane helix and/or the fourth extracellular domain are involved in ligand binding (230).

Several studies indicate that the binding site of OT antagonists is different from the agonist binding site. Studies with chimeric receptors provided evidence that the binding site for the peptide OT antagonist (151) was formed by the transmembrane helices 1, 2, and 7, with a major contribution to binding affinity by the upper part of helix 7 (see Fig. 5). These regions did not participate in OT binding (463). Most mutations affecting agonist binding affinities (398) have little effect on antagonist binding affinities (42).

GPCRs may also be constitutively active, in the absence of any agonist. This was first shown for the -adrenergic receptor where mutations in the third intracellular loop, or simply overexpression of the receptor, resulted in constitutive receptor activation. The human V2 receptor mutant D136A (396) and the human OT receptor mutant R137A (166) represent such constitutively active receptors. Both positions are located within the conserved DRY motif (DRC in the OT receptor) at the cytoplasmic side of transmembrane domain 3. The invariably conserved Arg has been hypothesized to be constrained in a hydrophilic pocket formed by conserved polar residues in transmembrane domains 1, 2, and 7 (see "polar pocket" site in Fig. 5) (166, 424). Receptor activation was suggested to involve protonation of the Asp in this motif causing Arg to shift out of the polar pocket leading to cytoplasmic exposure of buried sequences in the second and third intracellular loops. In accordance with this hypothesis, mutating Asp in this motif resulted in increased agonist-independent activity of some receptors including the V2 receptor (208, 396). Although for the V2 receptor (487) as for some other receptors, mutations of the Arg residue within this motif result in uncoupled receptor forms, the human OT receptor mutant R137A possesses an increased basal activity (166). Thus, in this mutant, conformational constraints are released that normally stabilize the wild-type OT receptor in its inactive ground state. Activation of the OT receptor might occur similarly as proposed for the _1B-adrenergic receptor, i.e., by the opening of a solvent-exposed site in the cytosolic domains that has been hypothesized to be involved in G protein recognition (166, 503).

OT receptors are functionally coupled to G_q/11 class GTP binding proteins that stimulate together with G the activity of phospholipase C- isoforms. This leads to the generation of inositol trisphosphate and 1,2-diacylglycerol. Inositol trisphosphate triggers Ca²⁺ release from intracellular stores, whereas diacylglycerol stimulates protein kinase C, which phosphorylates unidentified target proteins. Finally, in response to an increase of intracellular [Ca²⁺], a variety of cellular events are initiated. For example, the forming Ca²⁺-calmodulin complexes trigger activation of neuronal and endothelial isoforms of nitric oxide (NO) synthase. NO in turn stimulates the soluble guanylate cyclase to produce cGMP. In smooth muscle cells, the Ca²⁺-calmodulin system triggers the activation of myosin light-chain kinase activity which initiates smooth muscle contraction, e.g., in myometrial or mammary myoepithelial cells (495). In neurosecretory cells, rising Ca²⁺ levels control cellular excitability, modulate their firing patterns, and lead to transmitter release. Further Ca²⁺-promoted processes include gene transcription and protein synthesis.

In most cell systems studied so far, OT-induced intracellular Ca²⁺ increase is greater in the presence of extracellular Ca²⁺ than that in its absence. This suggests that OT has also effects on calcium influx through voltage-gated or receptor-coupled channels. The effect was nifedipine insensitive (495). OT was also shown to inhibit Ca²⁺/Mg²⁺-ATPase activity in sarcolemmal membranes from the rat uterine myometrium (532). This could sustain transient increases in intracellular Ca²⁺ concentrations and thereby prolong the effects of OT. In rat, guinea pig, and human myometrial cells, the OT-stimulated phosphoinositide hydrolysis was suggested to be mediated by pertussis toxin-sensitive and/or pertussis toxin-resistant G proteins (20, 359, 452). OT-stimulated GTPase and phospholipase C activities were attenuated by incubation with an antibody directed against the COOH termini of G_q and G₁₁ in rat and human myometrial cells (314). In human myometrium, the coupling of OT receptors to a ~80-kDa G protein with transglutaminase activity, termed G_h, was proposed from experiments with solubilized OT receptor-G protein ternary complexes (38). Phospholipase C-1 was suggested as the effector for this kind of signal transduction (435). In Chinese hamster ovary (CHO) cells expressing the rat OT receptor, OT stimulated increases in intracellular [Ca²⁺], extracellular signal-related kinase-2 (ERK-2) phosphorylation, and PGE₂ synthesis (279, 539). OT also induces PGE₂ synthesis in uterine endometrial and amnion cells. In cultured uterine myometrial cells, OT caused tyrosine phosphorylation of mitogen-activated protein (MAP) kinase through an islet-activating protein-sensitive G protein (421). Solubilization experiments in combination with pertussis toxin sensitivity assays indicated that rat OT receptors can couple to both G_q/11 and G_i proteins in transfected CHO cells as well as in pregnant rat myometrium (539, 540).

Which are the receptor domains conferring G protein specificity? Functional analysis of V1a/V2 hybrid receptors demonstrated that the second intracellular loop of the V1a receptor and the third intracellular loop of the V2 receptor each are required and sufficient for efficient coupling to G_q/11 and G_s, respectively (344). Cytoplasmic loops 2 and 3 are also proposed to be implicated in receptor G protein coupling for many other GPCRs. In case of the OT receptor, this appears to be more complex. Several intracellular domains of the receptor could be involved in the specificity and/or efficacy of coupling to G_q/11. This was concluded from the finding that various coexpressed intracellular receptor domains interfered with the OT-stimulated inositol phosphate production (470, 495). Hoare et al. (241) provided evidence that proximal parts of the COOH terminus of the rat OT receptor are required for coupling to G_q (Fig. 5). Whereas OT receptors with COOH-terminal truncations of 22 and 39 residues showed no effect on receptor function, the OT receptor lacking 51 COOH-terminal residues revealed an interesting phenotype: OT-induced intracellular [Ca²⁺] transients could be produced, although the phosphoinositide pathway was apparently not activated. However, it remained unclear which signals could mediate this Ca²⁺ release from intracellular stores. The 51 mutant receptor had a reduced affinity for OT and was uncoupled from G_q- mediated pathways. A coupling of this receptor to G_i was concluded, since the OT-induced Ca²⁺ transients were sensitive to pertussis toxin and to a G sequestrant. Because the 39 mutant was still able to couple to both G_q/11 and G_i, the sequence comprising the residues 339-350 of the rat OT receptor is required for interaction with G_q/11, but not G_i (241) (Fig. 5). Because of the high conservation of the COOH terminus of the OT receptor between various species, similar signal transduction mechanisms may also occur for OT receptors from species other than rats. It is possible that the fidelity of receptor G protein interaction is decreased when OT receptors are strongly upregulated, e.g., in myometrium near term. Moreover, it is known that phosphorylation of GPCRs not only induce their desensitization but may also modify their coupling specificity (123).

When receptors are persistently stimulated with agonists, they desensitize. This process can occur by numerous mechanisms operating at the transcriptional, translational, and protein levels. Rapid, i.e., within seconds to minutes, homologous desensitization of GPCRs consists of two steps, phosphorylation and subsequent arrestin binding. The receptor uncouples from G proteins and undergoes endocytosis, internalization, or sequestration. Receptor sequestration is viewed as an early step in the downregulation of receptors that occurs after prolonged (hours to days) agonist stimulation and that may either end in degradation within lysosomes or in recycling back to the plasma membrane. These processes have been best studied for the adrenergic receptors (330). Birnbaumer and co-workers (52, 252, 253) have analyzed the internalization process for the vasopressin V1a and V2 receptors in some detail.

Like most other GPCRs, OT receptors may undergo rapid homologous desensitization following persistent agonist stimulation (162). Within 5-10 min after agonist stimulation, >60% of the human OT receptors expressed in HEK 293 fibroblasts were internalized (Gimpl and Fahrenholz, unpublished data), similar to as found for the human V2 receptor expressed in the same system (450) and in LLC-PK₁ cells (276). Internalization of OT receptors occurs mainly by a clathrin-dependent pathway. But when stably expressed in HEK 293 cells, a fraction of OT receptors (10-15% of total) is localized in caveolae-like membrane microdomains (210). The internalization mechanism for this receptor population has not been examined. The internalized OT receptor is not recycled back to the cell surface (Gimpl and Fahrenholz, unpublished data). This indicates that the OT receptor behaves more like the V2 receptor and unlike the V1a receptor that rapidly recycles back to the cell surface (252). Using mutagenesis experiments and chimeric receptor constructs, Innamorati et al. (253) identified a serine cluster (Ser-362 to Ser-364) in the COOH-terminal tail of the V2 receptor acting as a retention signal for the internalized V2 receptor. The human OT receptor contains 17 potential phosphorylation sites including two serine clusters in its COOH terminus. One may speculate that these clusters also contribute to prevent the recycling of the internalized OT receptor (Fig. 5).

Exposure of human myometrial cells to OT for up to 20 h resulted in an almost 10-fold reduction in OT binding capacity (451). Although the total amount of OT receptor protein appeared not to be affected by OT treatment for up to 48 h, the OT receptor mRNA was reduced, which may be due to transcriptional suppression and/or destabilization of mRNA (451). When HEK 293 cells expressing the human OT receptor were treated for 18 h with high (µM) concentrations of OT, ~50% of the initial binding capacity remained at the cell surface (278). In WRK1 cells, OT was able to induce a desensitization of the vasopressin (VP) receptors when present for 18 h (89). Up- or downregulation of receptors could also be affected by yet ill-defined cross-talk mechanisms. Evidence for a cross-talk between the corticotropin-releasing hormone (CRH) and OT signal transduction pathways was provided in human myometrial cells at term (217). Furthermore, stimulation of ₂-adrenergic receptors causes heterologous upregulation of OT receptors in the nonpregnant estrogen-primed rat myometrium. In this system, a threefold increase in OT receptor mRNA, an ~100% rise in receptor binding, and an augmented contractile response of isolated uterine strips to OT were observed (156).

F.  Effects of Steroids

1.  Cholesterol

Both solubilized and membrane-associated OT receptors require at least two essential components for high-affinity OT binding: divalent cations such as Mn²⁺ or Mg²⁺ and cholesterol. Compared with many other GPCRs, the GTP sensitivity of the agonist binding to the OT receptor is rather modest. All attempts to purify functional OT receptors have been unsuccessful to date. With the use of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) as detergent, it is possible to solubilize functional OT receptors from different sources (174, 234, 301, 527). However, a common observation is that following solubilization, OT receptors lose characteristic binding properties, the affinity for OT becomes lower, and/or additional low-affinity state receptors appear in the extract. Unfortunately, low-affinity [e.g., dissociation constant (K_d) >10-50 nM] receptor populations cannot be characterized adequately by conventional radioligand binding assays. The necessity to separate free from bound ligand concomitantly leads to a dissociation of low-affinity ligand-receptor interactions. Solubilization with CHAPSO is known to lead to a substantial cholesterol depletion of the soluble extract, and we could demonstrate that substitution with cholesterol markedly enhanced the OT binding of soluble OT receptors (165, 301, 302). This became first evident in reconstitution of soluble OT receptors using liposomes of defined composition. A saturable high-affinity OT binding was obtained only with liposomes that contained a critical amount of cholesterol (301). Moreover, when OT receptors were expressed in insect cells, which naturally have plasma membranes with low cholesterol content, the receptors are mainly in a low-affinity state (K_d >100 nM). After addition of cholesterol to the culture medium, a fraction of OT receptors is converted from a low- to a high-affinity state (K_d ~1 nM) (212). The low-affinity state was identified as a physiological active receptor state, and the conversion of the affinity states to each other is, at least to a certain degree, reversible. The interaction of cholesterol with OT receptors is of high specificity and is not due to mere changes of membrane fluidity (209). Furthermore, cholesterol stabilizes both membrane-associated and solubilized OT receptors against thermal denaturation (210). Taken together, our data suggest a direct and cooperative molecular interaction of cholesterol with OT receptors. Cholesterol acts as an allosteric modulator and stabilizes the receptor in a high-affinity state for agonists and antagonists. In many but not in all cell systems, populations of high- and low-affinity OT receptors have been observed (119, 135, 209, 456). This could reflect uneven cholesterol distributions within the plasma membrane of these cells. We would expect that high-affinity state OT receptors are preferentially localized in cholesterol-rich subdomains of the plasma membrane. Likewise, receptor heterogeneity with respect to affinity states should be highest in cell systems with abundant cholesterol-rich domains such as rafts or caveolae structures, e.g., in myometrial cells at term (70). In fact, we recently provided evidence for a partial enrichment of high-affinity OT receptors in cholesterol-rich plasma membrane domains in HEK 293 fibroblasts stably expressing the human OT receptor (210, 211).

Divalent metal ions like Mg²⁺ are long known to increase the response of target cells to OT and to shift the dose-response curve to the left. Thus addition of Mg²⁺ was found to increase both the OT binding capacity and the affinity state of the OT receptor (525). This is surprisingly similar to what cholesterol does. In addition, Mg²⁺ has been proposed to display its effect on the OT receptor interaction by influencing positive cooperativity (457). Mg²⁺ increases the potency of OT analogs in stimulating uterine contractions whereby the effects of Mg²⁺ were observed to be inversely related to the potency of the peptide. Relatively inactive peptides like 7-glycine OT became significantly more potent when the Mg²⁺ concentration bathing the uterine smooth muscle in vivo was increased from 0 to 0.5 mM (530). Conclusively, cholesterol and Mg²⁺ are essential allosteric modulators of the OT receptor and may be involved in the regulation of OT-mediated signaling functions (see Fig. 6).

View larger version (51K): [in this window] [in a new window]   Fig. 6. Schematic model of nongenomic inhibitory effects of progesterone. Progesterone inhibits both the signal transduction of Gq coupled receptors (as shown here for the OT receptor) and the intracellular trafficking of cholesterol. Principally, eukaryotic cells can obtain the required cholesterol (Chol, gray ellipses) by two sources: endogenously by de novo synthesis of cholesterol and exogenously by uptake of cholesteryl ester (CE)-rich low-density lipoprotein (LDL) particles via receptor-mediated (R) endocytosis. De novo synthesized cholesterol first arrives at cholesterol-rich domains in the plasma membrane (caveolae and/or "lipid rafts") that may function as cholesterol "sorting centers" within the plasma membrane, where most of the cellular cholesterol resides. Progesterone blocks several intracellular transport pathways of cholesterol (red bars) except for the LDL receptor-mediated uptake of cholesterol. Moreover, cholesterol esterification does not occur in the presence of progesterone, presumably due to the lack of cholesterol substrate for acyl-CoA:cholesterol acetyltransferase (ACAT). As a consequence, unesterified cholesterol accumulates in lysosomes (or late endosomes) and lysosome-like compartments (designated as "lamellar bodies") (marked by red background). The key enzyme for the cholesterol de novo synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA Red), is stimulated in the presence of progesterone (red arrow), but the cholesterol biosynthesis stops at the level of precursors (e.g., lanosterol). Enzymes involved in the conversion of cholesterol precursors reside in the endoplasmic reticulum (ER), and progesterone most likely prevents sterol precursors localized in the plasma membrane from reaching the ER-resident enzymes, thereby preventing their conversion to cholesterol. Overall, progesterone induces a state of cholesterol auxotrophy (383). However, after progesterone withdrawal, the accumulated precursors will be rapidly converted to cholesterol. Thus cells will become overloaded with cholesterol for a certain period of time, after which the cholesterol homeostasis will be reestablished. We hypothesize that these reversible progesterone-induced changes of the cholesterol trafficking could have a strong influence on signal transduction processes, particularly in case of the OT receptor (OTRH and OTRL, high-affinity and low-affinity OT receptor, respectively; receptor in blue; OT in yellow) (for further details see sect. IIIF).

Do these allosteric modulators play a role for the regulation of OT-related physiological processes? Some reports suggest that particularly in reproductive tissues, the cholesterol concentrations may be highly dynamic. With the use of freeze-fracture cytochemistry with the cholesterol-binding filipin, marked increases in cholesterol have been found in rat uterine epithelial cells at the time of blastocyst implantation (400). In the human placental syncytiotrophoblast basal membrane, Sen et al. (512) observed a steady decrease in cholesterol-to-phospholipid ratio in correlation with an increase in membrane fluidity during placental development. At term however, the cholesterol-to-phospholipids ratio in syncytiotrophoblast membranes was found to be increased compared with the cholesterol-to-phospholipid ratio in early placentas (365). Moreover, cholesterol-enriched caveolae structures are a conspicuous feature in the rat myometrium at term (70). We have provided evidence that cholesterol can modulate receptor function by both changes of the membrane fluidity and direct binding effects, e.g., in case of the OT receptor (209). Plasma membranes with lowered cholesterol content showed a decreased capacity (B_max) of binding sites and/or a decreased affinity (K_d) of ligand-receptor binding. Interestingly, Lopez et al. (345) reported that pregnancy in humans was associated with increases in both density and affinity of OT receptors. To draw further conclusions, correlation studies are required using tissues in which both the membrane cholesterol content and the OT receptor activity will be measured at the same time.

Progesterone is considered to be essential to maintain the uterine quiescence. Grazzini et al. (218) recently postulated that progesterone specifically binds to the rat OT receptor with high affinity (K_d ~20 nM) and thereby inhibits the receptor function. In case of the human OT receptor, a direct inhibitory interaction [inhibitory constant (K_i) ~30 nM] with a progesterone metabolite, 5-pregnane-3,20-dione, has been reported by the same authors. They claimed that progesterone could act as a negative modulator of the OT receptor and thus offered a plausible mechanism of how progesterone could contribute to uterine quiescence. However, these findings could not be reproduced in several other laboratories including our own (81). Instead, we found that high concentrations of progesterone (>10 µM) attenuated or blocked the signaling of several GPCRs, including the OT receptor. The progesterone effects occurred within minutes, were reversible, and could not be blocked by a protein synthesis inhibitor (81). Overall, the action of progesterone was more cell type specific than receptor specific. The progesterone doses that are required to affect the signaling function of receptors are much higher than the progesterone levels found in plasma or in nonsteroidogenic tissues such as the myometrium. In steroidogenic tissues, however, huge amounts of progesterone have been measured. Near term, the human placenta secretes upward of 300 mg of progesterone daily. The progesterone content of this organ was shown to be 7 µg/g wet tissue (520). In human corpus luteum, progesterone concentrations reached peak levels of ~25 µg/g tissue shortly after ovulation and in the early luteal phase (545). These values are within the range of the progesterone concentrations that were effective in our study (81). Thus, in steroidogenic cells as well as in their environment, progesterone might nongenomically influence the signaling of receptors. The molecular mechanisms underlying this progesterone action are not understood. A well-known progesterone binding protein is the multidrug resistance P-glycoprotein (471). In addition to their role in detoxification, P-glycoproteins are involved in intracellular cholesterol transport. It is known that progesterone markedly interferes with the intracellular transport (and metabolism?) of cholesterol (see model in Fig. 6). At concentrations in the micromolar range, it inhibits both the cholesterol esterification and the transport of cholesterol to and from the plasma membrane (343). In particular, progesterone reduces the cholesterol pool residing in caveolae (521). Paradoxically, at the same time, progesterone stimulates the activity of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the key enzyme of de novo cholesterol biosynthesis. Hence, cholesterol precursors like lanosterol begin to enrich in the membranes of the cell (383). As mentioned above, the OT receptor needs a cholesterol-rich microenvironment to become stabilized in its high-affinity state (210). Because the cholesterol precursors, particularly lanosterol, are completely inactive to support the OT receptor in its high-affinity state (209), the responsiveness of the OT system may not be fully operative during the continuous presence of high progesterone concentrations. According to this scenario, progesterone withdrawal would restore the cholesterol transport so that the highly enriched amounts of cholesterol precursors would now become rapidly converted to cholesterol. This would lead to a sudden rise of cholesterol and should push the responsiveness to OT since low-affinity OT receptors could now be converted into their high-affinity state (302). According to this postulated mechanism, progesterone could affect the signaling of all those receptors that are functionally dependent on cholesterol. It is important to note that the nongenomic actions of progesterone including its influence on the cholesterol transport require progesterone concentrations in the micromolar range. This suggests that the described effects may be limited to the steroidogenic tissues and to their environment. Most likely, progesterone acts in these tissues via both genomic and nongenomic pathways (summarized in Fig. 6) together with other steroids to control receptor activity.

	IV. THE PERIPHERAL OXYTOCIN SYSTEM

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A.  Female Reproductive System

1.  Uterus

The pregnant uterus is one of the traditional targets of OT. OT is one of the most potent uterotonic agents and is clinically used to induce labor. Accordingly, the development of highly specific OT antagonists may be of therapeutic value for the prevention of preterm labor and the regulation of dysmenorrhea (358, 594).

The OT gene was found to be expressed in the rat uterine epithelium at term. The estrogen-induced elevation of OT mRNA levels was restricted to 3 days and reached peak levels that exceeded hypothalamic OT mRNA levels by a factor of 70 (327). In rats, OT gene expression was shown to be present in placenta and amnion (328) and in humans in amnion, chorion, and decidua (104). However, in most studies, significant increases of OT before the onset of labor have not been detected, neither in maternal plasma nor in intrauterine tissues. On the other hand, some findings suggest that there is a relationship between the pattern of OT secretion and advancing pregnancy. In rhesus monkeys, it was shown that maternal but not fetal OT concentrations were positively correlated with nocturnal uterine activity and progressively increased during late pregnancy and delivery (239).

Around the onset of labor, uterine sensitivity to OT markedly increases. This is associated with both an upregulation of OT receptor mRNA levels and a strong increase in the density of myometrial OT receptors, reaching a peak during early labor (188, 299). This has been demonstrated both in the rat and in the human species, in which receptor levels rise during early labor to 200 times that in the nonpregnant state (189). Thus, at the onset of labor, OT can stimulate uterine contractions at levels that are ineffective in the nonpregnant state. After parturition, the concentrations of OT receptors rapidly decline. In rats, the uterine OT receptor mRNA levels decreased more than sevenfold within 24 h (624). Possibly, the downregulation of the OT receptors may be necessary to avoid unwanted contractile responses during lactation when OT levels are raised.

Gonadal steroids play an important role in the regulation of uterine OT receptors. In the days preceding birth, the ratio of plasma progesterone to estrogen falls. These changes in the steroid concentrations may occur in most mammals. At least in humans, progesterone withdrawal has not been determined. The steep drop of circulating progesterone occurs after placental delivery. Alteration of sex steroid metabolism in the fetoplacental unit appears to occur in women and primates (374). As shown in bovine and in sheep, maturation of the fetal hypothalamus leads to an increased secretion of CRH, which in turn stimulates the pituitary to secrete ACTH (188). Subsequently, ACTH stimulates the fetal adrenal to release cortisol. Additionally, OT-induced contractures of the myometrium in late pregnancy could lea