Design and Characterization of Superpotent Bivalent Ligands Targeting Oxytocin Receptor Dimers via a Channel-Like Structure.

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.

Design, synthesis, and pharmacological characterization of fluorescent peptides for imaging human V1b vasopressin or oxytocin receptors.

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys(1),Leu(4),Lys(8)]vasopressin (d[Leu(4),Lys(8)]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu(4),Lys(Alexa 647)(8)]VP (3) and d[Leu(4),Lys(11-aminoundecanoyl-Alexa 647)(8)]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.

Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents.

Oxytocin (OT) and vasopressin (AVP) mediate their biological actions by acting on four known receptors: The OT (uterine) and the AVP V(1a) (vasopressor), V(1b) (pituitary), V(2) (renal) receptors and a fifth putative AVP V(1c)? (vasodilating) receptor. This presentation will summarize some highlights of the recent progress, in the design and synthesis of selective peptide agonists, antagonists, radioiodinated ligands, fluorescent ligands and bivalent ligands for these receptors. Here we present published and unpublished pharmacological data on the most widely used agonists, antagonists and labelled ligands. The pharmacological properties of promising new selective OT antagonists and V(1b) agonists are also presented. This review should serve as a useful guide for the selection of the most appropriate ligand for a given study. The current status of non-peptide OT and AVP antagonists and agonists is also summarized. The relative merits of peptide and non-peptide AVP and OT agonists and antagonists as: (1) research tools and (2) therapeutic agents will be evaluated. Many of the receptor selective peptide agonists and antagonists from this and other laboratories are far more widely used as pharmacological tools for studies on the peripheral and central effects of OT and AVP than their non-peptide counterparts. In addition to OT and to a lesser extent AVP (pitressin), a number of OT and AVP analogues; such as carbetocin (OT agonist) dDAVP (desmopressin, V(2) agonist), terlipressin (V(1a) agonist), felypressin (V(1a) agonist) and atosiban (Tractocile OT antagonist) are also in clinical use. Despite much early promise, no non-peptide V(1a) or OT antagonists are currently in clinical trials. While a number of orally active non-peptide V(2) antagonists (Vaptans); notably, Tolvaptan, Lixivaptan and Satavaptan, are currently in Phase III clinical trials; to date, only the mixed V(2)/V(1a), antagonist Conivaptan (Vaprisol), has been approved by the US FDA for clinical use (by i.v. administration), for the treatment of euvolemic and hypervolemic hyponatremia in hospitalized patients. Promising new non-peptide V(1b) and OT antagonists, as well as non-peptide V(2) and OT agonists are now in pre-clinical development.

Pharmacological and physiological characterization of d[Leu4, Lys8]vasopressin, the first V1b-selective agonist for rat vasopressin/oxytocin receptors.

Recently, we synthesized and characterized the first selective V(1b) vasopressin (VP)/oxytocin receptor agonist, d[Cha(4)]arginine vasopressin. However, this agonist was only selective for the human receptors. We thus decided to design a selective V(1b) agonist for the rodent species. We started from previous observations showing that modifying [deamino(1),Arg(8)]VP in positions 4 and 8 altered the rat VP/oxytocin receptor selectivity. We synthesized a series of 13 [deamino(1),Arg(8)]VP analogs modified in positions 4 and 8. Among them, one seemed very promising, d[Leu(4), Lys(8)]VP. In this paper, we describe its pharmacological and physiological properties. This analog exhibited a nanomolar affinity for the rat, human, and mouse V(1b) VP receptors and a strong V(1b) selectivity for the rat species. On AtT20 cells stably transfected with the rat V(1b) receptor, d[Leu(4), Lys(8)]VP behaved as a full agonist on both phospholipase C and MAPK assays. Additional experiments revealed its ability to induce the internalization of enhanced green fluorescent protein-tagged human and mouse V(1b) receptors as expected for a full agonist. Additional physiological experiments were performed to further confirm the selectivity of this peptide. Its antidiuretic, vasopressor, and in vitro oxytocic activities were weak compared with those of VP. In contrast, used at low doses, its efficiency to stimulate adrenocorticotropin or insulin release from mouse pituitary or perfused rat pancreas, respectively, was similar to that obtained with VP. In conclusion, d[Leu(4), Lys(8)]VP is the first selective agonist available for the rat V(1b) VP receptor. It will allow a better understanding of V(1b) receptor-mediated effects in rodents.

Design and synthesis of the first selective agonists for the rat vasopressin V(1b) receptor: based on modifications of deamino-[Cys1]arginine vasopressin at positions 4 and 8.

The neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) mediate a wide variety of peripheral and central physiological and behavioral effects by acting on four different G-protein coupled receptors, termed V1a (vascular), V1b (pituitary), V2 (renal), and OT (uterine). We recently reported that d[Cha4]AVP (A), d[Leu4]AVP (B), d[Orn4]AVP (C), and d[Arg4]AVP (D) have high affinity and are selective agonists for the human V1b receptor. However, peptides A-D were subsequently shown to be potent antidiuretic agonists in the rat and are, thus, not selective V1b agonists in the rat. Peptides A-D served as leads for the studies reported here. They were modified at position 8 by Lys, ornithine (Orn), diaminobutyric acid (Dab), and diaminopropionic acid (Dap) to give d[Cha4,Lys8]VP (1), d[Cha4,Orn8]VP (2), d[Cha4,Dab8]VP (3), d[Cha4,Dap8]VP (4), d[Leu4,Lys8]VP (5), d[Leu4,Orn8]VP (6), d[Leu4,Dab8]VP (7), d[Leu4,Dap8]VP (8), d[Orn4,Lys8]VP (9), d[Orn4,Orn8]VP (10), d[Arg4,Lys8]VP (11), d[Arg4,Orn8]VP (12), and d[Arg4,Dab8]VP (13). All peptides were synthesized by the Merrifield solid-phase method. Their binding and functional properties were evaluated in rat AVP V1a, V1b, and V2 receptors and on the rat OT receptor expressed either in native tissues or in stably transfected cells. They were also examined in rat vasopressor, antidiuretic, and in in vitro (no Mg++) oxytocic assays. Functional studies performed on chinese hamster ovary cells expressing the different AVP/OT receptors confirm that d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP (3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8) are the first selective agonists for the rat V1b receptor. These selective V1b agonists are promising new tools for studies of the role of the V1b receptor in the rat.

Design and synthesis of potent, highly selective vasopressin hypotensive agonists.

We report here the solid-phase synthesis and vasodepressor potencies of a new lead vasopressin (VP) hypotensive peptide [1(beta-mercapto-beta,beta-pentamethylenepropionic acid)-2-0-ethyl-D-tyrosine, 3-arginine, 4-valine, 7-lysine, 9-ethylenediamine] lysine vasopressin, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4), Lys(7), Eda(9)]LVP (C) and 21 analogues of C with single modifications at positions 9 (1-13), 6 (14), 2 (16-20) and combined modifications at positions 6 and 10 (15) and 2 and 10 (21). Peptides 1-13 have the following replacements for the Eda residue at position 9 in C: (1) Gly-NH(2); (2) Gly-NH-CH(3); (3) Ala-NH(2); (4) Ala-NH-CH(3), (5) Val-NH(2); (6) Cha-NH(2); (7) Thr-NH(2); (8) Phe-NH(2); (9) Tyr-NH(2); (10) Orn-NH(2); (11) Lys-NH(2); (12) D-Lys-NH(2); (13) Arg-NH(2). Peptide 14 has the Cys residue at position 6 replaced by Pen. Peptide 15 is the retro-Tyr(10) analogue of peptide 14. Peptides 16-20 have the D-Tyr(Et) residue at position 2 in C replaced by the following substituents: D-Trp (16); D-2-Nal (17); D-Tyr(Bu(t))(18); D-Tyr(Pr(n)) (19); D-Tyr(Pr(i)) (20). Peptide 21 is the retro-Tyr(10) analogue of peptide 20. C and peptides 1-21 were evaluated for agonistic and antagonistic activities in in vivo vasopressor (V(1a)-receptor), antidiuretic (V(2)-receptor), and in in vitro (no Mg(2+)) oxytocic (OT-receptor) assays in the rat, and, like the original hypotensive peptide, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4)]AVP (A) (Manning et al., J. Peptide Science 1999, 5:472-490), were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure (BP) maintained at 100-120 mmHg. The effective dose (ED), in microg/100 g i.v., the dose required to produce a vasodepressor response of 5 cm(2) area under the vasodepressor response curve (AUC) during the 5-min period following the injection of the test peptide, was determined. The EDs measure the vasodepressor potencies of the hypotensive peptides C and 1-21 relative to that of A (ED = 4.66 microg/100 g) and to each other. The following ED values in microg/100 g were obtained for C and for peptides 1-21; C 0.53; (1) 2.41; (2) 1.13; (3) 1.62; (4) 0.80; (5) 1.83; (6) 1.56; (7) 2.12, (8) 2.58; (9) 1.40; (10) 0.88; (11) 0.90; (12) 0.85; (13) 0.68; (14) 0.99; (15) 1.05; (16) 0.66; (17) 0.54; (18) 0.33; (19) 0.18; (20) 0.15; (21) 0.14. All of the hypotensive peptides reported here are more potent than A. Peptides 20 and 21 exhibit a striking 30-fold enhancement in vasodepressor potencies relative to A. With a vasodepressor ED = 0.14, peptide 21 is the most potent VP vasodepressor agonist reported to date. Because it contains a retro-Tyr(10) residue, it is a promising new radioiodinatable ligand for the putative VP vasodilating receptor. Some of these new hypotensive peptides may be of value as research tools for studies on the complex cardiovascular actions of VP and may lead to the development of a new class of antihypertensive agents.

Position 4 analogues of [deamino-Cys(1)] arginine vasopressin exhibit striking species differences for human and rat V(2)/V(1b) receptor selectivity.

Arginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G-protein coupled receptors, termed V(1a) (vascular), V(1b) (pituitary) and V(2) (renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V(1b) receptor, we recently reported the human V(1b), V(1a), V(2) and OT receptor affinities of the following position 4 substituted analogues of [deamino-Cys(1)] arginine vasopressin (dAVP)-(1) d[Leu(4)]AVP, (2) d[Orn(4)]AVP, (3) d[Lys(4)]AVP, (4) d[Har(4)]AVP, (5) d[Arg(4)]AVP, (6) d[Val(4)]AVP, (7) d[Ala(4)]AVP, (8) d[Abu(4)]AVP, (9) d[Nva(4)]AVP, (10) d[Nle(4)]AVP, (11) d[Ile(4)]AVP, (12) d[Phe(4)]AVP, (13) d[Asn(4)]AVP, (14) d[Thr(4)]AVP: (15) d[Dap(4)]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V(1b) receptor. Furthermore, peptides 1-4 exhibit high selectivities for the human V(1b) receptor with respect to the V(1a), V(2) and OT receptors and, with d[Cha(4)]AVP, in functional tests, are the first high affinity selective agonists for the human V(1b) receptor (Cheng LL et al., J. Med. Chem. 47: 2375-2388, 2004). We report here the pharmacological properties of peptides 1-4, 5 (from a resynthesis), 7, 9-13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro: no Mg(++)) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V(1b), V(1a), V(2) and OT receptor affinities of peptides 1-5 and the rat V(2) receptor affinities for peptides: 7-15.The antidiuretic activities in units/mg of peptides 1-15, are: 1=378; 2=260; 3=35; 4=505; 5=748; 6=1150; 7=841; 8=1020; 9=877; 10=1141; 11=819, 12=110; 13=996; 14=758; 15=1053. Peptides 1-4 exhibit respectively the following rat and human (in brackets) V(2) receptor affinities: 1=3.1 nm (245 nm); 2=3.4 nm (1125 nm); 3=24.6 nm (11,170 nm); 4=0.6 nm (1386 nm). Their rat V(1b) receptor affinities are 1=0.02 nm; 2=0.45 nm; 3=9.8 nm; 4=0.32 nm. Their rat V(1a) receptor affinities are 1=1252 nm; 2=900 nm; 3=1478 nm; 4=32 nm. Their rat oxytocin (OT) receptor affinities are 1=481 nm; 2=997 nm; 3=5042 nm; 4=2996 nm. All four peptides have high affinities and selectivities for the rat V(1b) receptor with respect to the rat V(1a) and OT receptors. However, in contrast to their high selectivity for the human V(1b) receptor with respect to the human V(2) receptor, they are not selective for the V(1b) receptor with respect to the V(2) receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V(2) receptors. Peptides 1-4 are promising leads to the design of the first high affinity selective agonists for the rat V(1b) receptor.

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.

Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4.

The glutamine(4) residue in [deamino-Cys(1)]arginine vasopressin (dAVP) was replaced by a broad series of aliphatic, aromatic, polar, and charged amino acids to give the following peptides: d[Gly(4)]AVP (1), d[Ala(4)]AVP (2), d[Abu(4)]AVP (3), d[Nva(4)]AVP (4), d[Nle(4)]AVP (5), d[Leu(4)]AVP (6), d[Ile(4)]AVP (7), d[Thi(4)]AVP (8), d[Phe(4)]AVP (9), d[Tyr(4)]AVP (10), d[Trp(4)]AVP (11), d[Asn(4)]AVP (12), d[Ser(4)]AVP (13), d[Thr(4)]AVP (14), d[Dap(4)]AVP (15), d[Dab(4)]AVP (16), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), d[Arg(4)]AVP (19), d[Har(4)]AVP (20), and d[Glu(4)]AVP (21). All peptides were synthesized by solid-phase methods using BOC chemistry for all but one peptide (8), which required the use of Fmoc chemistry. The binding and functional properties of these position 4 substituted analogues of dAVP (d[X(4)]AVP) and the previously reported d[Cha(4)]AVP (Derick et al. Endocrinology 2002, 143, 4655-4664) were evaluated on human arginine vasopressin (AVP) V(1a), V(1b), and V(2) receptors and on the human oxytocin (OT) receptor expressed in living Chinese hamster ovary (CHO) cells. Binding studies revealed that broad modifications of the fourth residue of dAVP do not significantly alter affinity for the human V(1b) receptor. Only aromatic (Phe, Tyr, Trp) or negatively charged (Glu) residues reduce V(1b) affinity. By contrast, the human V(1a) and more particularly the human V(2) and the OT receptors are more sensitive to many of these modifications. Thus, the replacement of the Gln(4) residue of dAVP by aliphatic (Leu, Cha) or positively charged (Orn, Lys, Arg, Har) amino acids led to analogues exhibiting drastic reductions of their affinity for the human V(1a), V(2), and OT receptors. Consequently, in addition to the previously reported d[Cha(4)]AVP, peptides 6 and 17-20 display excellent selectivities for the human V(1b) receptor. The key structural requirement responsible for optimal V(1b) selectivity appears to be the length and branching of the aliphatic side chain of the fourth residue of dAVP. Functional studies performed on CHO cells expressing the different human AVP/OT receptors confirm the V(1b) selectivity of peptides 6, 17, 18, 20, and d[Cha(4)]AVP. However, d[Arg(4)]AVP (19), which triggers an excellent coupling between the human V(2) receptor and adenylyl cyclase, was found to exhibit both V(1b) and V(2) agonism in functional tests. More interestingly, these functional experiments revealed that, depending on the AVP/OT receptor, a given d[X(4)]AVP analogue may behave as a full agonist or as a partial agonist. This strongly suggests that the fourth residue of dAVP plays an important role in the coupling between the hormone-receptor complex, the heterotrimeric G protein, and the effectors. In conclusion, the synthesis of these d[X(4)]AVP analogues led to the discovery of new V(1b) agonists with high affinity and greatly enhanced selectivities. Thus, in addition to d[Cha(4)]AVP, d[Leu(4)]AVP (6), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), and d[Har(4)]AVP (20) are useful new tools for studying the structure and the function of the human V(1b) receptor.

Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin and vasopressin V(1)(a) receptors.

The fluoresceinyl (Flu) group has been linked by an amide bond to the side chain amino group at position 8 of (a) two oxytocin (OT) antagonists, to give d(CH(2))(5)[Tyr(Me)(2),Thr(4),Orn(8)(5/6C-Flu),Tyr-NH(2)(9)]VT (Orn(8)(5/6C-Flu)OTA) (1) and desGly-NH(2),d(CH(2))(5)[D- Tyr(2),Thr(4),Orn(8)(5/6C-Flu)]VT (2), and (b) eight Lys(8) and Orn(8) analogues of potent OT agonists, to give d[Lys(8)(5/6C-Flu)]VT (3), d[Thr(4),Lys(8)(5/6C-Flu)]VT (4), [HO(1)][Lys(8)(5/6C-Flu)]VT (5), [HO(1)][Thr(4),Lys(8)(5/6C-Flu)]VT (6), d[Orn(8)(5/6C-Flu)]VT (7), d[Thr(4),Orn(8)(5/6C-Flu)]VT (8), [HO(1)][Orn(8)(5/6C-Flu)]VT (9), and [HO(1)][Thr(4),Orn(8)(5/6C-Flu)]VT (10). The tetramethylrhodamyl (Rhm) group was attached to the precursor peptide of 9 to give [HO(1)][Orn(8)(5/6C-Rhm)]VT (11). All 11 fluorescent peptides were evaluated in human OT and vasopressin V(1a) (vasoconstrictor), V(1b) (pituitary), and V(2) (antidiuretic) receptor binding and functional assays. With K(d) = 6.24, 217, >10000, and >10000 nM for the OT, V(1a), V(1b), and V(2) receptors, peptide 1 is a potent and selective fluorescent OT antagonist and may be useful for specifically labeling OT receptors while peptide 2 exhibits low affinities for all the receptors. The fluorescent peptides 3-10 are all very potent agonists for the human OT receptor. They exhibit the following K(d) values (nM) for the human OT, V(1a), V(1b), and V(2) receptors, respectively: (3) 0.29, 57, 124, >10000; (4) 1.8, 25.5, 150, >10000; (5) 0.34, 13.7, 66, nd (not determined); (6) 0.32, 17.3, 53, >10000; (7) 0.25, 107, 393, >10000; (8) 0.40, 30, 282, >10000; (9) 0.18, 12.2, 126, nd; (10) 0.17, 11.8, 87, >1000; (11) 0.092, 7.36, nd, nd. Peptide 7 exhibits both a high affinity and a high selectivity for human OT receptors. Peptides 7 and 11 were utilized to study the internalization of the OT receptor-ligand complex. Preliminary studies indicate that this process is similar to that observed for the vasopressin V(1a) receptor and differs from that observed for vasopressin V(2) receptors. Some or all of the fluorescent OT antagonists and agonists reported here are very promising new fluorescent ligands for labeling cells which express the human OT receptor and are also useful tools to follow endocytosis of the receptor-ligand complex.