Structure-activity relationship study of position 4 in the urotensin-II receptor ligand U-II(4-11)

In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands.     

Structure-activity relationships of human urotensin II and related analogues on rat aortic ring contraction

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an L-alanine or by a D-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4-11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or D-isomer substitutions in UII(4-11) or in UII(4-11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their D-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4-11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure-activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.     

Muscarinic cholinergic and histamine H1 receptor binding of phenothiazine drug metabolites

In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, alpha 1- and alpha 2 adrenoceptors in rat brain were previously reported from our laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using 3H-quinuclidinyl benzilate and 3H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. Their histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine.     

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

Fluorescent peptide hormones: development of high affinity vasopressin analogues.

Highly potent and specific peptide hormone analogues with fluorescent reporter groups are current research goals. Until now, however, only moderately potent analogues have been described. We report here several types of vasopressin (VP) analogues with different fluorophores attached to the peptide. In a first series, fluorophores were attached to the free epsilon amino function of [des-amino1-lysine8]VP (dLVP), producing agonistic analogues. In a second series, reporter groups were added to the N-terminal of open-chain antagonist structures. The biological activities of these analogues were assessed by two different sets of experiments: 1) The measurement of their binding affinities towards the V1a-vasopressin receptor subtype from WRK1 cells or rat liver membrane preparations; 2) Their ability to stimulate the phospholipase C activity in WRK1 cells. As expected, a simple acylation of fluorophores to dLVP resulted in a considerable loss of affinity. If however, the Lys8 side chain was extended through double Schiff-base formation with glutaraldehyde-ethylenediamine followed by reduction to an aminoalkyl aminoalkylamine, single fluorophores could be added without loss of affinity compared to VP. The open-chain analogues, on the other hand, while displaying weak affinity, nevertheless exhibited pure antagonistic behavior.     

des-Met carboxyl-terminally modified analogues of bombesin function as potent bombesin receptor antagonists, partial agonists, or agonists.

In the present study we examined the effect of carboxyl-terminal modifications of des-Met14-bombesin (Bn) on Bn receptor affinity in murine 3T3 cells, rat and guinea pig pancreatic acini, and the ability to initiate biologic responses by synthesizing 18 des-Met14-Bn(6-13) analogues. With guinea pig acini and 3T3 cells, affinity was affected by the chain length of the alkyl moiety (R) added to [D-Phe6]Bn(6-13)NH2R with relative potencies: propyl greater than ethyl greater than butyl = hexyl greater than heptyl greater than free amide, whereas in rat acini affinity was not increased by the chain length. In each cell system the affinity of the alkylamide was not increased by insertion of a phenyl group in the alkyl side chain, by making the analogue more neuromedin B-like or by addition of a reduced peptide bond. The affinity in each cell system was increased by additions of other electron releasing groups to the COOH-terminal carboxyl group such as [D-Phe6]Bn(6-13)ethyl or methyl ester, or hydrazide. In guinea pig pancreas and 3T3 cells, 12 analogues were antagonists, 1 a full and 5 partial agonists. In rat pancreas, 8 were antagonists, 5 full agonists, and 5 partial agonists. Potent antagonists in each cell system were the methyl and ethyl ester, hydrazide, and ethylamide analogues. In 3T3 cells or guinea pig pancreas, agonist activity of the alkylamide was critically dependent on the chain length, whereas with rat pancreatic Bn receptors any alkylamide longer than the ethylamide had agonist activity. In all three cell systems any alteration that made the alkylamide more neuromedin B-like caused agonist activity. These results demonstrate that the nature of the substitution on the carboxyl terminus of des-Met14-Bn analogues is critically important, not only for determining Bn receptor affinity, but also for determining the ability to initiate a biologic response. In contrast to previous studies, the present results demonstrate that the presence of the COOH-terminal amino acid in position 14 of Bn is not essential for initiating a biologic response. Several des-Met14-Bn analogues were potent partial agonists, whereas others such as the hydrazide or ethyl ester are very potent antagonists.     

New bradykinin analogues acylated on the N-terminus: effect on rat uterus and blood pressure

Our previous studies suggested that acylation of the N-terminus of several known B2 antagonists with various kinds of bulky acyl groups consistently improved their antagonistic potency in rat blood pressure assay. On the other hand, our earlier observations also seemed to suggest that the effects of acylation on the contractility of isolated rat uterus depended substantially on the chemical character of the acyl group, as we observed that this modification might either change the range of antagonism or even transform it into agonism. Bearing all this in mind, we decided to synthesize seven new analogues of bradykinin by N-terminal acylation with various acyl groups of a moderately potent B2 antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. The analogues were tested in vitro for their blood pressure-lowering and uterotonic activities. The modifications either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, all seven substituents negatively influenced the interaction with the rat uterine receptors. Our results may be helpful for designing new B2 agonists and antagonists.     

Modifications in the bradykinin main chain are not necessary for antagonistic activity in rat blood pressure assay.

Previous studies demonstrated that the presence of a bulky acyl substituent at the N-terminus of B(2) antagonists significantly influenced the interaction of the peptide with B(2) receptors, thus increasing potency in the blood pressure test. Reported results also suggested that even minor changes in the structure of analogues might be of importance in designing more potent B(2) antagonists. On the other hand, it was learned that the effects of acylation might vary substantially with the chemical character of the acyl group. It seemed that either the positive or the negative charge on the N-terminal acyl group influenced the activity of the analogues because a suppressed antagonistic potency due to these modifications was observed.Bearing all these findings in mind, it was decided to check how N-terminal acylation would affect the pharmacological activity of bradykinin. Of the many acylating agents tested previously on B(2) antagonists, it was decided to use acridin-9-ylacetic acid (Ana) and anthracen-9-ylacetic acid (Ata). The potencies of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous BK in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by BK.     

Bisquaternary caracurine V and iso-caracurine V salts as ligands for the muscle type of nicotinic acetylcholine receptors: SAR and QSAR studies

The binding constants (K(i) values) of 24 caracurine V and 6 iso-caracurine V analogues for the muscle type of nicotinic ACh receptors (nAChR) from Torpedo californica were determined in a binding assay using (+/-)-[(3)H]epibatidine as a radioligand. The allyl alcohol group present in the iso-caracurine V ring system was found to be essential for high binding affinity. The most potent compounds are the dimethyl and di-(4-nitrobenzyl)-iso-caracurinium V salts 29 (18 nM), and 31 (79 nM), respectively. Compound 29 and the corresponding diallyl analogue 30 (350 nM) exhibited similar binding affinities as the equally substituted neuromuscular-blocking agents toxiferine I (14 nM) and alcuronium (234 nM), respectively. The SAR results were confirmed by QSAR studies, which additionally revealed that the presence of hydrogen-bond acceptor groups close to the quaternary nitrogen, is detrimental for the nicotinic binding affinity. The diallyl- and dimethylcaracurinium V salts 13 and 27, respectively, which are known to be among the most potent allosteric modulators of M(2) receptors (EC(50)=10 and 8nM, respectively), exhibited rather low nicotinic binding affinities for muscle type nAChR (K(i)=1.5 and 5.2 microM, respectively). Such a large difference in affinity suggests that it is possible to develop compounds with high muscarinic allosteric potency and low or negligible affinities for (alpha1)(2)beta1gammadelta nAChR. Additionally, the iso-caracurine V analogues with binding affinities comparable to those of (+)-tubocurarine and alcuronium could become a new class of neuromuscular-blocking agents.     

Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes

Thirteen natural bombesin-like peptides and 14 synthetic analogues were submitted to parallel bioassay on 9 smooth muscle preparations in order to determine their relative potency, in comparison to bombesin and litorin. The natural peptides of the bombesin subfamily showed a uniformly high or moderate potency on all preparations. However, synthetic bombesins of shorter chain length (hepta- and octapeptides) manifested a good potency only on the rat uterus preparation. Among the peptides of the litorin and phyllolitorin subfamilies, only litorin and ranatensin presented a full spectrum of potency, equalling or even surpassing that of bombesin. All other natural and synthetic members of the two subfamilies showed a sharply dissociated spectrum of potency on the different smooth muscle preparations. The only exception was the rat urinary bladder and, in part, the chicken intestine, on which the peptides displayed a uniformly high potency, comparable to, or even greater than that of bombesin. The present results help to explain structure/activity relationships and suggest the probable existence, in the periphery, of multiple bombesin receptor subtypes.     

Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues

Background

Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/Principal Findings

A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/Significance

Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.     

Validation of a histamine H3 receptor model through structure-activity relationships for classical H3 antagonists

Histamine H(3) receptor is a G protein-coupled receptor whose activation inhibits the synthesis and release of histamine and other neurotransmitters from nerve endings and is involved in the modulation of different central nervous system functions. H(3) antagonists have been proposed for their potential usefulness in diseases characterized by impaired neurotransmission and they have demonstrated beneficial effects on learning and food intake in animal models. In the present work, a 3D model of the rat histamine H(3) receptor, built by comparative modeling from the crystallographic coordinates of bovine rhodopsin, is presented with the discussion of its ability to predict the potency of known and new H(3) antagonists. A putative binding site for classical, imidazole-derived H(3) antagonists was identified by molecular docking. Comparison with a known pharmacophore model and the binding affinity of a new rigid H(3) antagonist (compound 1, pK(i)=8.02) allowed the characterization of a binding scheme which could also account for the different affinities observed in a recently reported series of potent H(3) antagonists, characterized by a 2-aminobenzimidazole moiety. Molecular dynamics simulations were employed to assess the stability and reliability of the proposed binding mode. Two new conformationally constrained benzimidazole derivatives were prepared and their binding affinity was tested on rat brain membranes; compound 9, designed to reproduce the conformation of a known potent H(3) antagonist, showed higher potency than compound 8, as expected from the binding scheme hypothesized.     

Combined tachykinin receptor antagonist: synthesis and stereochemical structure-activity relationships of novel morpholine analogues

We report herein the synthesis and stereochemical structure-activity relationships of novel morpholine analogues 12 and 13 with regards to NK1, NK2 and NK3 tachykinin receptor binding affinity. An essential requirement for more potent binding affinities was controlled by absolute configuration. (S,R)-12 and (S,R)-13 exhibited high binding affinities for NK1, NK2 and NK3 receptors.     

New acylated bradykinin analogues: effect on rat blood pressure and rat uterus.

It was previously reported that acylation of the N-terminus of several known B(2) antagonists with various types of bulky acyl groups consistently improved their antagonistic potency in the rat blood pressure assay. On the other hand, earlier results seem to suggest that the effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character of the acyl group, as it was observed that this modification may either change the range of antagonism or even transform it into agonism. Bearing all this in mind, three new analogues of bradykinin were designed by modifying the moderately potent B(2) antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. New analogues were obtained by acylation of the N-terminus of the above peptide with succinic acid, 12-aminododecanoic acid and 4-aminobenzoic acid in order to confirm whether either the positive or the negative charge on the N-terminal end of the peptide is responsible for the transformation of activity. The activity of analogues was assessed on blood pressure and in uterotonic in vitro tests. The modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, the three substituents, depending on their chemical character, differently influenced the interaction with the rat uterine receptors. The results may be of value in the design of new B(2) agonists and antagonists.     

Stereoselectivity and affinity in molecular pharmacology. IV. A search for eudismic-affinity correlations among angiotension II analogues

In order to establish the applicability of eudismic analysis (correlation between potency and the ratio of isomer potencies), the extensive data reported on the biological activities of angiotensin II analogues were examined. For a series of 13 heptapeptides epimeric at the N-terminal residue, apparent correlations were found for pressor activity in vivo, but not for 10 of them on the basis of their myotropic activity under in vitro conditions. The analogues belongning to sub-groups displaying correlations have side chains of sufficiently different nature so that their grouping is questionable. 3 octapeptides epimeric at the second residue displayed an excellent correlation for their pressor activity. The demonstrated differential metabolic susceptibility of these epimers, however, casts further doubt on the usefulness of the data as a measure of their potency. The lack of a correlation under in vitro conditions would indicate that eudismic-affinity correlations (EAC) may be limited to epimers which differ less in their properties than these angiotensin analogues. Nevertheless, until better receptor affinity estimates become available, the observed correlations may be useful in advancing our knowledge of the angiotensin II receptor in particular, and of stereoselective molecular recognition in general.     

Structure-activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A.

A series of analogues of neurokinin A(4-10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), all compounds except [Asp5]NKA(4-10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4-10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4-10) is in fact neurokinin B(4-10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4-10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4-10). However, [Glu5]NKA(4-10) and [Gln5]NKA(4-10) were no different from NKA(4-10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.     

Receptors for angiotensin: a critical analysis.

Angiotensin exerts numerous contractile and secretory effects by activating specific receptors. Recent pharmacological findings obtained with this peptide in various laboratories are analyzed, using the order of potency of agonists and the affinity of competitive antagonists as criteria for the classification of receptors for angiotensin in several systems. The analysis is restricted to experiments in which biological effects have been measured. Desensitization (the third criterion for classification of receptors) is discussed and a new protocol is proposed for its utilization. The analysis reveals that receptors for angiotensin in intestinal and vascular smooth muscles, in the heart, and in the vas deferens are all of the same type, while the receptors mediating the release of catecholamines from the adrenal medulla and those subserving the steroidogenic action on the adrenal cortex remain still unidentified. The recently proposed role of ATI as mediator of renin in the adrenal medulla is not substantiated by pharmacological findings with decapeptide antagonists. Moreover, the utilization of ATI as an agonist to determine the order of potency of angiotensins and the use of SQ 20881 as an inhibitor of the converting enzyme have shown serious limitations and should be reconsidered. The hypothetical role of ATIII as mediator of the renin-angiotensin system in the adrenal cortex, at least in other species than the rat, appears to be supported by the high affinity of heptapeptide antagonists for the adrenocortical receptor. However, these antagonists have generally been compared with [Sar1,Ala8]-ATII, A compound which is definitely inadequate for evaluating the affinities of octapeptides in the adrenal cortex. Therefore most of the data supporting the role of ATIII in this system have to be carefully reconsidered. Analogues of ATII are proposed for using as agonists and as antagonists instead of the natural angiotensins (for determining the order of potency of agonists) and instead of [Sar1,Ala8]-ATII (for measuring the affinities of competitive antagonists).     

Characterization of beta-adrenergic receptors in the rat vas deferens using [3H]-dihydroalprenolol binding

The beta-adrenergic antagonist, [3H]-dihydroalprenolol ([3H] DHA), binds to membranes prepared from the rat vas deferens in a specific and saturable manner. Scatchard and Hill plot analysis indicates a single class of binding sites with no evidence of cooperative interactions. The specific binding sites have a high affinity (Kd = 0.3 nM) and a maximal occupancy estimated to be 460 fmoles [3H]-DHA bound/g wet tissue weight. Beta-adrenergic agonists and/or antagonists inhibit [3H]-DHA binding to rat vas deferens membranes in a stereospecific manner and with a relative order of potency expected for beta-adrenergic receptors of the beta2 subtype. The receptor affinities of various beta-adrenergic antagonists in the rat vas deferens determined using inhibition of [3H]-DHA binding correlated with their receptor affinities determined physiologically using antagonism of isoproterenol-induced inhibition of neurogenic contractions in-vitro.