Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus. Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae, CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria. The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10(-9)-10(-6) M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus. CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10(-9)-10(-6) M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor, however, the CavTKs induced no significant effect on the heart.Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut.In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.     

Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies

Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK.     

(35)S]GTPgammaS binding stimulated by endomorphin-2 and morphiceptin analogs

The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.     

Novel analogs of alloferon: Synthesis,conformational studies,pro-apoptotic and antiviral activity

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1_MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala⁹]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys⁹]-, [Phg⁹]-, [Lys¹²]-, and [Phe¹²]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.     

Interactions of selected insect neuropeptides with synthetic lipids.

Interactions of the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr), its [beta-cyclohexyl-(4-O-methyl)2]-L-alanine analog, and the leucopyrokinin [2-8]-fragment (Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2), with synthetic phospholipids (DPPC, DMPE, DMPG) were studied using the microcalorimetric method. Most pronounced changes of the lipid thermotropic behavior were observed with DMPG/leucopyrokinin [2-8]-fragment mixtures. Proctolin itself was less active with all the lipids studied. The results obtained suggest that the studied peptides interact with the head group region of lipid bilayer.     

Proctolin antagonists bind to [(3)H]proctolin binding sites in the locust hindgut

Proctolin caused dose-dependent (1-200 nM) contraction of the isolated hindgut of S. gregaria which was abolished by [alpha-methyl-L-tyrosine(2)]-proctolin (1 microM). In comparison, cycloproctolin (5 microM) reduced the proctolin maximum response by 41%. Hindgut homogenates contained [(3)H]proctolin binding sites with a K(d) value of 660 nM, a B(max) value of 23.8 pmol/mg protein and a Hill coefficient of 0.934. Cycloproctolin (IC(50,) 220 nM; K(i), 204 nM), unlabeled proctolin (IC(50) 680 nM) and [alpha-methyl-L-tryosine(2)]-proctolin (IC(50) 3.1 microM, K(i), 2.9 microM) but not SchistoFLRFamide (1 nM-10 microM) were capable of displacing bound [(3)H]proctolin.     

Effect of corazonin and crustacean cardioactive peptide on heartbeat in the adult American cockroach (Periplaneta americana)

Changes in the frequency of cardiac pulsations have been monitored in the decapitated body of adult P. americana before and 5 h after the injections of [Arg(7)]-corazonin and CCAP, using newly invented touch-free, noninvasive optocardiographic methods. Relatively large dosages of these peptides (10(-6) M concentrations in the body) had no effect on the rate of the heartbeat beyond the Ringer control limits. It has been concluded, therefore, that Corazonin and CCAP, which are currently cited in the literature as "the most potent cardiostimulating peptides" in insects, have no effect on the physiological regulation of cardiac functions in the living body.     

Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs

In this brief overview we give the historical background on the discovery of myostimulatory neuropeptides in cockroaches. Related peptides were later found in other insect groups as well. We summarize the current knowledge on primary structures, localization, physiological and pharmacological effects of the different cockroach neuropeptides, including kinins, sulfakinins, pyrokinins, tachykinin-related peptides, periviscerokinins, corazonin, and proctolin. In addition, we briefly comment on the development of mimetic pseudopeptide analogs in the context of their possible use in insect pest management.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity.

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.     

Alpha,beta-dehydrophenylalanine containing cecropin-melittin hybrid peptides: conformation and activity.

Synthesis and conformational studies of a cecropin-melittin hybrid pentadecapeptide CA(1-7)MEL(2-9), and its three alpha, beta-dehydrophenylalanine (DeltaPhe) containing analogs in water-TFE mixtures are described. DeltaPhe is placed at strategic positions in order to preserve the amphipathicity of the molecule. The wild type CAMEL0 and its three analogs, containing one, two and three DeltaPhe residues namely CAMELDeltaPhe1, CAMELDeltaPhe2 and CAMELDeltaPhe3 respectively were synthesized in solid phase and their conformation determined by CD and NMR. CAMELDeltaPhe2 and CAMELDeltaPhe3 peptides exhibit the presence of 3(10)-helix and beta-turns in the former and only turns in the latter. CAMELDeltaPhe1 peptide was found to have a largely extended conformation. Antibacterial and hemolytic activities of the peptides were also evaluated. CAMELDeltaPhe2 peptide is maximally potent against both Staphylococcus aureus ATCC 259230 and Escherichia coli ATCC 11303. CAMELDeltaPhe1 with a single DeltaPhe at the center shows minimal hemolysis.     

Functional Consequences of Peptide Cotransmission in Arthropod Muscle

While the widespread occurrence of peptidergic neurons has beenamply demonstrated, their physiological significance, particularlywith regard to co-release of peptides and conventional transmitters,remains a topic of considerable interest. The innervation ofarthropod muscle by proctolinergic neurons provides favorableopportunities for analysis of cotransmitter actions and theirphysiological consequences. Three uniquely identified neuronsare described, each of which releases the neuropeptide proctolin(Arg-Tyr- Leu-Pro-Thr) as a cotransmitter at a well-definedmuscle target. Activity in the neurosecretory LW neuron increasesthe force and frequency of insect heartbeat in a manner similarto that obtained by bath application of the peptide. This neuronmay release peptide hormones together with multiple cardioacceleratorypeptides at the heart to achieve, through increased blood flow,rapid changes in hormonal state. Two motoneurons co-releaseproctolin with a conventional neurotransmitter to produce bothdirect and joint actions on muscle targets. The cockroach Dsmotoneuron induces slow, sustained tension in the coxal depressormuscle in the absence of depolarization, and slows the relaxationof fast twitch events caused by L-glutamate. In the crayfishtonic flexor muscle, proctolin release from the f 1 motoneuronamplifies depolarization-dependent tension, but does not affectthe time course of relaxation. Proctolinergic cotransmissionmay be an adaptation for amplification of synaptic input and/ormaintenance of long-term tension, both of which increase theefficiency of muscle activation by motoneurons.     

Structure function analysis of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor

Proctolin is a pentapeptide (arg-tyr-leu-pro-thr) found in nervous tissues throughout the phylum Arthropoda. Initially described as a peptidergic neuromuscular transmitter, it now appears that proctolin is a major arthropod neurohormone modulating nervous activity, muscle tonus and contractile force. Structure-function studies with synthetic analogues demonstrate diverse peptides which retain agonistic activity, but few exhibit a high degree of affinity for the cockroach hindgut receptor compared with proctolin (Kdapp = 2 x 10(-8) M). High affinity agonists (Kdapp less than or equal to 10(-7) M) are limited to [phe2]-proctolin, [lys1]-proctolin and specific N-terminal additions. In this regard the hindgut receptor differs in its ligand specificity from that reported for the locust extensor tibia receptor. Using the analogue studies to predict sequences which may act as agonists, we have examined the known vertebrate peptide hormones for proctolin-like sequences. A possible relationship between vasoactive intestinal peptide, proctolin and erythrophore concentrating hormone is critically evaluated.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

New Chimeric Analogues of Galanin: Synthesis and Effects on the Glucose-Induced Insulin Secretion

The solid-phase synthesis and in vitro assays on the glucose-induced insulin secretion from rat pancreatic islets of Langerhans with six new chimeric peptides were performed. All the peptides were built up of the N-terminal galanin (GAL) fragment or its analogues, linked to the C-terminal portion of substance P (SP) analogues or scyliorhinin I (SCY-I) analogues. Two strong antagonists of the inhibitory effect of galanin on the glucose-induced insulin release were found: [cycloleucine⁴]GAL(1-13)-SP(5-11)-amide and GAL(1-13)-[L-norleucine¹⁰]SCY-I(3-10)-amide.     

Potent V2 vasopressin antagonists with structural changes at their C-terminals

A variety of structural changes were made in the C-terminals of four potent antidiuretic (V2) antagonists. The parent analogs were all derivatives of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)]arginine-vasopressin, d(CH2)5AVP, namely d(CH2)5[D-Phe2,Ile4]AVP, d(CH2)5[D-Ile2,Ile4]AVP, d(CH2)5[D-Tyr(Et)2, Val4]AVP and d(CH2)5[D-Tyr(Et)2,Ile4]AVP. A number of amino acid amides were substituted for the C-terminal 9-glycinamide without reducing their V2-antagonistic potencies in rats. Many non-amino acid structures were also tolerated at the C-terminals of these antagonists and this end of these peptides can be prolonged without interfering with antagonistic potencies. Such altered V2-antagonists may be useful for the development of radioactive ligands, affinity labels and in affinity columns for studies on antidiuretic receptors. These C-terminal modifications also provide useful information for the further development of potent and specific V2-antagonists which can be valuable pharmacological tools and also promise to become useful clinically for the treatment of excessive water retention.     

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.     

Solution conformation of Substance P antagonists-[D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP by CD, NMR and MD simulations

Substance P (SP) is an important neuropeptide involved in pain transmission and induction of inflammation. Its antagonists are being extensively investigated for their non-narcotic analgesic and anti-inflammatory activity. With a view towards better understanding the structural requirements of these analogs for efficient interaction with the SP receptor, the conformation of three SP antagonists [D-Arg1, D-Trp7,9, Leu11]-SP, [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP and [D-Pro2, D-Trp7,9]-SP has been studied by CD, NMR and molecular dynamics (MD) simulations. All three peptides exhibit a high dependence of structure on the solvent. The molecules tend to adopt beta-turns in solvents like DMSO and H2O and form helices in a hydrophobic environment. A direct relation between the helix forming potential of these antagonists with their receptor binding potency has been observed.