弓状核中阿片受体在细胞介素所致发热效应中的作用

本研究旨在了解弓状核内的阿片受体在体温调节中的作用。研究使用细胞介素ＩＬ１β做致热源。以自动推进器向ＳＤ雄性大鼠弓状核微量注射１μ１ＩＬ１β。在给药前３０ｍｉｎ分别向弓状核微量注射通常阿片受体拮抗剂纳洛酮（Ｎａｌ）、阿片受体μ、δ和κ各自特异性拮抗剂ＣＴＡＰ、ＮＴＩ和ｎｏｒＢＮＩ做预处理,用生理盐水（Ｓａｌ）做对照。结果表明：ＩＬ１β所致的升体温效应能被Ｎａｌ和ＣＴＡＰ阻断,提示弓状核中的阿片受体（主要是μ受体）参与或介导了ＩＬ１β的致热效应;δ和κ受体特异性拮抗剂阻断ＩＬ１β所致的体温升高效应不明显。提示δ和κ阿片受体参与体温调节的可能性较小。对照ＡＲＨ和ＰＯＡＨ中阿片受体在ＩＬ１β所致发热中的作用可发现：二者作用极为相似,这一结果有力地支持了弓状核是体温调节中枢重要组成部分的观点。     

Involvement of spinal kappa opioid receptors in the antagonistic effect of dynorphins on morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.     

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.     

Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone.

The pharmacological profile of naltrindole (NTI) and three of its analogues, N-methyl-NTI (N-Me-NTI), oxymorphindole (OMI) and naltriben (NTB) were studied in antinociceptive assays. The compounds were found to have agonist activities that appear to be mediated mainly by kappa opioid receptors because norbinaltorphimine (nor-BNI), the selective kappa opioid receptor antagonist inhibited their effects significantly. All of the compounds, behaved as antagonists at doses that were lower than those that produced agonist effects and they possessed a profile that was very selective for inhibiting the antinociceptive activities of delta opioid receptor agonists. Differential antagonism by NTB of the activities of DSLET and DPDPE was demonstrated.     

Thermic response of selective muscarinic agonists and antagonists in rat

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of the opioid system in the central antisecretory action of alpha-2 adrenoceptor agonists in rat

The aim of the present study was to analyse the role of the central alpha-2 adrenoceptors in the regulation of gastric acid secretion in pylorus ligated rats. It was found that the intracerebroventricularly (icv.) injected presynaptic alpha-2 adrenoceptor agonist clonidine and the alpha-2A adrenoceptor subtype selective stimulant oxymetazoline exerted a dose dependent inhibition on gastric acid secretion. The antisecretory ED(50) values for clonidine and oxymetazoline were 20 and 7.5 nmol/rat icv., respectively. The antisecretory effect of these compounds was antagonised by the presynaptic adrenoceptor antagonist yohimbine (50 nmol/rat icv.) indicating that the action is mediated through central presynaptic alpha-2 adrenoceptors. Moreover, naloxone (50 nmol/rat icv.)--non-selective opioid antagonist--and naltrindole (0.5 nmol/rat icv.)--delta-opioid receptor selective antagonist--also decreased the antisecretory effect of clonidine and oxymetazoline suggesting that the endogenous opioid system is likely to be involved in the central antisecretory action of alpha-2 adrenoceptor stimulants.     

Peripheral effect of a kappa opioid receptor antagonist on nociception evoked by formalin injected in TMJ of pregnant rats

The effect of sex hormones on orofacial pain modulation is poorly understood. Therefore, this study aimed to investigate the effect of hormonal changes as a result of pregnancy, as well as that of the kappa (kappa) opioid receptor antagonist on female rats' sensitivity to the temporomandibular joint (TMJ) formalin test. Initially, female rats at estrus and pregnant females on day 19 of pregnancy received a 50 microl formalin (1.5%) injection in the right TMJ. The pregnant females showed a reduction in nociceptive responses to the TMJ formalin test when compared with those at estrus. Then, the selective kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), was co-administered with the formalin. Next, additional groups received the kappa (200 microg) receptor antagonist or 0.9% NaCl 24 hours prior to the periarticular injection of formalin. Co-administration of nor-BNI with formalin into the TMJ region had no significant effect. The pre-injection of selective kappa-opioid receptor antagonist, nor-BNI, significantly enhanced the nociceptive behavioral responses in pregnant females. When applied in the contralateral TMJ, nor-BNI did not affect the magnitude of the nociceptive response induced by formalin. It can be concluded that: 1) The increase of the sex hormone levels, as result of pregnancy, induces a reduction of nociceptive behavioral responses to the TMJ formalin test; 2) the peripheral kappa opioid receptor activation, by endogenous opioid agonists release, is involved in the antinociception to TMJ formalin test, induced by pregnancy.     

The cardiovascular responses to mu opioid agonist and antagonist in conscious normal and obese rats

Beta-endorphin decreases blood pressure in normal rats but increases blood pressure in obese rats. Since beta-endorphins can bind both mu opioid and kappa-opioid receptors we investigated the effect of a mu specific receptor agonist, D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and a mu specific antagonist, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) on cardiovascular responses in conscious control and obese rats. Rats were also implanted with telemetry transmitters and intracerebroventricular (ICV) cannulas for recording and peptide administration. The mu agonist, DAMGO, increased blood pressure (BP) in control rats. DAMGO also increased BP in obese rats but only at high concentrations. The heart rate responses paralleled the MAP responses. CTAP, the mu antagonist, paradoxically increased the MAP in both control and obese rats. The responsiveness to the mu agonist and antagonist was greater in controls. In other animals the brains were excised and the ventral medial hypothalamic area removed and mu receptor expression determined using PCR. The expression of mu opioid receptors was increased in obese rats. We conclude that the mu opioids can stimulate cardiovascular responses, but the excitatory responsiveness was not increased in conscious obese rats.     

Drug design and synthesis of epsilon opioid receptor agonist: 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative epsilon opioid receptor

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative epsilon opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative epsilon opioid receptor (IC(50) = 71.71nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine 5'-triphosphate analog, guanosine 5'-(gamma-thio)-triphosphate (GTPgammaS), to the mouse pons/medulla membrane via the activation of putative epsilon opioid receptor. Moreover, TAN-821 given intracerebroventricularly (i.c.v.) produced a marked antinociception in the tail-flick test (ED(50) = 1.73 microg) and the hot-plate test (ED(50) = 2.05 microg) in a dose-dependent manner. The antinociception induced by TAN-821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative epsilon opioid receptor partial agonist beta-endorphin [1-27], but not a mu opioid receptor antagonist beta-FNA, a delta opioid receptor antagonist NTI, or a kappa opioid receptor antagonist nor-BNI. The present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative epsilon opioid receptor.     

Pharmacological effects of naltriben as a ligand for opioid mu and kappa receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.     

Spinal administration of selective opioid antagonists in amphibians: evidence for an opioid unireceptor

In mammals, opioids act by interactions with three distinct types of receptors: mu, delta, or kappa opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective mu, delta, or kappa opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, beta-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested mu, delta, and kappa opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor.     

Cardiovascular responses to central administration of mu and kappa opioid receptor agonist and antagonist in normal rats

The response to centrally administered beta-endorphin has been characterized by decreasing sympathetic nervous activity and decreased cardiovascular tone. We investigated the effect of the central administration of both mu and kappa opioid receptor agonist and antagonists on cardiovascular responses. The administration of the mu agonist, DAMGO (0.2nmol) increased the mean arterial pressure (MAP) and stimulated iliac vasoconstriction while higher doses (2 and 20nmol) decreased MAP and stimulated iliac vasodilation. The administration of the kappa receptor agonist, Dynorphin decreased the MAP and stimulated superior mesenteric vasodilation. beta-Funaltrexamine reduced MAP and superior mesenteric vasodilation while nor-binaltorphimine increased MAP and iliac and superior mesenteric vasoconstriction. We conclude that mu receptor activation decrease or increase MAP depending on the mu agonist concentration. However, kappa receptor activation is consistently associated with a decrease in MAP.     

Synthesis of a novel 6,14-epoxymorphinan derivative and its pharmacology

A novel 6,14-epoxymorphinan benzamide derivative (NS22) was synthesized, which showed opioid kappa receptor agonistic activity in the [(35)S]GTPgammaS binding assay. The antinociceptive effect of NS22 was evaluated in the tail-flick and the hot-plate test. This compound showed a potent antinociceptive activity in mice by s.c. administration, which was attenuated with nor-BNI (selective opioid kappa receptor antagonist).     

Inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) on gastrointestinal transit

The inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) (TAPA), a highly selective mu(1)-opioid receptor agonist, on mouse gastrointestinal transit was compared with that of morphine and [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]-enkephalin (DAMGO). When administered intracerebroventricularly 5 min before the oral injection of charcoal meal, TAPA (10-100 pmol), morphine (0.25-4 nmol), and DAMGO (20-80 pmol) dose-dependently inhibited gastrointestinal transit of charcoal. The inhibitory effect of each mu-opioid receptor agonist was completely antagonized by naloxone, a nonselective opioid receptor antagonist. The inhibitory effects of morphine and DAMGO were significantly antagonized by both beta-funaltrexamine, a selective mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist. In contrast, the inhibitory effect of TAPA was not affected at all by beta-funaltrexamine, naloxonazine, nor-binaltorphimine (a selective kappa-opioid receptor antagonist), or naltrindole (a selective delta-opioid receptor antagonist). These results suggest that the inhibitory effect of TAPA on gastrointestinal transit may be mediated through an opioid receptor mechanism different from that of morphine and DAMGO.     

Selective kappa antagonist properties of nor-binaltorphimine in the rat MES seizure model

The opioid antagonist properties of nor-binaltorphimine (nor-BNI; 17,17'-Bis(cyclopropylmethyl)-6,6',7,7'-tetradehydro-4,5:4', 5'-diepoxy-6,6'-(imino) [7,7'-bimorphinan]-3,3',14,14'-tetrol) were evaluated in vivo in the rat maximal electroshock (MES) seizure model. Following s.c. or i.c.v. pretreatment, nor-BNI selectively antagonized the anticonvulsant effects of the kappa opioid U50, 488, significantly increasing its ED50 by 2.3 and 4.5 fold, respectively. In contrast, pretreatment with nor-BNI (s.c. or i.c.v.) failed to antagonize the anticonvulsant effects of the selective mu opioid, DAMGO. At the doses and injection routes used, nor-BNI itself had no apparent effect on overt behavior or MES-induced convulsions. These data support the earlier suggestion that the anticonvulsant effects of U50,488 are mediated by kappa opioid receptors and confirm 1) the selectivity of nor-BNI as a kappa antagonist and 2) its applicability as a pharmacological tool in the differentiation of multiple opioid receptors.     

An orexigenic role for mu-opioid receptors in the lateral parabrachial nucleus

The pontine parabrachial nucleus (PBN) has been implicated in regulating ingestion and contains opioids that promote feeding elsewhere in the brain. We tested the actions of the selective mu-opioid receptor (mu-OR) agonist [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) in the PBN on feeding in male rats with free access to food. Infusing DAMGO (0.5-4.0 nmol/0.5 microl) into the lateral parabrachial region (LPBN) increased food intake. The hyperphagic effect was anatomically specific to infusions within the LPBN, dose and time related, and selective for ingestion of chow compared with (nonnutritive) kaolin. The nonselective opioid antagonist naloxone (0.1-10.0 nmol intra-PBN) antagonized DAMGO-induced feeding, with complete blockade by 1.0 nmol and no effect on baseline. The highly selective mu-opioid antagonist d-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 1.0 nmol) also prevented this action of DAMGO, but the kappa-antagonist nor-binaltorphimine did not. Naloxone and CTAP (10.0 nmol) decreased intake during scheduled feeding. Thus stimulating mu-ORs in the LPBN increases feeding, whereas antagonizing these sites inhibits feeding. Together, our results implicate mu-ORs in the LPBN in the normal regulation of food intake.     

Mobilization of calcium from intracellular stores as one of the mechanisms underlying the antiopioid effect of cholecystokinin octapeptide.

In enzymatically dissociated brain cells prepared from neonatal rats, KCl produced a significant increase in [Ca2+]i and this increase could be prevented by verapamil or nifedipine, known to block voltage-sensitive calcium channels. The opioid receptor agonists ohmefentanyl (OMF, mu agonist), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist), and 66A-078 (kappa agonist) produced a marked suppression of the Ca2+ influx induced by high K+ depolarization. The suppressive effect of OMF, DPDPE, and 66A-078 on the high K(+)-induced increase in [Ca2+]i was markedly reversed by their respective antagonists beta-funaltrexamine (beta-FNA), ICI174864, and nor-binaltorphimine (nor-BNI). Cholecystokinin octapeptide (CCK-8), at concentrations of 0.3, 3.0, and 30 nM, dose-dependently mobilized Ca2+ from intracellular stores. While CCK-8 30 nM did not affect significantly the increase of [Ca2+]i following high K+, it did reverse the suppression of the high K(+)-induced increase in [Ca2+]i by the mu agonist OMF and the kappa agonist 66A-078, but not that by the delta agonist DPDPE. The results suggested that while opioid ligands suppress [Ca2+]i by blocking voltage-operated Ca2+ influx, the antiopioid effect of CCK-8 seems to be operated via mobilization of Ca2+ from intracellular stores.     

Effect of a mu-opioid receptor-selective antagonist on interleukin-6 fever

The endogenous opioid system has been found to be involved in fever caused by pyrogens. Recent work in our laboratory has demonstrated that the mu-opioid receptor is involved in interleukin-1beta (IL-1beta)- and in lipopolysaccharide (LPS)-induced fevers. In the present study, we have investigated the role of the mu-opioid receptor in the preoptic anterior hypothalamus (POAH) in fever induced by interleukin-6 (IL-6). Following stereotaxic implantation of a guide cannula into the POAH for microinjection, radio transmitters to monitor body temperature (Tb) continuously were inserted intraperitoneally. Adult male Sprague-Dawley rats were microinjected with 0.5 microg of the selective mu-opioid receptor antagonist, cyclic D-phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), into the POAH. Thirty min later, IL-6 (100 ng) was injected into the POAH. CTAP significantly blocked the IL-6 fever. CTAP alone had no effect on Tb during the 390-min recording period. These data indicate that mu-opioid receptors within the POAH mediate IL-6 fever and add to the increasing evidence that the opioid system is involved in the pathogenesis of fever in rats.     

Analgesic effect of interferon-alpha via mu opioid receptor in the rat

Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors.     

Structure-activity relationships of arodyn, a novel acetylated kappa opioid receptor antagonist.

We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn A-(1-11)NH(2), Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617-5619) is a kappa opioid receptor-selective peptide [K(i)(kappa) = 10 nm, K(i) ratio (kappa/mu/delta) = 1/174/583] which exhibits antagonist activity at kappa opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg(6) and Arg(7) are the most important residues for arodyn's nanomolar binding affinity for kappa opioid receptors. Ala substitution of the other basic residues (Arg(4), Arg(9) and Lys(11)) resulted in lower decreases in affinity for kappa opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala(10)]arodyn exhibits similar kappa opioid receptor binding as arodyn, it displays higher kappa vs. mu opioid receptor selectivity [K(i) ratio (kappa/mu) = 1/350] than arodyn because of a twofold loss in affinity at mu opioid receptors. Surprisingly, the Tyr(1) analog exhibits a sevenfold decrease in kappa opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr(1)]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing kappa opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe(1)]arodyn which exhibits high affinity [K(i)(kappa) = 4.56 nm] and exceptional selectivity for kappa opioid receptors [K(i) ratio (kappa/mu/delta) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A-(1-13)NH(2). Thus [NMePhe(1)]arodyn is a highly kappa opioid receptor-selective antagonist that could be a useful pharmacological tool to study kappa opioid receptor-mediated activities.