Κ1 Opioid Receptor Research Articles

Comparative Analysis of Infarct Size Limiting Activity of κ-Opioid Receptor Agonists in In Vivo Reperfused Heart.

A 45-min coronary artery occlusion followed by a 120-min reperfusion was performed in rats anesthetized with α-chloralose. The selective κ1-opioid receptor (OR) agonist U-50,488 was administered intravenously in doses of 0.1 or 1 mg/kg. The selective κ2-OR agonist GR-89696 was injected in a dose of 0.1 mg/kg. The selective κ1-OR agonists ICI-199,441 and ICI-204,448 were employed in the doses of 0.1 and 4 mg/kg, respectively. These drugs were injected 5 min prior to reperfusion. U-50,488 exerted the cardioprotective effect in a dose of 1 mg/kg, but it produced no effect on infarct size in a dose of 0.1 mg/kg. ICI-199,441 reduced the reperfusion injury to the heart. The infarct size limiting effects of U-50,488 and ICI-199,441 were prevented by preliminary injection of naltrexone or nor-binaltorphimine. It is concluded that infarct size limiting effects of U-50,488 and ICI-199,441 were mediated via activation of κ1-OR.

2-phenyl-1-(3-pyrrolidin-1-il-propyl)-1H-indole hydrochloride (SS-68): Antiarrhythmic and Cardioprotective Activity and Its Molecular Mechanisms of Action (Part II)

Introduction. In previous studies on different animal models, it was shown that compound N-(N-butylpyrrolidine)-2-phenylindole hydrochloride (SS-68) has a broad antiarrhythmic activity. The molecular mechanisms of the pharmacological action of SS-68 were chosen as the focus for this study.
 Materials and methods. The study of the molecular basis of the pharmacological action of SS-68 was based on 1) molecular docking with the determination of the affinity constant for κ1-opioid receptors; 2) recording the fluorescence of a culture of cardiomyocytes with the determination of the effect of SS-68 on ionic homeostasis; 3) determining the negative chronotropic action in vitro; 4) studying the effect of SS-68 on the transmembrane ion currents of isolated unidentified neurons of the large pond snail (Lymnaeastagnalis), orb snail (Planorbariuscorneus) and rat hippocampal neuron cultures.
 Results. 1) In experiments using molecular docking, the affinity of SS-68 for κ1-opioid receptors is significantly higher than that of butorphanol, but lower than that of (-)-U-50.488; 2) In spontaneously excited preparations of the right atrium, SS-68 causes an irreversible negative chronotropic effect. In experiments on atrial myocardium in rats, SS-68 is capable of demonstrating the ability to block M2 and M3-cholinergic receptors; 3) When studying the effects on cardiac myocyte ion currents, it was shown that SS-68 has moderate Na+, K+ and Ca2+ – blocking activity; 4) In the study of isolated neurons, it was shown that SS-68 influences the electrophysiology of neurocytes in a dose-dependent manner.
 Discussion. The study of the molecular basis of the action of SS-68 showed that this compound has a pleiotropic multitarget effect, which consists of, at least, the effect on Na+, Ca2+ and K+-homeostasis of cardiomyocytes and neurons, M2-, M3-cholinergic receptors, and κ1-opioid receptors.
 Conclusion. From the point of view of molecular pharmacology, SS-68 can be attributed to an antiarrhythmic drug with a mixed type of action.

Hypermethylation of the κ1 opioid receptor promoter in Chinese heroin and methamphetamine addicts.

Heroin and methamphetamine (METH) addiction continues to be a major social, economic and therapeutic problem worldwide. The opioid pathway may mediate the effects of addictive drugs. However, the potential correlation between the κ1 opioid receptor (OPRK1) and drug addiction has not yet been characterized. The aim of the present study was to investigate the potential association between methylation of the OPRK1 promoter and substance abuse. Bisulfite pyrosequencing technology was used to determine the levels of OPRK1 promoter methylation in 60 drug abusers (30 heroin and 30 METH addicts) and 52 controls, observed to exhibit no significant differences in age or gender. The results indicated that levels of OPRK1 promoter methylation were significantly higher in drug addicts when compared with controls (P=2.43×10−4). Significant correlations between OPRK1 promoter methylation and the length and frequency of drug use were also observed in male heroin addicts (length: r=0.661, P=0.007; frequency: r=−0.684, P=0.005). In addition, a luciferase reporter gene assay indicated that the OPRK1 promoter fragment was able to regulate gene expression (fold change between two groups >32.12, P≤0.0001). In conclusion, results of the present study indicate that methylation of the OPRK1 promoter contributes to the pathophysiology of drug addiction.

Panicolytic-like effects caused by substantia nigra pars reticulata pretreatment with low doses of endomorphin-1 and high doses of CTOP or the NOP receptors antagonist JTC-801 in male Rattus norvegicus.

Gamma-aminobutyric acid (GABA)ergic neurons of the substantia nigra pars reticulata (SNpr) are connected to the deep layers of the superior colliculus (dlSC). The dlSC, in turn, connect with the SNpr through opioid projections. Nociceptin/orphanin FQ peptide (N/OFQ) is a natural ligand of a Gi protein-coupled nociceptin receptor (ORL1; NOP) that is also found in the SNpr. Our hypothesis is that tectonigral opioid pathways and intranigral orphanin-mediated mechanisms modulate GABAergic nigrotectal connections. Therefore, the aim of this work was to study the role of opioid and NOP receptors in the SNpr during the modulation of defence reactions organised by the dlSC. The SNpr was pretreated with either opioid or NOP receptor agonists and antagonists, followed by dlSC treatment with bicuculline. Blockade of GABAA receptors in the dlSC elicited fear-related defensive behaviour. Pretreatment of the SNpr with naloxone benzoylhydrazone (NalBzoH), a μ-, δ-, and κ1-opioid receptor antagonist as well as a NOP receptor antagonist, decreased the aversive effect of bicuculline treatment on the dlSC. Either μ-opioid receptor activation or blockade by SNpr microinjection of endomorphin-1 (EM-1) and CTOP promoted pro-aversive and anti-aversive actions, respectively, that modulated the defensive responses elicited by bicuculline injection into the dlSC. Pretreatment of the SNpr with the selective NOP receptor antagonist JTC801 decreased the aversive effect of bicuculline, and microinjections of the selective NOP receptor agonist NNC 63-0532 promoted the opposite effect. These results demonstrate that opioid pathways and orphanin-mediated mechanisms have a critical role in modulating the activity of nigrotectal GABAergic pathways during the organisation of defensive behaviours.

Prospects for Creation of Cardioprotective and Antiarrhythmic Drugs Based on Opioid Receptor Agonists.

It has now been demonstrated that the μ, δ1, δ2, and κ1 opioid receptor (OR) agonists represent the most promising group of opioids for the creation of drugs enhancing cardiac tolerance to the detrimental effects of ischemia/reperfusion (I/R). Opioids are able to prevent necrosis and apoptosis of cardiomyocytes during I/R and improve cardiac contractility in the reperfusion period. The OR agonists exert an infarct‐reducing effect with prophylactic administration and prevent reperfusion‐induced cardiomyocyte death when ischemic injury of heart has already occurred; that is, opioids can mimic preconditioning and postconditioning phenomena. Furthermore, opioids are also effective in preventing ischemia‐induced arrhythmias.

Opioids as Triggers of the Adaptive Phenomenon of Ischemic Preconditioning of the Heart

Transient ischemia/reperfusion has been shown to evoke increases in enkephalin levels in myocardial tissue. Blockade of δ opioid receptors eliminates the cardioprotective effect of ischemic preconditioning both in vivo and in vitro. Inhibition of κ1 opioid receptors eliminates the cardioprotective effect of preconditioning only in vitro. Agonists of μ, δ1, δ2, and κ1 opioid receptors imitate the cardioprotective effect of preconditioning. These results indicate that endogenous opioid peptides are triggers of ischemic preconditioning.

Prospects of Using of κ-Opioid Receptor Agonists U-50,488 and ICI 199,441 for Improving Heart Resistance to Ischemia/Reperfusion.

We studied the ability of the agonist of κ1-opioid receptors U-50,488 in doses of 0.1 and 1 mg/kg to simulate ischemic pre- and postconditioning of the heart and κ-opioid receptors ICI 199,441 in a dose of 0.1 mg/kg to simulate the antiarrhythmic effect of heart preconditioning. The duration of ischemia was 10 or 45 min and the duration of reperfusion was 10 min or 2 h. Administration of 1 mg/kg U-50,488 both before ischemia and 5 min before reperfusion produced a pronounced antiarrhythmic effect. U-50,488 injected 5 min before reperfusion 2-fold reduced the ratio of infarction to risk area. Administration of ICI 199,441 in a dose of 0.1 mg/kg 15 min before ischemia produced a potent antiarrhythmic effect. Antiarrhythmic effect of κ-opioid receptor agonists depended on activation of κ-opioid receptors.

Role of Intracellular Calcium and Cyclic Nucleotides in Realization of Cardioprotective Effects of δ1- and κ1-Opioid Receptor Agonists

The role of cyclic nucleotides (cAMP, cGMP) and Ca(2+)-ATPase of the sarcoplasmic reticulum in the mechanism of cardioprotective effects of selective δ(1)- and κ(1)-opioid receptor agonists DPDPE and U-50488 was studied under conditions of global ischemia and reperfusion of isolated and perfused rat heart. Activation of both types of opioid receptors 2-fold reduced the reperfusion release of creatine phosphokinase. The cardioprotective effect of U-50488 was paralleled by a 2-fold decrease in cAMP content in the myocardium, while DPDPE did not modify the content of cAMP throughout the experiment. None of these substances changed the content of cGMP in the myocardium. The cardioprotective effect of DPDPE was not observed after inhibition of sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid. The cardioprotective effect of U-50488 was associated with reduction of cAMP level in the myocardium, while the cytoprotective effect of DPDPE was mediated by opioidergic modulation of Ca(2+) transport at the level of the sarcoplasmic reticulum.

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

Cardioprotective, inotropic, and antiarrhythmic effects of the selective agonist of k1-opioid receptors (k1-ORs) U-50.488H have been studied after 45-min global ischemia and 30-min reperfusion of isolated perfused rat hearts. The heart k1-ORs were stimulated by adding 0.1 or 1 mmol/l U-50.488H to the perfusion solution. The opioid did not affect the frequency of reperfusion arrhythmias. At a concentration of 0.1 mmol/l, it induced a twofold decrease in the reperfusion release of creatine phosphokinase (CPK), which positively correlated with a decrease in the myocardial cAMP level (r = 0.89, p < 0.01). Application of U-50.488H at a final concentration of 1 mmol/l did not change the cAMP level and CPK release. These results suggest that the cardioprotective effect of U-50.488H is due to a decrease in the level of cAMP in cardiomyocytes. Activation of k1-ORs decreased the frequency and force of myocardial contractions. It has been shown that the negative inotropic and chronotropic effects of U-50.488H are independent of changes in the myocardial cAMP level. A hypothesis is proposed that the absence of cardioprotective effect of 1 mM U-50.488H is a result of activation of nonopioid receptors in cardiomyocytes.

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

Role of κ1 opioid receptors and cAMP in regulation of cardiac tolerance to ischemia and reperfusion

Cardioprotective effect of κ1-opioid receptor activation and role of cAMP in its realization

The cardioprotective and antiarrhythmic effects of a selective kappa1-opioid receptor agonist U-50,488 were studied during experimental 45-min total ischemia and 30-min reperfusion of isolated rat heart. The opioid had no effect on the incidence and type of reperfusion arrhythmias. U-50,488 in a concentration of 0.1 microM inhibited reperfusion-induced release of creatine phosphokinase and decreased cAMP concentration in the myocardium by 2 times. These parameters remained unchanged after treatment with U-50,488 in a concentration of 1 microM. The cardioprotective effect of U-50,488 was probably associated with a decrease in cAMP concentration in heart cells. U-50,488 in a concentration of 1 microM produced no cardioprotective effect, which can be explained by its interaction with an unknown non-opioid receptor in cardiomyocytes.

Modification of κ-Opioid Receptor Agonist-Induced Antinociception by Diabetes in the Mouse Brain and Spinal Cord

The supraspinal and spinal antinociceptive effects of several κ-opioid receptor agonists were examined in diabetic and non-diabetic mice using the tail-flick assay. The antinociception induced by intrathecal (i.t.), but not intracerebroventricular (i.c.v.), CI-977, a highly selective κ1-opioid receptor agonist, in diabetic mice was less than that in non-diabetic mice. The antinociceptive effects of ICI-199,441 and R-84760, high potency κ1-opioid receptor agonists, given i.c.v., but not i.t., were attenuated in diabetic mice compared to those in nondiabetic mice. On the other hand, the antinociceptive effects of the new κ-opioid receptor agonist TRK-820, which has high affinity for κ2- and/or κ3-opioid receptors, injected both i.c.v. and i.t. in diabetic mice were markedly less than those in non-diabetic mice. These results indicate that the antinociceptive effects of those κ-opioid receptor agonists in diabetic mice are altered in a region-specific manner in the central nervous system (CNS). The dysfunction of κ-opioid receptor subtypes in diabetic mice may underlie this CNS region-specific variation in the effects of these κ-opioid receptor agonists.

Involvement of κ-opioid receptors and σ receptors in memory function demonstrated using an antisense strategy

Although antinociceptive effects of U-50,488H ( trans-(±)-3,4-dichloro- N-methyl- N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulfonate and (−)-pentazocine have been reported to influence κ-opioid receptors, the involvement of κ-opioid receptors in learning and/or memory is still controversial. We have recently reported that the memory improving effect of (−)-pentazocine was antagonized by σ1 receptor antagonist. In this study, we examined the effects of several antisense oligodeoxynucleotides (antisenses) to κ1-opioid receptors and σ1 receptor on memory and nociceptive function. Male ddY mice were treated subcutaneously (s.c.) with scopolamine (1.65 μmol/kg) and/or test drugs 30 min before a Y-maze test. U-50,488H significantly improved the scopolamine-induced impairment of spontaneous alternation behavior. Twenty micrograms of antisense targeting exons 2 and 3 of the κ1-opioid receptor significantly reversed the effects of U-50,488H, but antisense targeting exon 1 and mismatch sense did not. The antisense targeting exon 3 was most effective. These antisenses themselves did not affect normal mice, indicating that κ1-opioid receptors do not tonically regulate memory function. All three antisenses equally prevented U-50,488H-induced antinociceptive effects in the acetic-acid-induced writhing test. Pretreatment with antisense targeting σ1 receptors (AS-σ1) completely prevented the memory-improving effects of (−)- and (+)-pentazocine, although U-50,488H ameliorated the scopolamine-induced impairment of spontaneous alternation behavior in AS-σ1-treated mice. These results suggest that κ1-opioid receptors containing different exons have a distinct function in memory and nociceptive functions. Furthermore, κ-opioid receptors agonist showing analgesic effects act on κ-opioid receptors or σ receptors and play important roles only when memory function is impaired, but the two neuronal systems regulate memory function independently.

Opioid Modulation of Recurrent Excitation in the Hippocampal Dentate Gyrus

kappa opioid receptor activation inhibits granule cell-mediated excitatory neurotransmission in the hippocampal formation via a decrease in glutamate release from both perforant path and mossy fiber terminals. We now report a third, anatomically and pharmacologically distinct site of such kappa opioid inhibition within the hippocampus. Granule cell population responses to selective stimulation of an excitatory hilar pathway were decreased by the kappa(1) opioid receptor agonist U69,593, an effect blocked by the kappa(1) antagonist norbinaltorphimine. U69,593 also inhibited hilar path induced long-term potentiation (LTP) of granule cell responses. LTP in this pathway was also blocked by the NMDA receptor antagonist d-2-amino-5-phosphonovalerate, unlike granule cell mossy fiber LTP in CA3. The kappa opioid peptide dynorphin is present in hilar mossy fiber collaterals. Ultrastructural analysis of these collaterals demonstrated dynorphin-containing vesicles in asymmetric synapses formed between axon terminals and granule cell dendrites, suggesting direct granule cell-granule cell connections. Evoked release of endogenous dynorphin within the hilus was effective in reducing hilar excitation of granule cells, although this release, in contrast to the release of dynorphin in the dentate molecular layer, was not dependent on L-type calcium channels. No hilar path excitation was observed in the absence of bicuculline, suggesting a strong GABA(A)-mediated inhibition of this pathway. However, hilar path activity could be seen after LTP, with or without bicuculline. Thus, kappa opioids can inhibit granule cell recurrent excitation, likely via effects on excitatory mossy fiber collaterals. Such collaterals are thought to be important in mediating temporal lobe epilepsy.

Interaction between the tetracyclic antidepressant mianserin HCl and opioid receptors

The antinociceptive effects of the tetracyclic antidepressant mianserin and its interaction with various opioid receptor subtypes was evaluated. Mice were tested with a hotplate analgesia meter. Mianserin elicited an antinociceptive effect in a dose–dependent manner following doses from 1–25 mg/kg. As the mianserin dose increased beyond 30 mg/kg, latencies returned to baseline, yielding a biphasic effect. This effect of mianserin was antagonized by naloxone ( P<0.005), implying a possible opioid mechanism of action involved in the mianserin induced antinociceptive effect. When administered with various opioid antagonists, the sensitivity of mianserin to selective opioid antagonists was found significant for μ and κ1 opioid receptor subtypes ( P<0.005), but not for δ-receptor. At the next stage mianserin was administered together with various agonists of opioid receptors. When administered together with opiates, mianserin significantly potentiates analgesia at the μ, κ1 and κ3 opioid receptor subtype ( P<0.005) and to a lesser extent, at the δ opioid receptors. These results suggest a potential use of mianserin in the management of some pain syndromes. However, further research is needed in order to establish both the exact clinical indications and the effective doses of mianserin when prescribed for pain.

Effects of kappa opioid receptor-selective agonists on responses of pelvic nerve afferents to noxious colorectal distension.

The aim of this study was to examine the effects of kappa-opioid receptor selective agonists on responses of mechanosensitive afferent fibers in the pelvic nerve. Single-fiber recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root of the rat. A total of 572 afferent fibers in the S1 dorsal root were identified by electrical stimulation of the pelvic nerve; 252 (44%) responded to noxious colorectal distension (CRD; 80 mmHg). Of these 252 fibers that responded to CRD, 100 were studied further. All 100 fibers gave monotonic increases in firing to increasing pressures of CRD. Eighty-eight fibers had low thresholds for response (mean: 3 mmHg) and 12 fibers had high-thresholds for response (mean: 28 mmHg). Responses of 17 fibers also were tested after instillation of 5% mustard oil (MO) into the colon. The resting activity of 16/17 fibers significantly increased after MO instillation; 13 (77%) also exhibited sensitization of responses to graded CRD when tested 30 min after intracolonic MO instillation. The effects of kappa1-opioid receptor preferring agonists (U50,488H, U69,593 and U62,066), the kappa2-opioid receptor preferring agonist bremazocine, and the kappa3-opioid receptor preferring agonist naloxone benzoylhydrazone (nalBzoH) were tested on responses of 64 mechanosensitive afferent fibers to noxious CRD. All five agonists dose-dependently inhibited afferent fiber responses to noxious CRD. Doses producing inhibition to 50% of the control response to CRD did not differ among the five agonists, ranging from approximately 4 to 15 mg/kg. The effects of kappa1, kappa2, and kappa3 receptor agonists were attenuated by naloxone; two kappa-opioid receptor-selective antagonists were ineffective. There were no differences in the dose-response relationships of these drugs for fibers recorded from untreated and irritant-treated colons. Conduction velocities of the fibers remained unaffected after high doses of all tested agonists. In an in vitro study, U50,488 (10(-4) M) did not produce any significant change in the tension of colonic smooth muscle. These results document that responses of mechanosensitive pelvic nerve afferent fibers innervating the colon are inhibited by kappa-opioid receptor agonists having varying affinities for putative kappa-opioid receptor subtypes. The inhibitory effects of these drugs likely are mediated by an action at receptors associated with the afferent fibers. The receptor at which these effects are produced is kappa-opioid-like but clearly different from the kappa-opioid receptor characterized in the CNS and is perhaps an orphan receptor.

Preservation of κ1 opioid receptor recognition site density and regulation by G-proteins in the temporal cortex of patients with Alzheimer's disease

The pharmacological properties of the κ 1 opioid receptor were investigated in human post-mortem temporal cortical membranes from control and Alzheimer's disease brains, using the κ 1-selective radioligand [ 3H]U69593. [ 3H]U69593 bound to a single high affinity site population with no significant difference between control ( B max 31 ± 4.14 fmol/mg protein, K D 1.01 ± 0.26 nM) and Alzheimer's disease brains ( B max 37 ± 4.63 fmol/mg protein, K D 0.86 ± 0.08 nM). Competition studies with dynorphin B and α-neoendorphin gave flat inhibition curves with Hill coefficients of 0.31 ± 0.04 and 0.49 ± 0.09 in the control brains and 0.38 ± 0.05 and 0.48 ± 0.08 in the Alzheimer's disease brains, respectively. The pI 50 values for dynorphin B and α-neoendorphin were 8.73 ± 0.17 and 8.48 ± 0.09, respectively, in the control brains and 9.30 ± 0.22 and 8.70 ± 0.15 in the Alzheimer's disease brains. The guanine nucleotide analogue Gpp(NH)p inhibited binding by ca. 70% in both the control and Alzheimer's disease brains, the residual binding being sensitive to NaCl in both cases. These results indicate that the pharmacological properties and the functional integrity of G-protein coupling of the κ 1 receptor recognition site are preserved in Alzheimer's disease temporal cortex.

Distribution of κ Opioid Receptor mRNA in Adult Mouse Brain: An in Situ Hybridization Histochemistry Study

The distribution of the κ opioid receptor mRNA in adult mouse brain has been determined using the technique of in situ hybridization histochemistry. The mRNA for the κ opioid receptor was expressed in distinct areas throughout the brain. The telencephalon showed high levels of expression in the deeper layers of the parietal and temporal cortex, olfactory tubercle, nucleus accumbens, claustrum, endopiriform nucleus, nucleus of the vertical and horizontal limb of the diagonal band, and medial and central nuclei of the amygdala. In the diencephalon, κ opioid receptor mRNA was present in multiple medial thalamic nuclei including the centromedial, paraventricular, parafasicular, central, and peritenial nuclei, as well as in most hypothalamic nuclei including the ventromedial, periventricular, supraoptic, arcuate, and dorsomedial nuclei. The mesencephalon showed highest levels of κ receptor mRNA in the substantia nigra pars compacta, ventral tegmental area, zona incerta, interpeduncular nucleus, superior colliculus, inferior colliculus, central grey, and the raphe nucleus. In the metencephalon, κ opioid receptor mRNA was expressed in the parabrachial nuclei, locus coeruleus, dorsal and ventral tegmental nuclei, and the raphe pontine nuclei. The distribution of the κ receptor mRNA closely coincides with the localization of binding sites in rat brain for [3H]U-69,593, a specific κ1 opioid receptor ligand. The mRNA distribution also correlates with neuroanatomical sites of actions of κ agontists and distribution of the endogenous κ receptor ligand dynorphin.

Evidence for κ1 opioid receptor multiplicity in the guinea pig cerebellum

Both [ 3H]U69,593 alone and [ 3H]ethylketocyclazocine (EKC) in the presence of μ and δ blockers ([D-Ala 2,MePhe 4, Glyol 5] enkephalin (DAMGO) and [D-Pen 2,D-Pen 5]enkephalin (DPDPE)) label κ receptors in the guinea pig cerebellum. Dynorphin A(1–17) and nor-binaltorphimine (nor-BNI) potently competed the binding of both radioligands with Hill coefficients of approximately unity, strongly supporting a κ classification of binding. However, saturation studies revealed that the B max for [ 3H]EKC binding was 45% greater than that for [ 3H]U69593, suggesting that [ 3H]EKC might be labeling more than one site. Although nonlinear regression analysis of dynorphin A(1–17) and nor-BNI competition of [ 3H]EKC binding best fit the curves with a one site model, competitions by dynorphin B, dynorphin A(1–9) and α-neoendorphin revealed Hill coefficients less than unity and were best fit to a two site model. Kinetic analysis also supported [ 3H]EKC binding heterogeneity. Together, these studies imply that under these conditions [ 3H]EKC labels more than one site in the guinea pig cerebellum. The sensitivity of all specific [ 3H]EKC binding to the selective κ ligands dynorphin A(1–17) and nor-BNI indicates that both component are κ while the differing sensitivities of dynorphin B, α-neoendorphin and dynorphin A(1–9) for these components support our previous hypothesis of κ 1a and κ 1b binding subtypes.