Heterologous mu-opioid receptor adaptation by repeated stimulation of kappa-opioid receptor: up-regulation of G-protein activation and antinociception

The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.     

In-vitro investigation of oxazol and urea analogues of morphinan at opioid receptors

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.     

Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure

Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.     

Involvement of dynorphin and the kappa opioid receptor in feeding

Dynorphin-(1–17) produces a highly specific increase in food ingestion. Similar enhancement of food ingestion is found with dynorphin fragments (1–10), (1–11), (1–13) and (3–13) but not with (1–8) and (1–9). Dynorphin B (rimorphin) also enhances food intake. The highly specific kappa agonist U-50,488 also enhances food intake as do a number of other kappa-opiate receptor agonists. These studies provided further support for the role of a highly specific dynorphin-kappa opioid receptor in the modulation of feeding.     

Protection from cerebral ischemia by U-50,488E, a specific kappa opioid analgesic agent

U-50,488E is a novel analgesic agent with a specific agonist property on the kappa opioid receptor. It is found to protect against the lethal effect of temporary bilateral carotid occlusion (BCO) in Mongolian gerbils and rats of the Fischer strain. Pretreatment with U-50,488E in gerbils before 7 min of BCO reduced the development of behavioral hyperactivity and preserved the hippocampal neurons from ischemic death. This protective effect of U-50,488E resided predominantly in the levo-enantiomer which is also more potent as a kappa analgesic. Two other kappa opioid analgesics, ethylketocyclazocine and bremazocine, shared the effects of U-50,488E in the gerbils. Naloxone and dynorphin 1-13, on the other hand, were without protective effects in the same ischemic model. The ischemic protective effects of U-50,488E may involve the kappa opioid receptor.     

Enantioselective kappa opioid binding sites on the macrophage cell line, P388d1.

A kappa (kappa) opioid binding site has been characterized on the macrophage cell line, P388d1, using the kappa selective affinity ligand, [3H] (1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl) cyclohexyl] benzeneacetamide (-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((-)-U-50,488) blocks [3H](5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl]benzeneacetamide (U-69,593) binding to P388d1 cells with an IC50 = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide ((+)U-50,488) blocks [3H]U-69,593 binding to P388d1 cells with an IC50 = 7000 nM.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

Effects of U-50,488H and U-50,488H withdrawal on catecholaminergic neurons of the rat hypothalamus

Previous report from our laboratory showed that morphine produces a stimulatory effect of hypothalamic noradrenaline (NA) turnover concurrently with enhanced pituitary-adrenal response after its acute injection and during withdrawal. In the present work we have studied the effects of acute and chronic administration of the kappa agonist U-50,488H as well as the influence of U-50,488H withdrawal on the activity of hypothalamic NA and dopamine (DA) neurons and on the activity of hypothalamic-pituitary-adrenal (HPA) axis. A single dose of U-50,488H (15 mg/kg i.p.) significantly increased hypothalamic NA and decreased DA turnover at the time of an enhanced corticosterone release. Rats rendered tolerant to the kappa agonist by administration of U-50,488H twice a day for 4 days showed no changes in corticosterone secretion. Additionally, a decrease in both hypothalamic MHPG (the cerebral NA metabolite) production and NA turnover was observed, whereas DOPAC concentration and DA turnover were enhanced, which indicate the development of tolerance towards the neuronal and endocrine actions of U-50,488H. After naloxone (3 mg/kg s.c.) administration to U-50,488H-tolerant rats, we found neither behavioural signs of physical dependence nor changes in hypothalamic catecholaminergic neurotransmission. In addition, corticosterone secretion was not altered in U-50,488H withdrawn rats. Present data clearly indicate that tolerance develops towards the NA turnover accelerating and DA turnover decreasing effect of U-50,488H. Importantly and by contrast to mu agonists, present results demonstrate that U-50,488H withdrawal produce no changes in hypothalamic catecholamines turnover or in corticosterone release (an index of the hypothalamus-pituitary-adrenal activity), which indicate the absence of neuroendocrine dependence on the kappa agonist. As has been proposed, this would suggest that the mu and the kappa receptor be regulated through different cellular mechanisms, as kappa agonists have a lower proclivity to induce dependence.     

Antagonism of kappa opioid mediated effects in the rat by cyclo(Leu-Gly)

The effect of cyclo(Leu-Gly) on U-50,488H- induced pharmacological actions was determined in male Sprague-Dawley rats. Intraperitoneal (i.p.) administration of U-50,488H to rats produced analgesia (tail-flick) and increased urinary output. Cyclo (Leu-Gly) (1-4 mg/kg, s.c.) antagonized the analgesic response to U-50,488H (25 mg/kg; i.p.). A dose of 10 mg/kg (i.p.) of U-50,488H increased the spontaneous urinary output which was antagonized by cyclo (Leu-Gly) (1-4 mg/kg; s.c.). To determine whether cyclo (Leu-Gly) was acting as a kappa-opioid receptor antagonist, the effect of cyclo (Leu-Gly) on the binding of [3H]ethylketocyclazocine (EKC) to membranes of rat cerebral cortex and spinal cord was determined. The IC50 values of cyclo(Leu-Gly) in displacing [3H]EKC from its binding sites in cortex and spinal cord were 1.44 and 0.40 mM, respectively. Chronic administration of U-50,488H (25 mg/kg; i.p., b.i.d.) for 4 days induced tolerance to its analgesic effect. The latter was not affected by cyclo(Leu-Gly) (2 to 8 mg/kg; s.c.) given once a day for 4 days. It is concluded that cyclo(Leu-Gly) antagonizes acute actions of U-50,488H and that such effects of cyclo(Leu-Gly) are not mediated via a direct action on kappa-opioid receptors.     

Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The kappa-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other kappa-agonists Dynorphin-A (1-13) amide, and its protected analog D[Ala]2-dynorphin-A (1-13) amide also produced a significant increase in the formation of [3H]-IP's, whereas the mu-selective agonists [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin and morphine and the delta-selective agonist [D-Pen2,5]-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the kappa-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medulla. The results indicate that brain kappa- but neither mu- nor delta-receptors are coupled to the PI turnover response.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.     

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.     

Effects of selective opioid agonists on feline colonic transit.

The mu agonist morphine and the non-specific opioid antagonist naloxone both may accelerate feline colonic transit; the effects of morphine are dose dependent. Kappa and delta receptor function was studied in the present work. Colonic transit of a radionuclide marker instilled into the cecum was quantitated for 6 hr in a crossover study. The delta agonist [D-Pen2,D-pen5]enkephalin (1 mg/kg, i.m.) prolonged the cecum and ascending colon half-emptying time by 337% (P less than 0.05), and delayed the progression of the geometric center over time. The kappa agonist U-50,488 (1 mg/kg, i.m.) had no apparent effect on the cecum and ascending colon, but delayed filling of the descending colon. Loperamide, an antidiarrheal agent, also delayed colonic transit. Thus, selective opioid agonists have both site and functional differences in their effect on feline colonic transit.     

Study the mechanisms of U-50,488 to prevent the development of morphine tolerance in guinea pigs

Previously we have shown that low dose of [trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride] (U-50,488) could prevent the development of morphine tolerance in guinea pigs. In the present study we tried to investigate the role of glutamate and nitric oxide in this process. Male Hartley guinea pigs (200-300 g) were chronically treated s.c. with either saline or morphine (15 mg/kg) or morphine + U-50,488 (0.003 mg/kg) twice a day for 7 days. Antinociceptive activity was assessed by hot-plate test on the first, fourth and seventh day. Spinal cord slices (450 microm) were prepared 30 min after drug treatment on eighth day and [3H] glutamate and nitric oxide (NO) released were determined. We found that coadministration of U-50,488 (0.003 mg/kg) suppressed the development of morphine tolerance to antinociceptive effect as we reported before. The percentage of in vitro spinal release of [3H] glutamate by 100 microM morphine was significantly higher in the chronic morphine group than the control group. On the other hand, coadministration of U-50,488 with morphine for 7 days blocked this effect significantly. The basal NO level released from the spinal cord slices was significantly higher in chronic morphine group but not in chronic (morphine + U-50,488) group. In vitro morphine (100 microM) increased the NO level in control group and chronic (morphine + U-50,488) group and also further increased NO in chronic morphine group. From the NMDA-displaced [3H] glutamate binding in guinea pig spinal cord, we found that the Bmax decreased in chronic morphine group but not in the chronic (morphine + U-50,488) group. In conclusion, chronic morphine treatment may activate the NMDA receptors by increasing the release of glutamate which causes the increase of synthesis and release of NO and following uncertain mechanisms to induce the development of morphine tolerance. And the mechanisms of U-50,488 to prevent the development of morphine tolerance may involve the inhibition of glutamate released by chronic morphine and also the decrease of NO induced by chronic morphine.     

Prejunctional effects of opioids in the perfused mesentery of the rat and rabbit: interactions with alpha 2-adrenoceptors.

Prejunctional effects of opioids were examined in the perfused mesentery of two species: the rat and rabbit. Use of agonists selective for subtypes of mu, delta, and kappa opioid receptors produced no effect on contractile responses to adrenergic nerve stimulation in the rat perfused mesentery, except for small effects of the kappa agonist EKC, which may be non specific. In contrast, mu, delta and kappa receptors appear to be present in the rabbit. The mu selective agonist, DAMGO, kappa agonist, ethylketocyclazocine, and delta agonists, DPDPE and [Leu5]-enkephalin, all produced significant inhibition of contractile responses to transmural nerve stimulation. The inhibitory effect was greatest for ethylketocyclazocine. To test the possibility that prejunctional activation of alpha 2 adrenoceptors with endogenous norepinephrine might decrease the activity of prejunctional opioid receptors in the rabbit, inhibitory effects of delta and kappa selective agonists were tested in the presence of 10(-7) M yohimbine. Inhibitory responses of the kappa selective agonist ethylketocyclazocine were enhanced, while that of delta selective agonists [Leu5]-enkephalin and DPDPE remained unchanged when yohimbine was present. Thus, the effects of opioids vary and depend on the tissue and receptor subtypes they act upon. Furthermore, the enhanced inhibitory effect of opioid receptor activation in the presence of yohimbine is not found for all opioid receptors.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

3H]U-69593 labels a subtype of kappa opiate receptor with characteristics different from that labeled by [3H]ethylketocyclazocine

[3H]U-69593 is an opiate agonist that has been reported to bind in vitro with high affinity and selectivity to the kappa receptor subtype. The studies reported here were designed to determine the optimal conditions for labeling kappa receptors with [3H]U-69593 and to further characterize the binding site. The effects of temperature and NaCl on [3H]U-69593 binding were of particular interest because previous studies reported that [3H]ethylketocyclazocine ([3H]EKC) and [3H]bremazocine binding to kappa receptors was optimal at 4 degrees C in the presence of NaCl. Those conditions were not found to be optimal for [3H]U-69593 binding. Although the pharmacological specificity and Bmax of [3H]U-69593 binding was similar at room temperature and at 4 degrees C, the binding affinity was approximately three times lower at 4 degrees C than at room temperature. In addition, NaCl had an effect on [3H]U-69593 binding that was opposite that on [3H]EKC binding at 4 degrees C (100 nM DAGO and 100 nM DADLE were included in all [3H]EKC assays to prevent binding to mu and delta receptors), i.e. NaCl decreased, rather than increased, [3H]U-69593 binding at 4 degrees C. These differences between [3H]U-69593 and [3H]EKC binding at 4 degrees C were accentuated by a vast difference in the density of the binding sites [Bmax approximately equal to 12 fmol/mg protein for [3H]U-69593 vs approximately equal to 375 fmol/mg protein for [3H]EKC at 4 degrees C in the presence of NaCl) and suggested that [3H]U-69593 might bind selectively to a kappa receptor subtype. This concept was supported by competition experiments. In particular, the site labeled by [3H]EKC at 4 degrees C was found to be relatively insensitive (compared to [3H]U-69593 and [3H]EKC binding at room temperature) to the kappa agonist U-50488H, a close analog to U-69593. Based on these findings, we propose that [3H]U-69593 (and U-50488H) labels a kappa receptor subtype which differs from that labeled by [3H]EKC at 4 degrees C.     

The NK1 receptor is involved in the neurokinin-induced shape change of rabbit platelets.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK1 receptor agonist Ac [Arg6,Sar9,Met(O2)11]-substance P (6-11) induced shape change (EC50 = 3 and 6 nM, respectively), whereas the selective NK2 agonist [Nle10]-Neurokinin A (4-10) and the selective NK3 agonist [MePhe7]-Neurokinin B did not show any effect. Moreover, the specific NK1 receptor antagonist CP-96,345 selectively and dose-dependently counteracted the effect of substance P or of the NK1 receptor agonist (IC50 = 2 and 0.8 nM, respectively), whereas the selective NK2 receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK1 receptor agonist to become a proaggregating substance. These data therefore show that the NK1 receptor is solely involved in the neurokinin-induced shape change of rabbit platelets.