Characterization of K Opioid Receptors in Neurosecretosomes from Bovine Posterior Pituitary

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high-affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D-Ala2,D-Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of mu, delta, or benzomorphan (kappa 2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bind etorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 microM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the kappa type. Furthermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.     

Modulation of human neuroblastoma transplanted into nude mice by endogenous opioid systems

The role of endogenous opioid systems (endogenous opioids and opioid receptors) in human cancer was explored using an opioid antagonist paradigm and neuroblastoma cells (SK-N-MC) transplanted into nude mice. Mice inoculated with 2.5 X 10(6) neuroblastoma cells received daily injections of either 0.1 or 10 mg/kg naltrexone (=0.1 and 10 NTX groups) which blocked the opioid receptor for 6-8 hr/day or the entire 24 hr/day, respectively, or sterile water. The latency for appearance of a measurable tumor (5 mm diameter) in the 0.1 NTX group was 27% longer than controls (11 days), and the first death in this group occurred 33% later than controls (day 27). Mice inoculated with tumor cells in the 10 NTX group had an acceleration (18%) in the latency of tumor appearance and, 2 weeks after cell inoculation, 70% of the mice in this group had tumors, in contrast to 10% of the controls. At the termination of the experiment (day 45), only 33% of the 10 NTX group were alive, in contrast to 90% of the controls. Receptor binding assays using DAGO, DADLE, or EKC revealed specific saturable binding only for DADLE and EKC. NTX administration resulted in a 148-186% increase in density for both binding sites, but no changes in binding affinity. Measures of opioid levels showed that tumor tissue levels of both beta-endorphin and methionine-enkephalin were elevated 2.5 to 6.5 fold from control values in both NTX groups, whereas plasma beta-endorphin was subnormal by 4 to 6 fold. These results indicate that endogenous opioid systems regulate human neuro-oncogenesis, with opioids being active inhibitors of growth. Opioid antagonists up-regulate receptors and increase tissue levels of endogenous opioids and, under conditions in which the opioid antagonist is short-acting (e.g., 0.1 NTX), can have an exaggerated antitumor effect during the interval when the antagonist is no longer present.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Analgesic effects of ethylketocyclazocine and morphine in rat and toad

We have previously found rat and toad (Bufo marinus) brain to contain inverse ratios of benzomorphan-preferring (kappa/sigma) and morphine-preferring (mu) opioid receptor types. The aim of the present study was to compare in vivo pharmacologic activity of a benzomorphan, ethylketocyclazocine (EKC) and morphine sulfate (MS) in rat and toad. Footshock intensity thresholds for eliciting locomotion were determined and dose-response curves for EKC and MS analgesia were obtained. Drugs were injected subcutaneously. In rats (high mu, low kappa in brain), both compounds produced analgesia and displayed similar sensitivity to naloxone antagonism. The analgesic effects of EKC and MS may, therefore, be mediated by a common receptor type (mu) in this pain test in rats. In toads (high kappa, low mu in brain), MS produced naloxone-reversible analgesia at doses 20-fold higher than were effective in rats. Toads did not display EKC analgesia at doses below those producing motor impairment. Moreover, 50-fold higher doses were required to produce such impairment in toads. Thirty minutes following subcutaneous injection of 3H-EKC, similar concentrations were found in rat and toad brain. Uptake into brain is probably not a factor in the behavioral resistance of toads to EKC.     

A monoclonal antibody capable of modulating opioid binding to rat neural membranes

A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and reversible manner, as well as the binding of the delta-ligand [3H][D-Ala2,D-Leu5]enkephalin, the kappa-ligand [3H] ethylketocyclazocine, and 3H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 125I-labeled immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with 125I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites.     

Selective proliferation of brain kappa opiate receptors in spontaneously hypertensive rats

The binding of tritiated ligands for various opiate receptor subtypes to brain membranes prepared from spontaneously hypertensive rats and normotensive Wistar-Kyoto rats was determined. The density (Bmax) or the apparent dissociation constant (Kd) for the binding of the mu-ligand (naltrexone) and delta-ligand (Tyr-D-Ser-Gly-Phe-Leu-Thr) to brain membranes of hypertensive and normotensive rats did not differ. However, the Bmax for the binding of kappa-ligand (ethylketocyclazocine, EKC) to brain membranes after the suppression of mu and delta-sites by 100 nM each of unlabeled D-Ala2-MePhe4-Gly-ol5-enkephalin and D-Ala2-D-Leu5-enkephalin, respectively, was significantly greater in hypertensive rats compared to normotensive rats. The Kd values for the binding of 3H-EKC in the two groups did not differ. The binding of 3H-EKC in brain regions was in the order: hypothalamus greater than midbrain greater than striatum greater than cortex greater than pons + medulla. The increase in the binding of 3H-EKC in the brain of hypertensive rats compared to normotensive rats was due to increased binding in the hypothalamus and cortex. These results provide for the first time evidence of selective proliferation of kappa-opiate receptors in the brain of hypertensive rats, and suggest that brain kappa-opiate receptors may play an important role in the pathophysiology of hypertension.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.     

Modification of opioid ligand binding in the central and the peripheral nervous system by different buffers

The modification of binding parameters (equilibrium dissociation constant and binding capacity) of three opioid ligands (DADLE, Etorphine and EKC) on bovine adrenal medulla and rat brain membranes have been examined in three buffer systems: Tris-HCl 50 mM, Hepes-NaOH 10 mM and Tes-KOH 10 mM. Major differences of these parameters have been found: Hepes-NaOH provoked a diminution of the apparent number of binding sites, while a concomitant diminution of the KD and Bmax was observed in Tes-KOH buffer. Substitution of counterions in these two buffers produced further changes of binding characteristics: in Hepes buffer we have observed an abolition of 3H DADLE binding, an enhancement of 3H EKC binding and no modification of 3H etorphine binding characteristics. On the contrary an abolition of the specific binding of all three ligands in Tes buffer was found in the bovine adrenal medulla while minor changes were observed in rat brain. It is concluded that, inspite same disadvantages (substitution for bivalent cations and temperature dependence), Tris-HCl is the buffer of choice for the analysis of opioid binding site interactions.     

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): Evidence for an interaction between the two systems

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/ or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/ or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/ or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/ or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/ or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/ produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities. J. Cell. Biochem. 71:416–428, 1998. © 1998 Wiley-Liss, Inc.     

Affinity labeling of delta opioid receptors by an enkephalin-derivative alkylating agent, DSLET-Mal

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.     

Role of ADH in ethylketocyclazocine-induced diuresis: studies in the Brattleboro rat

Kappa opioids produce diuresis presumably through ADH. We investigated further the role of ADH in kappa-induced diuresis by utilizing the Brattleboro rat, a strain lacking endogenous ADH. Ethylketocyclazocine (EKC), a kappa opioid prototype, increased urine formation in Sprague-Dawley, but not in Brattleboro rats. Furthermore, EKC pretreatment abolished the antidiuretic response to ADH administered exogenously to Brattleboro rats. Our study suggests that, in addition to a fall in plasma ADH reported previously, kappa opioids have direct effects on the renal response to ADH.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lung opioid receptors: pharmacology and possible target for nebulized morphine in dyspnea

Opioid receptors are located throughout the respiratory tract. Yet, these have received relatively scant attention compared to other opioid receptors. The most abundant sites within the respiratory tract appear localized within the alveolar walls, other sites appear to line the smooth muscle within the trachea and main bronchi near the lumen. There is about 100-times greater [3H]morphine binding density within the bronchioles and lobes than in the main bronchi or trachea. In addition to the usual mu, delta and kappa types of opioid receptors, 'non-conventional' opioid binding sites have been suggested, although the function of these or of the other opioid receptors in the pulmonary tract is not known. However, they might explain the otherwise counterintuitive apparent utility of morphine treatment of dyspnea. Dyspnea is a common and distressing symptom in terminally-ill cancer patients and patients with chronic lung disease. It results from multiple causes, is difficult to treat and is a significant precipitating factor for late-stage hospital or hospice admissions. Nebulized morphine or other opioids have been reported to have beneficial effect, but the mechanism by which opioids might produce this seemingly contradictory effect is not clear. We review here lung opioid receptor distribution, pharmacology and possible clinical relevance in the treatment of dyspnea.     

A Monoclonal Antibody Recognizing K- but Not γ- and δ-Opioid Receptors

A monoclonal antibody (mAb), KA8 that interacts with the kappa-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified kappa-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (kappa-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain kappa-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when mu- and delta-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when mu-, delta-, and kappa 2-sites were blocked by 5 microM DADLE. However, the mu-specific [3H]DAGO and the delta-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the kappa- but not the mu- and delta-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites

Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors.     

Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of rat brain (mu-, delta-, kappa 2-receptors) and guinea pig cerebellum (kappa-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively). The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations (relative potencies of 0.8 and 8.8% on the GPI and MVD) while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

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.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)