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.     

Solubilization of adrenal medullary opioid receptors

The opioid-binding activity of digitonin extract of bovine adrenal medullary membranes was studied. [3H]Diprenorphine binding to the solubilized material was rapid and saturable. The dissociation constant of [3H]diprenorphine binding was 0.76 nM. Several opioids displaced the [3H]diprenorphine binding. The complex of [3H]diprenorphine and the solubilized binding sites was eluted as a single peak on a Sepharose 6B column and showed an apparent molecular weight of 200,000. These results indicate that active opioid receptors are solubilized with digitonin from bovine adrenal medullary membranes.     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human μ—opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi／0 proteins

Human mu-opioid receptor (HmuOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by mu-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by delta nor by kappa selective agonist. Na+ (100 mM) and GTP (50 microM) could reduce HmuOR agonists etorphine and Ohm affinity binding to the overexpressed HmuOR. mu-selective agonists DAGO and Ohm effectively stimulated [35S]GTP-gammaS binding (EC50 = 2.7 nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HmuOR overexpressed in Sf9 insect cells functionally coupled to endogenous G(i/o) proteins.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

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)     

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Using [³H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P₂ fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [³H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (K_d) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.     

Differences of binding characteristics of non-selective opiates towards mu and delta receptor types

[3H]ET (etorphine), which is considered either as an "universal" ligand or a mu agonist, interacts with identical affinities KD = 0.33-0.38 nM to hybrid cells and rabbit cerebellum, pure delta and mu-enriched opioid receptor preparations, respectively. In rat brain tissue, [3H]ET binding is inhibited by DAGO (Tyr-D-Ala-Gly-(Me)-Phe-Gly-ol), a mu selective agonist, in a competitive manner without apparent modification of the maximal number of sites. Furthermore, even at a DAGO concentration (300 nM) which should be sufficient to block [3H]ET interaction with mu sites, no variation in the total capacity of the tritiated ligand is observed. In contrast, DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), a delta-preferential agonist, blocks [3H]ET binding in rat brain at a concentration able to saturate delta-sites. At higher concentrations, where DTLET cross reacts with mu-sites, this ligand exhibits similar properties to those of DAGO. These data are very different from those obtained with [3H]EKC (ethylketocyclazocine), another "universal" ligand, the binding properties of which are easily explained by the occurrence in rat brain tissue of independent sites exhibiting pharmacological profiles of mu, delta and kappa sites. Our results underline the possible misinterpretation of binding data obtained by using [3H] etorphine as a non selective ligand.     

Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens.

Membranes of the rat vas deferens were shown to contain a high density of binding sites for [3H] alpha, beta-methylene ATP ([3H] alpha, beta-MeATP), a ligand selective for the P2X purinoceptor. Analysis demonstrated two classes, of high affinity (Kd = 1.8 nM, Bmax (maximum density) = 9.3 pmol/mg of protein) and of low affinity (Kd = 34 nM, Bmax = 29 pmol/mg of protein). The high affinity [3H] alpha, beta-MeATP binding sites were successfully solubilized with 2% digitonin: the Kd was then 1.6 nM. Both the association and dissociation of the receptor-ligand complex were rapid (half-time for association = 6.5 min). The rank order of potency of purinergic ligands in displacing [3H] alpha, beta-MeATP binding from the solubilized preparation was in accord with the pharmacological criteria for P2X purinoceptors. The receptor-detergent complex was separated by sucrose gradient ultracentrifugation from the ATPase enzymes also present in the preparation. The sedimentation coefficient of the receptor-detergent complex was 12.1 S. It was shown that [3H] alpha, beta-MeATP can function as a photoaffinity labeling reagent upon exposure to ultraviolet light; in the rat vas deferens membranes, it thus became cross-linked in a specific manner to a polypeptide of apparent molecular mass = 62,000 daltons, proposed to be the ligand-binding subunit of the functional P2X purinoceptor.     

A Kinetic Analysis of k-Opioid Agonist Binding Using the Selective Radioligand [3H]U69593

The interaction of the nonselective opioid ligand [3H]bremazocine and of the kappa-opioid [3H]U69593 with the kappa-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although [3H]U69593 apparently recognised only 70% of those sites labelled by [3H]bremazocine. Naloxone and the kappa-selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both [3H]bremazocine and [3H]U69593. Detailed analysis of the binding of [3H]U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5'-imidodiphosphate (GppNHp) inhibited binding of [3H]U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of [3H]U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the kappa-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

Opiate Receptor-Mediated Inhibition of Catecholamine Release in Primary Cultures of Bovine Adrenal Chromaffin Cells

Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg⁶,Phe⁷], and the synthetic peptide [d -Ala²,Me-Phe⁴,Met(O)^s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC₃₀ varying from 10^?7 to 1 × 10^?6M. The effect was stereospecific because levorphanol (IC₃₀= 7.5 × 10^?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala², d -Leu⁵]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC₅₀= 5 × 10^?7M) and naloxone (IC₅₀= 10^?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [³H]etorphine, [³H]dihydromorphine, [³H-d -Ala²,d -Leu⁵]enkephalin, [³H]ethylketazocine, and for [³H]N-allylnormetazocine, [³H]diprenorphine, and [³H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [³H]etorphine and [³H]diprenorphine was 10–70 times higher than the number of sites measured with the other ³H ligands. The rank order of potency of these compounds for the displacement of [³H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg⁶,Phe⁷] modulates the function of the nicotinic receptor in adrenal chromaffin cells.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.     

Studies on the effects of glucose in vitro and of the glycaemic state in vivo on the binding characteristics of mu, delta and kappa opiate receptors in mouse brain.

The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.     

Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats.

Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.     

Evidence for Disulfide Bonds in Membrane-Bound and Solubilized Opioid Receptors

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane-bound and digitonin-solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D-Ala2-D-Leu5-enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin-solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2. [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.