Solubilization of membrane bound opiate receptor from rat brain

Sonication of rat brain membranes for 9 minutes solubilized 35% of their stereospecific opiate binding activity; a second 9 minute sonication of the insoluble residue released an additional 21% of the original binding. The opiate binding properties of the solubilized material were highly similar to those of membrane bound receptor by a number of criteria, including affinity, effect of sodium, and the IC₅₀ of unlabeled opiates in displacing ³H-etorphine binding. Moreover, storage of the solubilized receptor fraction for two weeks at ?20°C did not significantly change the receptor binding. Sonication thus appears to be a useful first step in purifying the opiate receptor.     

Stability of opiate receptors of wet and lyophilized preparations of rat brain membranes

The effects of incubation of rat brain membranes at 0 degrees C on the specific binding of mu-ligands (naloxone, morphine) and the delta-ligand (D-Ala2, D-Leu5-enkephalin) to opiate receptors were studied. The effects of lyophilization of rat brain membranes on the properties of the opiate receptors were determined. The lyophilized brain membrane preparations revealed an extraordinarily high stability as compared to "wet" membranes. The experimental results suggest that morphine and D-Ala2, D-Leu5-enkephalin binding both to the high affinity and low affinity sites has different nature and point to the utility of stable and standard preparations of lyophilized membranes for the use in the receptor analysis of opiate and opioid peptides.     

Effects of Ethanol, Temperature, and Endogenous Regulatory Factors on the Characteristics of Striatal Opiate Receptors

Ethanol can alter the affinity of mouse striatal opiate receptors for their ligands, and the present studies were aimed at determining the importance of the receptor microenvironment for this effect of ethanol. Changing the temperature of the binding assay, and thus altering the properties of neuronal membrane lipids, resulted in changes in the observed affinity of striatal binding sites for dihydromorphine (DHM), but not for D-Ala2, D-Leu5-enkephalin (ENK). The changes in temperature also differentially altered the response of the two binding sites to ethanol. Two other factors that regulate opiate receptor affinity, Na+ and GTP, also affected the response to ethanol. High concentrations of ethanol were more effective at decreasing receptor affinity for both DHM and ENK when the binding assays were performed in the presence of GTP or Na+. In addition, at 37 degrees C and in the presence of GTP or Na+, DHM binding, but not ENK binding, was significantly inhibited by a low, physiologically attainable concentration of ethanol. Our results suggest that the response of opiate receptors to ethanol is influenced by the microenvironment of the receptors, including the physical state of the membrane lipids and/or by the nature of the interactions of receptors with "coupling proteins" within the membrane. The differential responses of mu and delta receptors to temperature and to ethanol suggest that these receptors reside in specific membrane environments. Under physiological conditions, several different factors may contribute to a selective action of ethanol on particular subtypes of opiate receptors.     

Monoclonal antibody to enkephalins with binding characteristics similar to opiate receptor

Monoclonal antibodies to enkephalins were established by immunization of mice with met-enkephalin, leu-enkephalin or both. Twenty-three clones with a high titer were classified into 6 types according to the binding properties to enkephalins and their derivatives. Antibody LM 239 showed binding characteristics similar to opiate receptor. It has a very high affinity to enkephalins and their derivatives which have a potent opioid activity, but a low affinity to enkephalin derivatives which devoid of opioid activity. The binding of 3H-met-enkephalin to the antibody was inhibited by naloxone and morphine, although the ID50 values were considerably higher than the Ka values of the alkaloids to opiate receptor.     

Heterogeneity of calcium channel agonist binding sites in the coronary artery

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.     

Localization of human placental opiate binding sites on the syncitial brush border membrane

Opiate binding sites were measured in different placental membrane fractions which were characterized by marker enzyme analysis and electron microscopic examination. The distribution pattern of opiate binding sites in the different fractions closely parallels that of placental alkaline phosphatase. These results clearly show thatopiate binding sites are mainly located on the syncitial brush border membrane. The opiate binding sites found on microvillus membrane fraction have the same pharmacological characteristics as the Kappa opiate binding site previously characterized on placental crude membrane fraction.     

Hyaluronate-Binding Proteins of Murine Brain

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.     

Effect ot thyroliberin on rat brain opiate receptors

The ability of thyroliberin to interact with opiate receptors of the rat midbrain and hypothalamus has been studied. It was shown by competitive displacement analysis that thyroliberin did not replace labeled opioid peptides in opiate receptor binding sites when added in vitro at concentrations of up to 10(-5) M. The specific binding of opioid peptides was increased by 10-20% in the presence of 10(-7)-10(-6) M thyroliberin. This effect was, probably, due to the rise in the affinity of high-affinity opiate receptors. At the same time the affinity of low-affinity binding sites was decreased. It is suggested that the antagonistic properties of thyroliberin are mediated by the modulation of the binding characteristics of enkephalin-low-affinity opiate receptors.     

Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain

The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [³H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with a low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes.     

In vitro effects of cytosolic inhibitor and opiates on the binding of [3H]oestradiol to nuclear type II binding sites of rat uterus and hypothalamus

The effect of cytosolic ultrafiltrates prepared from intact rat uteri, brain hemispheres and hypothalami and of some opiate analogues on oestradiol binding to nuclear type II sites in rat uterus and hypothalamus was studied. Opiate binding in nuclear fraction of rat uteri was also evaluated. Both uterine and hypothalamic low affinity nuclear oestradiol binding was inhibited by filtrate from uteri, while only hypothalamic nuclear binding was decreased in presence of hypothalamic filtrate. Filtrate from brain was ineffective on nuclear oestradiol binding of the studied tissues. Concentration dependent inhibition of uterine nuclear oestradiol binding could be demonstrated by some opiate analogues in vitro. Specific low affinity nuclear binding of opiate antagonist naloxone and agonist dihydromorphine was observed in rat uteri which could be inhibited by uterine filtrate and oestradiol but not by hypothalamic filtrate or other steroids. Present findings support the probable intracellular interplay of opiates and oestradiol action and suggest that cytosolic inhibitor factor might be involved.     

Studies on brain opiate receptor stability in vitro: inhibition of opiate receptor binding by adenosine-5'-diphosphate

Incubation of rat brain homogenates at 37° causes a time-dependent decrease in opiate receptor binding which does not occur with a washed membrane fraction. The supernatant fraction contains a heat-stable inhibitor which is partially destroyed by apyrase and completely removed by activated charcoal. ADP causes a similar inhibitory effect in homogenates, but not with washed membranes, which is characterized by a decrease in both opiate agonist and antagonist binding in the absence or presence of NaCl. The ADP inhibition is antagonized by ATP, α,β-methyleneADP, β-thioADP and EDTA. It is concluded that ADP, unlike the guanine nucleotides, facilitates the nonspecific degradation of opiate receptors by an endogenous soluble factor.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Evidence for a PGE-receptor in the rat kidney.

A membrane preparation derived from homogenates of the rat kidney has been shown to possess a high affinity for prostaglandins of the E-series. Other prostaglandins including PGI2 had characteristic but significantly weaker binding properties. A 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to be associated with the membrane fraction studied. However it was possible to distinguish between this and the "receptor" binding by kinetic studies and by the use of a new inhibitor highly specific for PGDH.     

Changes of [H]naloxone binding in oestrogen stimulated rat uterus

Effects of a single dose of oestradiol (Oe) on [³H]naloxone (Nal) binding in ovariectomized rat uterus were studied. Specific [³H]Nal binding was assessed by saturation analysis in 800 g supernatants and pellets of uterine homogenates. Two binding sites with higher (K_d 1nM) and lower affinity (K_d 15nM) for Nal were observed, their binding capacities and affinities have changed after Oe treatment in a time-dependent manner. The high affinity binding sites, detected only in the cytoplasmic fraction, disappeared after 1 h and only became detectable again at 24 h after hormone treatment, the lower affinity binding sites, after an initial drop, slowly increased, peaking at the 9th hour of hormone injection. The competition experiments indicate the involvement of different opiate receptor subpopulations in Oe induced changes. In the nuclear fraction, the B_max values started to increase at 15 h, reaching the highest level at 18 h. The K_d values of lower affinity sites, in both studied compartments, were increased, i.e. the affinity decreased in the second half of the examined period.     

Evidence for a PGE-receptor in the rat kidney

A membrane preparation derived from homogenates of the rat kidney has been shown to possess a high affinity for prostaglandins of the E-series. Other prostaglandins including PGI₂ had characteristic but significantly weaker binding properties. A 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to be associated with the membrane fraction studied. However it was possible to distinguish between this and the “receptor” binding by kinetic studies and by the use of a new inhibitor highly specific for PGDH.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.