Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

Action of detergents and pre- and postsynaptic localization of 3H-naloxone binding in synaptosomal membranes. A structural approach

3H-naloxone specific binding was carried out on synaptosomal membranes isolated from basal ganglia of the cat brain. A high- and a low-affinity site with Kd1 = 3.7 nM and Kd2 = 35 nM having B max 1 = 79 pmole/g protein and B max 2 = 224 pmole/g protein were found. The Hill number for the high- and low-affinity sites were, respectively, 1.01 and 0.86. Digitonin and Triton X-100 had an inhibitory effect on the binding at concentrations between 10(-5) and 10(-1)% (w/v). Deoxycholate and Nonidet P-40 also inhibited the binding of 3H-naloxone, but at 10(-4)% produced a 50% enhancement. After the binding to membranes, the 3H-naloxone receptor complex is stable to the action of Triton X-100 and dissociates slowly. In membranes bound with 10 nM 3H-naloxone and then submitted to 0.1-0.2% Triton X-100, in which only the presynaptic membrane disintegrates, the specific radioactivity is decreased. With a more drastic treatment that disintegrates the postsynaptic membrane, the 3H-naloxone binding to synaptosomal membranes is almost completely abolished. These results suggest that opiate receptors may be localized both pre- and postsynaptically in central synapses.     

Alpha interferon interaction with opiate receptors in the rat brain

The preferential interactions of alpha-interferon (alpha-IFN) with delta and mu opiate receptors were studied. alpha-IFN (specific antiviral activity 2 X 10(3) U/mg protein) was shown to inhibit in the competitive manner 3H-naloxone and 3H-D-ala2, D-leu5-enkephalin (3H-DADL) specific binding to opiate receptor subpopulations. alpha-IFN was much more effective in decreasing 3H-DADL than 3H-naloxone binding in opiate receptors: K1 values averaged 160 +/- 30 and 1150 +/- 80 U/ml, respectively. IFN effective concentrations inhibiting 50% of 3H-naloxone opiate receptor binding in the absence or presence of 100 mmol/l NaCl were similar, and the "sodium shift" value was equal to 1. The independence of alpha-IFN activity of the presence of NA+ cations suggests the antagonist character of alpha-IFN interaction with opiate receptors. Thus, alpha-IFN employed appears to be an alpha-selective ligand displaying the in vitro properties of "pure" morphine antagonists.     

Cocaine-induced changes in 3H-naloxone binding in brain membranes isolated from spontaneously hypertensive and Wistar-Kyoto rats

Effects of sub-acute cocaine treatment on 3H-naloxone binding to 6 brain regions were examined in spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Cocaine hydrochloride (3 mg/kg, i.v.) was given by bolus injection daily for five days. Rats were decapitated 24 hr following the final injection and crude membrane fractions prepared from the cortex (CT), hippocampus (HI), striatum (ST), hypothalamus (HY), midbrain (MB) and medulla/pons (MD). Binding of 3H-naloxone was consistent with a single site model in CT, HI, HY, MB and MD from vehicle-treated SHR and WKY. Cocaine treatment of SHR significantly decreased the maximal binding capacity (Bmax) of 3H-naloxone in the HI, ST and HY and the binding affinity was increased in HI. In contrast, a significant increase in Bmax was noted in CT and HI membranes isolated from cocaine-treated WKY. The binding affinity of 3H-naloxone to MB membranes of WKY was significantly decreased by cocaine treatment. The binding characteristics of 3H-naloxone in MD membranes were not different following cocaine treatment or between strains. Scatchard analysis indicated biphasic binding of 3H-naloxone binding to ST membranes from both SHR and WKY. Our results indicate that cocaine produces complex and differential changes in opiate receptors and, presumably, opioid peptide neuronal function in SHR and WKY.     

Dalargin-binding proteins of synaptic membranes from the rat brain: isolation and comparison of their properties with opiate receptor properties]

The proteins isolated from rat brain synaptic membranes were studied by affinity chromatography on dalargin-omega-aminohexyl-Sepharose 4B and specific elution with DAGO (Tyr-D-Ala-Gly-N-Me-Gly-ol). These proteins were shown to bind specifically 3H-naloxone (Kd = 6.6 nM; Bmax = 690 pmol/mg of protein). SDS electrophoresis of the dalargin-binding proteins termed as DBPDAGO revealed one major protein band with M(r) of 42 kDa and two minor bands with M(r) of 29 and 67 kDa. The glycoprotein component was found in DBPDAGO; their isoelectric properties were established (pI 5.4). The close similarity of DBP properties with those of isolated brain opiate receptors suggest them to be opiate receptor components.     

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.     

Changes in pain reactions and 3H-naloxone binding to opiate receptors of the hypothalamus and midbrain in rats after repeated swimming in cold water

The dynamics of nociceptive reactions and character of 3H-naloxone binding to hypothalamus and midbrain synaptic membranes were studied in rats subjected to repeated cold swim stress (3 min. daily during 3, 5 and 15 days). It was shown that an increase of latencies of background nociceptive reactions (hot-plate and tail-flick tests) was accompanied by an ambiguous changes of kinetic parameters of 3H-naloxone binding in the studied brain structures. The results suggest that an increase of antinociceptive systems tone under repeated cold swim stress may be caused by a dynamic transformation of opiate u-receptor apparatus in various brain structures.     

Long-term ethanol alters the binding of 3H-opiates to brain membranes

In order to examine whether ethanol treatment has selective or differential effects on brain binding sites for opiates, male Sprague Dawley rats were fed for 15 or 21 days with a complete liquid diet containing 6.5% ethanol (v:v) or an isocaloric amount of sucrose. The binding of 3H-DADL-enkephalin, 3H-dihydromorphine and 3H-naloxone to the brain membranes from rats treated with ethanol was increased. However, addition of ethanol directly in the incubation medium decreased the binding of 3H-DADL enkephalin and increased the binding of 3H-dihydromorphine to brain membranes from both control and ethanol treated rats. Direct exposure of brain membranes to ethanol caused no significant change in the binding of 3H-naloxone. Thus chronic ethanol ingestion alters the binding of opiate ligands to brain membranes. Furthermore, the direct effect of ethanol appears to be different for the different classes of opiate binding sites.     

Opiate receptor binding of naloxone in the rat brain: effect of sodium ions

The binding levels and opiate receptor binding parameters were determined for 3H-naloxone in rat brain in the presence of NaCl added in vitro. An addition of NaCl at concentrations of 5-35 mM to the reaction medium caused an increase in the level of the antagonist receptor binding. The maximal level of 3H-naloxone reception activation was observed in the presence of 10-20 mM NaCl and was, on the average, 25%. Both the increase in the NaCl dose in vitro and its decrease caused a gradual diminution of the Na+ effect. An analysis of opiate receptor saturation with 3H-naloxone revealed that the label interacted with one type of the binding sites irrespective of NaCl concentration. The affinity of receptor binding sites for 3H-naloxone increased already at NaCl concentration of 2.5 mM. In contrast, the apparent maximal number of binding sites did not change after NaCl addition at concentrations which coincided with the intracellular Na+ level but was decreased with an increase (up to 50-100 mM) in NaCl present in the reaction mixture. The results obtained point to the existence of two different binding sites that are coupled with the 3H-naloxone reactive opiate receptor.     

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.     

Buprenorphine: Characteristics of binding sites in the rat central nervous system

The binding of the mixed opiate agonist-antagonist ³H-buprenorphine to rat CNS membranes was stereospecific, saturable and had high affinity. Maximal specific binding of ³H-buprenorphine at 25°C was reached by 30 minutes and dissociation from the receptor was slow. ³H-Buprenorphine labelled a single class of high affinity binding sites (K_D = 0.86nM, B_max = 30.2pmole/g tissue). The B_max for ³H-buprenorphine was about two times that for the μ-opiate receptor drugs ³H-naloxone and ³H-dihydromorphine, and three times the B_max for the σ-opiate receptor ligand ³H-D-Ala², L-Met⁵-enkephalinamide. The regional distribution of ³H-buprenorphine binding was qualitatively similar to the distribution of ³H-naloxone and ³H-dihydromorphine binding. Changing the incubation temperature from 25°C to 37°C increased ³H-buprenorphine binding in all regions of the CNS yet decreased ³H-naloxone and ³H-dihydromorphine binding in most regions. These effects of increasing temperature were a result of changes in ³H-opiate affinity for the receptor with no significant changes in receptor number. Sodium chloride (154mM) enhanced both ³H-buprenorphine and ³H-naloxone binding, and decreased ³H-dihydromorphine binding. The potency of opiate alkaloids and peptides in displacing ³H-buprenorphine was relatively weak with IC₅₀ values ranging between 40nM and 600nM. Furthermore displacement curves were shallow, yielding curvilinear Scatchard plots. Buprenorphine was very potent in displacing ³H-naloxone (IC₅₀ = 0.52nM), ³H-dihydromorphine (IC₅₀ = 1.17nM) and ³H-D-Ala², L-Met⁵-enkephalinamide (IC₅₀ = 0.47nM). These findings suggest that buprenorphine binds to both μ- and δ-opiate receptors.     

Synthesis and binding of 3H-oxymorphazone to rat brain membranes

Oxymorphazone is a 14-hydroxydihydromorphinone derivative which contains a C-6 hydrazone group and hence could serve as an irreversible label for opioid receptors. 3H-oxymorphazone was synthesized by the reaction of 3H-oxymorphone with excess hydrazine. A specific radioactivity of 640 GBq/mmol (17,3 Ci/mmol) was achieved. Both the unlabelled compound and the tritiated ligand show high affinity to mu and kappa opiate receptor subtypes in rat brain membranes. Two binding sites were detected by equilibrium binding studies, with apparent Kd values of 0.62 nM and 28 nM. About 20% of the H-oxymorphazone specific binding is irreversible after reaction at 1 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand. No azine formation was detected. Preincubation of the membranes with unlabelled oxymorphazone resulted in an irreversible blockade of the high affinity 3H-naloxone binding sites.     

3H-diprenorphine is selective for mu opiate receptors in vivo

The displacement of 3H-diprenorphine from opiate receptors by mu-selective opiates was measured in the mouse striatum and thalamus in vivo. In addition, the regional distribution of opiate receptor binding using 3H-diprenorphine, 3H-naloxone and 3H-lofentanil was measured. The displacement of 3H-diprenorphine by naloxone and carfentanil in vivo showed no differences in the striatum and thalamus suggesting that 3H-diprenorphine binds only to one opiate receptor subtype in vivo. This finding is substantiated by the observation that the mu selective ligands 3H-naloxone and 3H-lofentanil have the same in vivo distribution of receptor binding as 3H-diprenorphine. The implication of these findings for PET imaging of opiate receptor subtypes is discussed.     

Effect of morphinone on opiate receptor binding and morphine-elicited analgesia

Specific binding of ³H-naloxone to opiate receptors was found to be irreversibly inactivated by morphinone. This inactivation exhibited pseudo-first-order kinetics. The presence of sulfhydryl compounds or morphine during incubation with morphinone proved good protection. Morphinone-pretreated mice blocked the analgesic effect of morphine. The possible mechanism for these observations is proposed as foolows: morphinone binds covalently to sulfhydryl group of opiate receptors, and inactivates irreversibly opiate binding sites, thus blocking the analgesic effect of morphine.     

Stereospecific opiate binding in living human polymorphonuclear leucocytes

Living human polymorphonuclear leucocytes were incubated with various opiate agonists and antagonists in radioreceptor assays. Binding of the opiate antagonists 3H-naloxone and 3H-diprenorphine and of the benzomorphan 3H-ethylketocyclazocine was found at 4 degrees C and at 37 degrees C, 3H-naloxone binding was stereospecific. Binding of the opiate agonist 3H-dihydromorphine was present at 37 degrees C but not at 4 degrees C and had a different time course as compared to the antagonists. At both temperatures no specific binding of the proteolytic stable analogue 3H-D-Ala-D-Leu-enkephalin was found. Autoradiography showed an unspecific accumulation of 3H-naloxone inside the cells and a specific localization of grains at the cell membrane.     

Phospholipid changes in synaptic membranes by lipolytic enzymes and subsequent restoration of opiate binding with phosphatidylserine.

A study has been made of the role of phosphatidylserine in stereospecific opiate binding to neural membranes, utilizing specific lipolytic enzymes to attack the lipid. At very low concentrations phospholipase A2 from bee venom will preferentially hydrolyze C22:6-fatty acid; and even after a few percent of the total phosphatidylserine is hydrolyzed, opiate binding is greatly inhibited. The addition of brain phosphatidylserine will restore opiate binding; however, when the inhibition approaches 50% restoration is only partial. Exposure of membranes to phosphatidylserine decarboxylase will partially inhibit opiate binding; and the binding returns to the control level after the addition of phosphatidylserine. The partial inhibition of opiate binding by low concentrations of Triton X-100, which presumably remove lipids, can be partially reversed by phosphatidylserine. The binding of 3H-naloxone, an opiate antagonist, is similar to agonists in its behavior towards phospholipases and phosphatidylserine; however, binding of naltrexone, also an antagonist, is far less responsive. It is concluded that the phosphatidylserine associated with the opiate receptor is the C18:0, 22:6-diacyl form, which is closely associated with protein.     

Sodium-induced alterations of opiate effects on the binding of 3H-dihydromorphine to mouse brain homogenates.

The inhibitory potency of opiate agonists on the stereo-specific binding of 3H-dihydromorphine in mouse brain homogenates was not affected by the presence of sodium ions. That of pure antagonists was greatly enhanced by NaCl whereas the inhibitory effects of mixed agonist-antagonists were reduced by NaCl, indicating that sodium ions might affect the agonist component more than the antagonist component of narcotic-antagonist analgesics. The inhibitory potency of the opiates tested in our system agrees with their potency in reducing the stereospecific binding of 3H-naloxone to rat membranes, the contractions of co-axially stimulated guinea pig ileum and their analgesic potency in animals and humans.     

Specific binding of mu- and delta-ligands by opiate receptors in the rat brain in the presence of reaferon

The ability of recombinant alpha 2-interferon (reaferon) to compete for opiate binding sites with mu- and delta-selective compounds was determined. Reaferon was found to inhibit the binding of 3H-D-ala2, D-leu5-enkephalin, and Ki value calculated was equal to 8.5 +/- 2.6 U.10(-3)/ml. The mu-agonists reception levels were decreased in the presence of reaferon at concentrations above 500 U/ml; the Ki values for 3H-morphine, 3H-dihydromorphine, 3H-RX 783006 were found to be 3.25 +/- 0.35, 4.28 +/- 0.81 and 6.51 +/- 1.27 U.10(-4)/ml, respectively. When reaferon was added into reaction medium at concentrations more than 5.10(3) U/ml the specific receptor binding of opiate antagonist 3H-naloxone was demonstrated to be increased and this effect was reversed with 100 mM NaCl. The existence of allosteric reaferon binding site which coupled with naloxone sensitive receptor was suggested to explain the results obtained.     

The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.     

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids.

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C18:0, C18:1 form (derived from myelin) and the C18:0, C22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Both phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.