二氢埃托啡对大鼠脑阿片受体的结合特性

本文在大鼠脑匀浆P_2膜上,观察了二氢埃托啡(DHE)对[~3H]纳洛酮,[~3H]DPDPE和[~3H]埃托啡(预先用30nmol/L吗啡和100nmol/L DADLE阻断μ和δ受体)与阿片受体结合的抑制强度。结果表明:DHE对[~3H]纳洛酮与阿片受体结合的抑制强度远远大于对[~3H]DPDPE和[~3H]埃托啡(预先阻断μ和δ受体后)。DHE对μ,δ和κ受体的相对亲和力之比为1951:2:1,提示DHE为μ受体相对选择性配体。     

The effect of two different buffers on the high affinity 3H-ethylketocyclazocine and 3H-SKF10047 binding to guinea pig brain membranes

In vitro mu and delta opioid receptor binding is known to be influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and postulated to be similar to mu opioid receptor binding. To investigate this postulate, we have studied how the high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a tritiated mu agonist, mu antagonist and delta agonist is altered when the radioreceptor binding assay incubation buffer is changed. The binding of 3H-ethylketocyclazocine and the mu antagonist (3H-naloxone) is highest in isotonic HEPES buffer, while the binding of the mu (3H-dihydromorphine) and delta (3H-D-ala-D-leu-enkephalin) agonist is highest in hypotonic Tris-HCl buffer. 3H-SKF10047 binding is similar in the two buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and tritiated mu and delta opioid ligands by seven unlabeled ligands is then compared in the two buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and tritiated mu ligand in hypotonic Tris-HCl buffer than in isotonic HEPES buffer. The potency of naloxone, nalorphine, SKF10047, D-ala-D-leu-enkephalin, cyclazocine and phencyclidine in inhibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the seven unlabelled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. In sum our results show that 1 nM 3H-ethylketocyclazocine binding is influenced by buffer change in a manner very similar to mu ligand binding, while the 1.2 nM 3H-SKF10047 binding is only slightly influenced by buffer change and therefore different from mu ligand binding.     

Binding of growth hormone-releasing hormones and enkephalin-derived growth hormone-releasing peptides to mu and delta opioid receptors in forebrain of rat

The purpose of this study was to compare the binding potency to opioid receptors of met-enkephalin-derived, hypophysiotrophic peptides with their reported growth hormone (GH)-releasing strengths in vitro and further, to determine the relative selectivity of each peptide for mu and delta opioid binding sites in the forebrain of the rat. A series of (GH)-releasing pentapeptides and hexapeptides (GHRP's), as well as rat (rGHRH) and human (hGHRH) growth hormone-releasing hormones were tested for preferential binding to specific opioid receptors. The site selectivity of each peptide was determined by its ability to compete for binding with synthetic ligands for mu (Tyr-D-Ala-Gly-MePhe-Gly-ol; DAGO) and delta ([D-Pen2,5]-enkephalin; DPDPE) opioid receptors. The various peptides differed in their selectivities for the two opioid receptors in that most of the GHRP's were mu-selective, while the naturally occurring GHRH's were delta-selective. Amidation of the C-terminal decreased delta selectivity. Besides affecting selectivity for the site, structural changes that enhanced GH-release by enkephalin-derived peptides also decreased their potency to compete for opioid binding sites. For example, dose-response curves for His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (SK&F 110679) inhibition of the binding of DAGO and DPDPE yielded IC50's of 6 and 20 microM, respectively. In contrast, Tyr-D-Trp-Gly-Phe-Met-NH2 (BI360), which is 1 X 10(3) times weaker than SK&F 110679 in releasing GH, had IC50's of 0.1 microM and 0.08 microM for inhibition of the binding of DAGO and DPDPE, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ethanol on the binding of conformationally rigid and labile ligands of opioid receptors to rat brain membranes

The effect of ethanol on the binding of conformationally rigid and labile ligands for mu and delta opioid receptors to rat brain membranes was determined. The mu ligands used for the studies were [3H]naltrexone and [3H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAGO), and delta ligands used were [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr-OH (DSTLE) and [3H]Tyr-D-Ala-Gly-Phe-Leu (DADLE). The binding of all the opioid ligands was inhibited by ethanol in a concentration-dependent manner. For mu ligands the inhibition was greater for [3H]DAGO binding than for the binding of [3H]naltrexone. For delta ligands, the inhibition by ethanol of the binding of [3H]DADLE was greater than that of [3H]DSTLE. Fourier-transform infrared (FT-IR) spectroscopy was used to determine the conformation of opioid peptides. The data indicated that the conformation of peptides was altered in the presence of ethanol. The results suggest that ethanol inhibits the binding of both mu and delta opioid ligands with greater inhibition observed with conformationally labile ligands. Finally, the alteration in the conformation of the peptide ligands by ethanol, in addition to denaturation of the receptor protein, may also account for the observed inhibitory effect of ethanol on brain opioid receptors.     

Modulation of mu, delta and kappa opioid receptors in rat brain by metal ions and histidine

The effect of zinc (Zn2+) and several other trace elements was studied on the binding of the opioid receptor agonists [3H] DAGO [( ([Tyr-D-Ala-Gly-Methyl-Phe-Glyol]-enkephalin)a, [3H] DSTLE ([Tyr-D-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H] EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC50 (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the Bmax (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of adrenal and sex hormones on opioid analgesia and opioid receptor regulation.

The role of endocrine factors on opioid analgesia (antinociception) and opioid receptors was studied in male and female Swiss-Webster mice. Morphine was more potent in male than in female mice, although this difference appears to be due to greater availability of morphine to the brain in males. Saturation binding studies indicated that the density and affinity of brain mu- and delta-opioid binding sites were equivalent in males and females. Males and females were implanted SC with naltrexone (NTX) or placebo pellets for 8 days, and then the pellets were removed. This treatment increased the density of mu and delta binding sites in brain and increased the potency of morphine for both sexes, although the increase in antinociceptive effects for males was greater than for females. Adrenalectomy (ADX) in male mice increased the potency of morphine and methadone but did not alter the brain levels of either drug. ADX did not alter brain opioid binding of either mu or delta ligands. When male ADX and control mice were treated with NTX, the potency of morphine and brain opioid binding sites were increased equivalently in both groups. Gonadectomy (GDX) in male mice tended to decrease morphine potency, although this was not found to be a very reliable effect. When male GDX and control mice were implanted with NTX, brain opioid binding was increased similarly in both groups, although morphine potency was increased less in GDX mice. Overall, these studies show that sex differences and hormones of the adrenals and gonads in male mice do not alter brain opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of N-formyl-methionyl-leucyl-phenylalanine (fMLP) binding to isolated human neutrophils

Short chain aliphatic alcohols have previously been found to enhance fMLP binding to human neutrophils, presumably by non-specific mechanisms involving increased membrane hydrophobicity or decreased microviscosity. We report the discovery of highly potent fMLP binding enhancers with 30,000 times the potency of the alcohols. Activity of the compounds is specific since slight changes in structure drastically alter their ability to enhance binding and since closely related compounds inhibit rather than enhance binding. The effects of experimental compounds on enhancement or inhibition of fMLP binding are paralleled by similar actions on fMLP-stimulated shape change of neutrophils.     

Species differences in mu- and delta-opioid receptors.

The mu opioid receptor ligand [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAGO) and delta opioid receptor ligand [D-Pen2,D-Pen5]enkephalin (DPDPE) show similar specificity in competition binding studies in whole brain homogenate in rat and mouse. However, in saturation studies, the density and affinity of DPDPE binding sites were substantially greater in the mouse. There was no difference between the mouse and rat in the density and affinity of DAGO sites. Results from dose-response studies for analgesia using the same ligands administered i.c.v. in both species paralleled the binding studies. DAGO was approximately 2 times more potent in the mouse compared to the rat; while DPDPE was more than 15 times more potent in the mouse. Thus, binding capacity and affinity differences appear to be related to the functional potency of the mu and delta ligands in the two species. These results suggest that the difference in potency of DPDPE between rat and mouse is related to the differences in brain delta opioid receptors.     

Guanine nucleotide-mediated inhibition of opioid agonist binding. Modulatory effects of ions and of receptor occupancy

We have analysed the potency of GTP, GDP and their analogues in reducing [3H]DADLE binding to opioid receptors in NG 108-15 cell membranes. Under conditions where non-specific hydrolysis and transphosphorylation is inhibited, the following rank order of potency was found: GDP greater than or equal to GTP gamma S greater than GTP greater than GDP beta S greater than or equal to GDPNH2 greater than GppNHp much greater than GMP. Remarkably, the slopes for the inhibition curves of GTP, GDP and their thiosubstituted analogues, but not of GDPNH2 and GppNHp, were extremely shallow, indicating either negative cooperativity or the existence of two states for the guanine nucleotide binding proteins, that both can mediate the effect of nucleotides on agonist receptor binding. The potencies of the different guanine nucleotide analogues, except that of GppNHp, were increased by the presence of sodium or chloride ions in the assay medium. Magnesium also affected GTP-mediated inhibition of opioid agonist binding since it decreased the IC50 of the nucleotide and steepened the slope of the inhibition curve. The IC50s of nucleotides and the slopes of their inhibition curves were also dependent on the extent of receptor occupancy by the agonist. From these data we conclude that (1) either diphospho- or triphosphonucleotides can regulate agonist binding. (2) Magnesium, sodium and chloride, by acting at different components of the receptor/G protein complex produce similar effects on nucleotide mediated regulation of agonist binding. (3) A mutual influence exists between receptor occupancy by agonists and G protein-mediated guanine nucleotide effect on the receptor.     

3H]ethylketocyclazocine binding to NCB-20 hybrid neurotumor cells

Ethylketocyclazocine (EKC) binds to two sites on NCB-20 neuroblastoma X Chinese hamster brain hybrid cells (KDH = 2 nM, Bmax = 21,000 sites/cell; KDL = 27 nM, Bmax = 140,000 sites/cell. The high-affinity site has been characterized as a delta opiate receptor. The low-affinity site is relatively benzomorphan-specific; opioid peptides, morphine, etorphine, and naloxone do not compete at it. Rank order of potency among benzomorphans is (+)-EKC greater than Mr 2267 greater than (+)-ketocyclazocine greater than (+)-SKF 10047 greater than bremazocine greater than cyclazocine. Among other drugs of interest that inhibit [3H]EKC binding are phencyclidine and its analogues, Ki values for which are 0.2-40 microM. Stereoselectivity is the reverse of other opioid receptors: (+)-EKC much much greater than (-)-EKC, Mr 2267 greater than Mr 2266, (+)-SKF 10047 greater than (-)-SKF 10047. The site is sensitive to trypsin, but not to N-ethylmaleimide. Binding is insensitive to nucleotides, slightly sensitive to physiological concentrations of sodium, magnesium, and manganese ions and to EDTA but not EGTA.     

Hybrid bivalent ligands with opiate and enkephalin pharmacophores

Bivalent ligands consisting of oxymorphamine and [D-Glu2]enkephalin pharmacophores linked through a spacer attached to the 6-amino group of the former and D-Glu of the latter were synthesized in an effort to investigate the possible coexistence of mu and delta recognition sites in the same opioid receptor complex. Of the two bivalent ligands (1,2) synthesized, only 1 had substantially greater antinociceptive potency in mice than its monovalent analogues (1a, 1b). Testing of 1, 1a, and 1b in the guinea pig ileum preparation (GPI) revealed a potency profile similar to that found in vivo, whereas no correlation was observed in the mouse vas deferens (MVD). Binding data indicated the same rank-order affinities at delta receptors as the opioid activities in the GPI and in mice. However, mu binding exhibited no relationship with activity. These results are consistent with the simultaneous occupation of mu and delta by a single bivalent ligand 1, but they are also in harmony with the interaction of 1 with an opioid receptor and an accessory binding site.     

Characterization of opioid, sigma, and phencyclidine receptors in the neuroblastoma-brain hybrid cell line NCB-20

Opioid, sigma, and phencyclidine (PCP) receptors were characterized in the mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20. Quantitative receptor assays under equilibrium binding conditions with highly specific radioligands demonstrated the presence of delta, but not mu or kappa, opioid receptors on NCB-20 cell membranes. NCB-20 cells were shown to possess two distinct sites specific for sigma opioids and PCP derivatives. One site was labeled by (+)-[3H]N-allylnormetazocine [(+)-[3H]SKF-10,047] (Kd = 69 nM; Bmax = 4100 fmol/mg of protein). The rank order of potency of drugs at this site was (+)-3-(3-hydroxy-phenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than haloperidol greater than (+)-SKF-10,047 greater than (+/-)-ethylketocyclazocine greater than (+/-)-bremazocine greater than N-[1-(2-thienyl) cyclohexyl]piperidine (TCP) greater than dexoxadrol. This site is similar in its ligand selectivity to the haloperidol-sensitive sigma receptor of rat brain. The other site was labeled by the potent phencyclidine derivative [3H]TCP (Kd = 335 nM; Bmax = 9300 fmol/mg of protein). This density is equivalent to approximately 60,000 sites/cell. The rank order of potency of drugs at this site was TCP greater than (+)-3-PPP greater than PCP greater than dexoxadrol greater than haloperidol greater than cyclazocine greater than levoxadrol greater than (+)-SKF-10,047; mu and delta ligands were inactive. This site is similar to the rat brain PCP receptor. The NCB-20 cell line is the only cultured cell line that has been demonstrated to have PCP receptors.     

Inhibition of dog kidney Na+, K+-ATPase activity by procaine, tetracaine and dibucaine

The potency of local anesthetics as inhibitors of Na+, K+-ATPase and K+- NPPase activities correlated with lipid solubility. The order of potencies was: dibucaine greater than tetracaine much greater than procaine. Na+-ATPase activity was remarkably more sensitive to inhibition by tetracaine and procaine, and inhibitory potency did not correlate with lipid solubility. The order of potencies for inhibition of Na+-ATPase activity was: tetracaine greater than dibucaine greater than procaine. We examined interactions between the local anesthetics and monovalent cations in an attempt to explain this observation. Inhibition of Na+-K+-ATPase by tetracaine and dibucaine was competitive with respect to Na+, and inhibition of Na+-ATPase activity by all three agents was competitive with respect to Na+. Inhibition of Na+, K+-ATPase activity by procaine and tetracaine was competitive with respect to K+, and inhibition of K+- NPPase activity by all three agents was competitive with respect to K+. Dibucaine, the most lipid soluble agent, was equipotent as an inhibitor of all three activities and was generally less effective as a competitor with respect to activation by monovalent cations. These results suggest that dibucaine may interact nonspecifically with membrane lipids to inhibit enzyme activity whereas less lipid soluble agents, such as tetracaine and procaine, may interact more selectively with cation binding sites. It appears that the presence of K+ in the assay medium specifically decreases the inhibitory potency of tetracaine and procaine. Direct competition between these agents and K+ may prevent inhibition or, alternately, the presence of K+ may convert the enzyme to a conformation less susceptible to inhibition by agents of low to intermediate lipid solubility.     

Affinity labeling of mu opioid receptors by sulfhydryl alkylating derivatives of morphine and morphinone.

After reduction of a disulfide bond at or near the mu opioid binding site in rat brain membranes, incubating membranes with 14 beta-bromoacetamido derivatives of either morphine, dihydromorphine, morphinone, or dihydromorphinone resulted in the irreversible inhibition of mu opioid binding to rat brain membranes. Without the addition of the disulfide bond-reducing reagent dithiothreitol, these affinity ligands bound reversibly to opioid binding sites. Binding to either delta or kappa opioid binding sites was not altered by alkylation of the membranes with the affinity ligands. The percentage of irreversible inhibition of mu opioid binding was dependent on the time and temperature of the incubation of membranes with the affinity ligands and on the concentrations of dithiothreitol and the affinity ligands. Incubating membranes with morphine afforded almost complete protection from alkylation of the mu opioid binding site. Naloxone and the l-isomer levorphanol also protected the site from alkylation, whereas the d-isomer dextrorphan and the kappa-selective opioid U50,488H did not protect the site. The mu-selective peptide [D-Ala2, (Me)Phe4,Gly(ol)5]enkephalin was the peptide that afforded the greatest protection. These studies have shown that, after the reduction of a disulfide bond at or near the mu opioid binding site, this sulfhydryl group can be specifically alkylated, resulting in the affinity labeling of the mu opioid binding site.     

Design of peptidomimetic delta opioid receptor antagonists using the message-address concept

Highly selective nonpeptide ligands with potent delta opioid receptor antagonist activity have been developed using the message-address concept. This approach envisaged the delta opioid receptor to contain two major recognition subsites; a message subsite which recognizes the pharmacophore, and an address subsite that is unique for the delta receptor type and confers selectivity. The message and address components of the delta-selective enkephalins were postulated to be Tyr1 and Phe4, respectively, with Gly2-Gly3 functioning as a spacer. The message component of the target compounds in this study was derived from naltrexone and related structures. An indole system was fused to the C ring of naltrexone as a mimic of the address component. The benzene moiety of indole was viewed as the delta address component, mimicking the phenyl group of Phe4, and the pyrrole portion was used as a rigid spacer. Members of the series (1-23) were evaluated for opioid antagonist activity on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Naltrindole (NTI, 1) was the most potent member of the series, with Ke values of approximately 0.1 nM at delta receptors. The antagonism by NTI was approximately 220- and 350-fold greater at delta than at mu and kappa opioid receptors. The binding of NTI and selected members of the series to guinea pig brain membranes was qualitatively consistent with their pharmacologic antagonist activity profiles in the MVD and GPI, but the Ki values were not in the same rank order. The selectivity of NTI arises mainly as a consequence of increased affinity at delta receptors. Thus, the Ke and Ki values of NTI were 1/530 and 1/90 that of the delta antagonist enkephalin analogue, ICI 174864. In contrast to NTI, ICI174864 derives its selectivity through greatly decreased recognition at mu and kappa receptors. The implications of the high affinity and selectivity of NTI as a consequence of its conformational rigidity are discussed. It is suggested that any attempt to model a receptor-bound conformation of an opioid peptide should consider affinity and potency at multiple receptor sites rather than selectivity alone.     

The effect of selected organic solvents on intact human red cell membrane acetylcholinesterase in vitro

Human erythrocytes were exposed to different concentrations of aromatic hydrocarbons, chlorinated aliphatic hydrocarbons, and alcohols in vitro to study the effects of these agents on the activity of acetylcholinesterase (AchE), a membrane integral protein. Aromatic hydrocarbons were in general more potent AchE inhibitors than chlorinated aliphatic hydrocarbons and alcohols at +37 degrees C. The influence of decreasing the temperature to +15 degrees C and +5 degrees C was more prominent on the effect of aromatic hydrocarbons than on the effect of chlorinated aliphatic hydrocarbons and alcohols. In general, however, the decrease in the incubation temperature increased the AchE-inhibiting effect of organic solvents. The lipid solubility and molecular structure, among other factors, may determine the AchE inhibitory potency of organic solvents. Changes in membrane AchE may be one of the factors affecting membrane fluidity, which is considered to determine membrane stabilization. The primary site of action of the membrane-stabilizing agents may involve a membrane protein.     

Affinity of Z-105 to the 1,4-dihydropyridine type calcium channel and several other receptor bindings in the central nervous system]

The affinity of a 1,4-dihydropyridine (DHP) type calcium channel blocker, NZ-105 ((+/-)-2-[benzyl (phenyl) amino] ethyl 1,4-dihydro-2, 6-dimethyl-5- (5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan- 2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride ethanol), on the DHP-binding site in the central nervous system and various receptor sites were compared with nicardipine and diltiazem by the use of a receptor binding assay technique. NZ-105 exhibited a displacement effect against [3H]nimodipine in the rat brain DHP-binding site with a potency similar to that of nicardipine. Nicardipine also inhibited the specific binding of several other [3H]-labelled ligands to their receptor such as adrenergic alpha 1, alpha 2, beta, dopamine D1, D2, opioid mu, delta, and kappa-type receptors. Diltiazem also showed a similar inhibitory property. However, NZ-105 showed only weak inhibition against the binding to these receptors. These results suggest that Z-105 has strong affinity to the DHP-binding site in voltage-dependent calcium channels with higher specificity.     

New models for the evaluation of opioid effects in the guinea-pig ileum.

The pharmacology of morphine and opioid peptides was studied in the guinea-pig ileum by examining their inhibitory effects on propulsive peristaltic activity and on the cooling-induced longitudinal contraction. In these experiments, dose-response curves were recorded. The rank order of potency in inhibiting peristalsis was found to be: dermorphin greater than FK 33-824 greater than dynorphin-(1-17) greater than dynorphin-(1-13) greater than delta-receptor-peptide greater than morphine greater than [Leu] enkephalin, whereas the rank order in inhibiting cooling-induced contractions was found to be: dynorphin-(1-13) congruent to FK 33-824 congruent to dermorphin greater than delta-receptor peptide greater than morphine. Naloxone antagonized the maximally effective dose of each of the opioid agents. In view of the differences between the abilities of these opioids to inhibit propulsive peristaltic activity, these models seem to be valuable for the examination of inhibitory opioid effects in the gut.     

Characterization of specific binding sites for [3H](d)-N-allylnormetazocine in rat brain membranes

Binding of [3H](d)-N-allylnormetazocine ([3H](d)-NANM) to rat brain membranes is stereospecific, reversible, and saturable (Bmax = 260 fmol/mg of protein) and manifests moderately high affinity (Kd = 20 nM). The rank order of potency among opioidbenzomorphans and phencyclidine (PCP) analogs for competition for [3H](d)-NANM-binding sites is as follows: (d)-NANM = PCP-3-OH greater than (d)-cyclazocine greater than N-ethylphenylcyclohexylamine greater than PCP greater than (l)-cyclazocine = dextrorphan greater than (d/l)-ethylketocyclazocine greater than (d/l)-bremazocine greater than (1)-NANM greater than 1-phenylcyclohexylamine greater than levorphanol. Other opioid ligands, relatively selective for each of the types of opioid binding sites other than sigma, such as morphine (mu), H-Tyr-D-Ala(Me)Phe-NH-CH2-OH (mu), D-Ala2-D-Leu5-enkephalin (delta), tifluadom (kappa), and U 50488 (kappa) as well as etorphine and naloxone were all unable to compete with [3H](d)-NANM for specific binding even at a concentration of 1 microM. Regional distribution studies of [3H](d)-NANM-binding sites show high density in the hippocampus, thalamus, hypothalamus, and amygdala and low density in cerebellum and nonfrontal neocortex membranes of the rat brain. These binding sites are very sensitive to protein-modifying enzymes and reagents such as trypsin and N-ethylmaleimide and to heat denaturation. These results provide direct biochemical evidence for the existence of distinct (d)-NANM-binding sites in rat brain. In addition, this study supports the view that PCP and several of its analogues and the dextrorotatory isomers of psychotomimetic benzomorphans may act at a common recognition site in rat central nervous system.     

Sodium regulation of agonist binding at opioid receptors. II. Effects of sodium replacement on opioid binding in guinea pig cortical membranes

We have examined the effects of sodium on the binding of opioid agonists to mu-, delta-, and kappa-receptors in guinea pig cortical membranes. Concentration curves for sodium indicated that maximal inhibition of mu binding by this cation was about 60% and maximal inhibition for delta binding was about 70%, whereas that for kappa binding was only about 20%. The concentration of sodium required for half-maximal inhibition of binding to all three sites was about 10-30 mM, corresponding to the intracellular sodium concentration. The nature of the sodium effect was further characterized by saturation analysis of binding to each of the three receptor types by comparing results obtained in the presence of 120 mM sodium with those obtained with equimolar replacement of sodium by another cation. Two radiolabeled agonists with different structural characteristics were tested for each binding site. In the presence of sodium, the affinity of the labeled agonists for mu sites was approximately 2-3-fold less than in its absence, but the density of binding sites was not changed. At kappa sites, sodium reduced agonist affinity slightly but, again, did not alter the number of binding sites. In contrast, sodium reduced the apparent density of delta-binding sites while leaving the agonist affinity unchanged. Competition against antagonist binding to delta sites indicated that, in the presence of sodium, a higher proportion of sites was in a lower affinity state, as reflected by the biphasic nature of the agonist displacement curve. In contrast, the effect of sodium on displacement of antagonist from mu sites was to of sodium on displacement of antagonist from mu sites was to lower the affinity of the agonist. Competition against antagonist binding to kappa sites also showed a reduction in agonist affinity by sodium, but no change in number of receptors. The results indicate that sodium may differentially regulate agonist binding to opioid receptor types and that this regulation may occur at an intracellular site. The kappa site appears to be less sensitive to sodium than the mu and delta sites.