A kinetic model of the interaction of stable GTP analogs with a system of opioid receptors

The effect of a stable GTP analog, GppNp, on the agonist binding to rat brain opioid receptors was studied. It was shown that the nucleotide used at low concentrations activates, and at high concentrations inhibits the ligand interaction with the mu-, delta- and kappa-receptors. The inhibiting effect of GppNp on the formation of the morphine and D-Ala2, D-Leu5-enkephalin complexes with high affinity opioid receptor binding sites is due to the decrease of the ligand affinity for the corresponding sites. A kinetic model of the GppNp effect on high affinity binding sites stipulating that in the course of nucleotide binding the GTP-binding protein dissociates and that the N-protein alpha-subunits thereby formed are liberated into the surrounding solution, was proposed. It was demonstrated that GppNp can modulate the properties of opioid receptors in the absence of the ligand in a system and the inhibiting effect of GppNp depends on the concentration of membrane preparation.     

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.     

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

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

Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

Altered Microviscosity at Brain Membrane Surface Induces Distinct and Reversible Inhibition of Opioid Receptor Binding

In synaptosomal membranes from rat and monkey brain cortex, the addition of petroselenic (18:1, cis-delta 6) acid, oleic (18:1, cis-delta 9) acid, and vaccenic (18:1, cis-delta 11) acid or their corresponding methyl esters at 0.5 mumol/mg of membrane protein caused a similar 7-10% decrease in the microviscosity of the membrane core, whereas at the membrane surface the microviscosity was reduced 5-7% by the fatty acids but only 1% by their methyl esters. Concomitantly, the fatty acids, but not the methyl esters, inhibited the specific binding of the tritiated mu-, delta-, and kappa-opioids Tyr-D-Ala-Gly-(Me)Phe-Gly-ol (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE), and U69,593, respectively. As shown with oleic acid, the sensitivity of opioid receptor binding toward inhibition by fatty acids was in the order delta greater than mu much greater than kappa, whereby the binding of [3H]DPDPE was abolished, but significant inhibition of [3H]U69,593 binding, determined in membranes from monkey brain, required membrane modification with a twofold higher fatty acid concentration. Except for the unchanged KD of [3H]U69,593, the inhibition by oleic acid involved both the Bmax and affinity of opioid binding. Cholesteryl hemisuccinate (0.5-3 mumol/mg of protein), added to membranes previously modified by fatty acids, reversed the fluidization caused by the latter compounds and restored inhibited mu-, delta-, and kappa-opioid binding toward control values. In particular, the Bmax of [3H]-DPDPE binding completely recovered after being undetectable.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

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

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

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.     

Evidence for delta opioid receptor subtypes regulating adenylyl cyclase activity in rat brain

Opioid agonists selective for mu- or delta opioid receptors inhibit adenyl yl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.     

Dermorphin gene sequence peptide with high affinity and selectivity for delta-opioid receptors

Skin of the frog Phyllomedusa sauvagei contains a cDNA sequence that codes for the selective mu-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide (DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for delta-type opioid receptors in rat brain synaptosomes. The IC50 values for DGAP on mu- and delta-receptors were only 28 microM and 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nM for delta-receptors and greater than 1 microM for mu-receptors yielding a very high delta selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for delta-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2 residue and is a selective mu-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem. 264, 354-362), exhibits a SR of 0.0055 similar to that for the conventional mu-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for mu- and delta-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.     

Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.     

Differential regulation of multiple neuroreceptors in a somatic cell hybrid by inhibitors of glycoprotein processing

Specific binding of [3H][D-Ala2,D-Leu5]enkephalin, [3H]ethylketocyclazocine, 5-[3H]hydroxytryptamine, and [3H]spiperone was examined in neuroblastoma-brain hybrid cell line NCB-20 following exposure to inhibitors of N-linked protein glycosylation (tunicamycin, TM) and oligosaccharide processing (swainsonine, SW). TM treatment reduced ligand binding at delta- and sigma-opiate receptors and neuroleptic binding sites (20 to 50% of control), with no discernible effect on the binding properties of 5HT1-serotonin receptors. In contrast, exposure to SW resulted in a three-fold increase in binding capacity of sigma-receptors, while decreasing receptor affinity for ligand. SW treatment did not alter ligand interactions with either sigma-receptors or neuroleptic binding sites, but did reduce specific binding of serotonin to 5HT1-receptors. The effects of TM and SW on distinct receptor subpopulations were further demonstrated by attenuation of opiate and serotonin-mediated regulation of intracellular cyclic AMP.     

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.     

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.     

Multiple opiate receptors on neurons of the mammalian central nervous system. In vivo and in vitro studies

Experiments were performed in rat spinal cord cells in vivo and on hippocampal pyramidal cells in vitro. These investigations suggest that acute and chronic treatment renders the neurons subsensitive to opiate alkaloids without altering their sensitivity to opioid peptides. The experiments performed in the dorsal horn of the spinal cord provide evidence that in this structure mu- and delta-receptors may also be localized on the same cell. The evidence for the existence of distinct types of opiate receptors as originally proposed by (1) and suggested by the differing pattern of opiate and opioid peptide activity in various assay systems has been substantiated by investigations involving the selective development of tolerance and the protection of a particular receptor subtype by chemical manipulation. Furthermore, they have been characterized by the use of low concentrations of radiolabelled agonists and antagonists and through the ability of GTP to influence differentially their binding to the opiate receptor (for refs. see: 2). Recently autoradiographic techniques were able to provide direct evidence by mu- and delta-receptors in the mammalian brain (3; 4; 5; 6; and cits. therein). The presence of multiple opiate receptors located on the same cell is suggested by the present study.     

Cation dependence of opioid receptor binding supports theory on membrane-mediated receptor selectivity

A quantitative analysis of the binding of dynorphin A-(1-9)-nonapeptide to the opioid Kappa-receptors of the guinea pig cerebellum [Paterson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6216-6220] shows that changes in electrostatic surface accumulation of the ligand fully account for the observed suppression of binding by a series of univalent and divalent salts. Binding to mu- and delta-receptors, on the other hand, is subject to additional ion-specific effects. These observations support the membrane locations for the receptor sites proposed by the "membrane compartments" theory for opioid receptor selection.     

Regulation of Central Cholecystokinin Recognition Sites by Guanyl Nucleotides

Guanyl nucleotides affected the binding of radiolabeled cholecystokinin (CCK) octapeptide to rodent cortical binding sites. Micromolar quantities of a stable GTP analogue, guanylyl-imidodiphosphate (GppNp), resulted in a plateau where binding was decreased by 30%. In the presence of 25 microM GppNp, binding analysis revealed a decrease in affinity (increase in KD), without an apparent effect on the maximal number of binding sites. Ki values for CCK-related peptides shifted up to 1.6-fold. The rate of peptide association decreased by threefold, and the rapid component of peptide dissociation increased. The collective data suggest that a class of central CCK binding sites is linked to nucleotide regulatory proteins. The evidence is discussed with regard to multiple receptor populations and to possible interconversions between receptor types.