Modification by Cholecystokinin Octapeptide of the Binding ofμ-, δ-, and K-Opioid Receptors

Previous study has shown that cholecystokinin (CCK) octapeptide (CCK-8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium-labeled agonists for the mu-[(tryrosyl-3,5-3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAGO], delta- ([tyrosyl-3,5-3H][D-Pen2,5]enkephalin ([3H]DPDPE], and kappa- ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK-8. In the competition experiments, CCK-8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 mumol/L. In the saturation experiments, CCK-8 at concentrations of 0.1 nmol/L to 1 mumol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK-8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK-8 inhibits the binding of mu- and kappa-opioid receptors via the activation of CCK receptors.     

Guanine nucleotides inhibit binding of agonists and antagonists to soluble opiate receptors

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.     

Agonist-Selective Protection of the Opioid Receptor-Coupled G Proteins from Inactivation by 5''-p-fluorosulphonylbenzoyl Guanosine

The guanine nucleotide analogue, 5'-p-fluorosulphonylbenzoyl guanosine (FSBG), can react covalently with GTP-binding proteins (G proteins). In rat brain membranes, FSBG causes a time-dependent loss of beta,gamma-imido[8-3H]guanosine 5'-triphosphate binding sites. Using 1 mM FSBG, the guanyl nucleotide modulation of opioid agonist binding is abolished, whereas the guanyl nucleotide sensitivity of neurotensin binding is retained. The action of FSBG can be prevented by the presence of opioid agonists, but not the antagonist naloxone. Iodoacetamide treatment of membranes in the presence of agonist, but not antagonist, can attenuate the action of FSBG in blocking guanyl nucleotide modulation of opioid agonist binding. These results suggest that FSBG covalently modifies essential thiol groups, whose exposure to the reagent is modified by agonist occupancy of the receptor, on a species of G protein linked to opioid receptors, but not on a species of G protein linked to neurotensin receptors. Thus, FSBG may have selectivity for the forms of Gi or Go, proteins associated with opioid receptors.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

八肽胆囊收缩素对抗mu和Kappa型受体介导的镇痛作用

以往的资料表明,八肽胆襄收缩素(CCK-8)能对抗阿片镇痛,本实验进一步分析 CCK-8对抗哪一类型阿片受体激动剂的镇痛作用。给大鼠脊髓蛛网膜下腔(I.T.)注射 CCK-8(剂量4ng到1.0μg)既不产生痛敏也不产生镇痛。I.T.注射特异性的μ受体激动剂 PL01710 ng 或 k 受体激动剂 NDA P500 ng 引起的镇痛作用可被注射 CCK-8 4ng 所对抗。而L.T.注射δ受体激动剂 DPDPE(6.5,13.0和26.Oμg)引起的镇痛作用不能被 CCK-8(4ng,40ng I.T.)所对抗。但 CCK-8对抗 PL017和 NDAP 镇痛的作用可被 I.T.CCK 受体拮抗剂 proglumide(3μg)所翻转。以上结果表明,I.T.注射 CCK-8可有效地对抗μ和 k 受体介导的镇痛,并且这种对抗作用是经 CCK 受体介导而实现的。     

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.     

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.     

Opioid Receptors in Magnesium-Digitonin-Solubilized Rat Brain Membranes Are Tightly Coupled to a Pertussis Toxin-Sensitive Guanine Nucleotide-Binding Protein

Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively.     

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.     

Solubilization and characterization of CCK receptors from mouse pancreas

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.     

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

Purification of the pancreatic cholecystokinin receptor

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 less than CCK-33 less than desulfated CCK-8 less than CCK-4) and binding affinity. Scatchard transformations of binding data for the purified receptor preparation were best fit by linear plots compatible with a single class of binding sites with Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.     

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.     

Association of enkephalin catabolism inhibitors and CCKB antagonists: A potential use in the management of pain and opioid addiction

The overlapping distribution of opioid and cholecystokinin (CCK) peptides and their receptors (μ and δ opioid receptors; CCK-A and CCK-B receptors) in the central nervous system have led to a large number of studies aimed at clarifying the functional relationships between these two neuropeptides. Most of the pharmacological studies devoted to the role of CCK and enkephalins have been focused on the control of pain. Recently the existence of regulatory mechanisms between both systems have been proposed, and the physiological antagonism between CCK and endogenous opioid systems has been definitely demonstrated by coadministration of CCK-B selective antagonists with RB 101, a systemically active inhibitor, which fully protects enkephalins from their degradation. Several studies have also been done to investigate the functional relationships between both systems in development of opioid side-effects and in behavioral responses. This article will review the experimental pharmacology of association of enkephalin-degrading enzyme inhibitors and CCK-B antagonists to demonstrate the interest of these molecules in the management of both pain and opioid addiction. Special issue dedicated to Dr. Eric J. Simon.     

(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands

(S)-4-(Carboxamido)phenylalanine (Cpa) is examined as a bioisosteric replacement for the terminal tyrosine (Tyr) residue in a variety of known peptide ligands for the mu, delta and kappa opioid receptors. The Cpa-containing peptides, assayed against cloned human opioid receptors, display comparable binding affinity (Ki), and agonist potency (EC50) to the parent ligands at the three receptors. Cpa analogs of delta selective peptides show an increase in delta selectivity relative to the mu receptor. Cpa is the first example of an amino acid that acts as a surrogate for Tyr in opioid peptide ligands, challenging the long-standing belief that a phenolic residue is required for high affinity binding.     

Sodium regulation of opioid agonist binding is potentiated by pertussis toxin

Pretreatment of intact NG108-15 cells with pertussis toxin suppresses opioid inhibition of cyclic AMP accumulation mediated by the inhibitory guanine nucleotide-binding regulatory protein, Ni, which apparently also mediates the inhibitory nucleotide effects on opioid against binding. The toxin treatment had no effect on opioid agonist binding measured in NG108-15 cell membranes without sodium present. However, the toxin potentiated the inhibitory effect of sodium on agonist binding, leading to an agonist-specific reduction of opioid receptor affinity in the presence of sodium in the binding reaction. The potency of the stable GTP analog, GTP gamma S, to reduce agonist binding in the presence of sodium was little changed in membranes prepared from pertussis toxin-treated cells compared to control membranes, whereas the potency of the stable GDP analog, GDP beta S, was magnified. The data indicate that ADP-ribosylation of Ni by pertussis toxin potentiates sodium regulation of opioid agonist binding and that the communication between Ni and opioid receptors is not lost by the covalent modification of Ni.     

Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer.

Dimerization of some G protein-coupled receptors has recently been demonstrated, but how widespread this phenomenon might be and its functional implications are not yet clear. We have utilized biophysical and biochemical techniques to evaluate whether the type A cholecystokinin (CCK) receptor can form oligomeric complexes in the plasma membrane and the impact of ligand binding and signaling on such complexes. We investigated the possibility of bioluminescence resonance energy transfer (BRET) between receptor constructs that included carboxyl-terminal tags of Renilla luciferase or yellow fluorescent protein. Indeed, co-expression of these constructs in COS cells resulted in the constitutive presence of a significant BRET signal above that in a series of controls, with this signal reduced by co-expression of competing non-tagged CCK receptors. The presence of an oligomeric complex of CCK receptor molecules was confirmed in co-immunoprecipitation experiments. Occupation of CCK receptors with agonist ligands (CCK or gastrin-4) resulted in the rapid reduction in BRET signal in contrast to the enhancement of such a signal reported after agonist occupation of the beta(2)-adrenergic receptor. These effects on CCK receptor oligomerization were concentration-dependent, correlating with the potencies of the agonists. A smaller effect was observed for a partial agonist, and no effect was observed for antagonist occupation of this receptor. Agonist-induced reduction in BRET signal was also observed for pairs of CCK receptors with a donor-acceptor pair situated in other positions within the receptor. Manipulation of the phosphorylation state of CCK receptor using protein kinase C activation with phorbol ester or inhibition with staurosporine had no effect on the basal level or agonist effect on CCK receptor oligomerization. This provides the first evidence for CCK receptor oligomerization in living cells, with insights that the active conformation of this receptor dissociates these complexes in a phosphorylation-independent manner.     

Novel delta opioid receptor agonists exhibit differential stimulation of signaling pathways

A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for the δ opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity (IC₅₀ = 5.8 nM), excellent selectivity for the δ opioid receptor over the alternative μ and κ opioid receptors, full agonist efficacy in receptor down-regulation and MAP kinase activation assays, and low-efficacy partial agonist activity in stimulation of GTPγS binding. The apparent discrepancy observed in these functional assays may stem from different signaling pathways involved in each case, as found previously for other G-protein coupled receptors. More biological studies are underway to better understand the differential stimulation of signaling pathways by these novel compounds.