Different types of opiate agonists interact distinguishably with mu, delta and kappa opiate binding sites

The present studies were undertaken to evaluate whether different types of opiate agonists interact in a distinguishable manner with mu, delta and kappa opiate binding sites. Two approaches were employed: (a) the well known effects of metal ions on opiate agonist binding affinities of subsite selective ligands were studied at mu, delta and kappa sites in rat brain homogenates. Binding parameters were obtained by simultaneous computeranalysis of displacement curves using the prototypic ligands dihydromorphine (DHM), (D-Ala2, D-Leu5) enkephalin (DADL) and ethylketocyclazocine (EKC) of the mu, delta and kappa binding sites respectively. The results show that the effects of metal ions depend not only on the binding site, but also on the ligand under investigation. (b) The interaction of the delta agonist DADL with the mu agonist DHM was investigated at mu binding sites by characterizing the type of competition occurring between the two ligands. The interaction was of the noncompetitive type. It therefore appears that the various opiate agonists either interact preferentially with different parts of a larger receptor site area or bind to topographically distinct sites on a single receptor molecule which are coupled allosterically.     

Effects of 5,5''-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

N-acetyl-beta-endorphin1-31 antagonizes the suppressive effect of beta-endorphin1-31 on murine splenocyte proliferation via a naloxone-resistant receptor.

High affinity binding sites for beta-endorphin1-31 (beta-EP) have been observed on transformed mononuclear cells such as the human U937 monocyte-like cell line and the murine EL4-thymoma line, and on normal murine splenocytes. Binding of beta-EP at these sites is resistant to competition by naloxone and other opiate receptor ligands but sensitive to N-acetyl-beta-endorphin1-31 (N-Ac), cations and GTP-gamma-sulfate. Thus, the following studies were done to determine the functional significance of binding beta-EP and N-Ac. beta-EP suppressed phytohemagglutinin (PHA)-stimulated [3H]thymidine uptake in a dose-dependent, naloxone-insensitive fashion. beta-Endorphin1-27, (des)-tyrosine beta-endorphin2-31, or N-Ac failed to duplicate the suppressive effect of beta-EP. However, N-Ac, which is equipotent to beta-EP at displacing 125I-beta-EP bound to murine splenocytes or U937 cells, antagonized the suppressive effect of beta-EP. Taken together with previous binding studies, the present observations suggest that beta-EP effects receptor-mediated responses on normal immunocytes that do not depend on the activation of neuronal-like opiate receptors which are naloxone-sensitive. N-Ac, which shows minimal binding to such brain opiate receptors, is a potent functional antagonist of the naloxone-insensitive immunocyte receptor for beta-EP.     

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.     

Enzymatic Reconstitution of Brain Membrane and Membrane Opiate Receptors

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.     

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.     

The development of opiate tolerance may dissociate from dependence

Experiments on opiate sensitive peripheral tissue preparations such as the mouse vas deferens and the guinea-pig ileum have demonstrated the ability to induce very high degrees of selective tolerance towards particular opiate agonists. Interestingly, the highly tolerant mouse vas deferens failed to display any sign of dependence as judged by the inability of naloxone to precipitate a withdrawal sign. In analogy, the present studies on the guinea-pig ileum revealed a striking dissociation in the degree of tolerance and dependence developed. Opiate receptor binding studies on both tissues point to distinct differences in the opiate-induced effector mechanisms. It is concluded that adaptational changes upon chronic opiate receptor activation may occur at multiple sites within the effector system of the opiate receptor.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

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

The Spectral Sensitivities of Single Cells in the Median Ocellus of Limulus

The spectral sensitivities of single Limulus median ocellus photoreceptors have been determined from records of receptor potentials obtained using intracellular microelectrodes. One class of receptors, called UV cells (ultraviolet cells), depolarizes to near-UV light and is maximally sensitive at 360 nm; a Dartnall template fits the spectral sensitivity curve. A second class of receptors, called visible cells, depolarizes to visible light; the spectral sensitivity curve is fit by a Dartnall template with λ_max at 530 nm. Dark-adapted UV cells are about 2 log units more sensitive than dark-adapted visible cells. UV cells respond with a small hyperpolarization to visible light and the spectral sensitivity curve for this hyperpolarization peaks at 525–550 nm. Visible cells respond with a small hyperpolarization to UV light, and the spectral sensitivity curve for this response peaks at 350–375 nm. Rarely, a double-peaked (360 and 530 nm) spectral sensitivity curve is obtained; two photopigments are involved, as revealed by chromatic adaptation experiments. Thus there may be a small third class of receptor cells containing two photopigments.     

Spectral sensitivity of the green photoreceptor of winged pea aphids

Intracellular recordings are obtained from photoreceptors in the retina of winged (alate) pea aphids Acyrthosiphon pisum (Harris). The responses to monochromatic light, applied in 10‐nm steps over the range 320–650 nm, reveal that all recordings are from green receptors and the spectral sensitivity function of these photoreceptors peaks at 518 nm. A comparison between the spectral sensitivity of the green receptors and extracellular electroretinogram recordings suggests that additional sensitivity to the short‐wavelength light (ultraviolet and/or blue) is also likely to be present in the compound eye of pea aphids. An analysis of the pea aphid genome, comparing its translated nucleotide sequences with the those of the opsin genes of other insect species, supports this electrophysiological finding, although it could not be established whether A. pisum, in addition to the green receptor, has both blue and ultraviolet receptors in the compound eye. The implications of these results for the visual ecology of herbivorous insects are discussed.     

Characterization of type 2 opiate receptors

Recent evidence suggests that the Type 1 opiate receptor (in rat striatal patches) is a mobile receptor which is able to adopt a mu, delta, or kappa opiate receptor ligand selectivity pattern under appropriate conditions. In this paper, we have investigated such a possibility for Type 2 opiate receptors which are visualized diffusely over rat striatum. Ligand selectivity analysis suggested that the Type 2 opiate binding site is equivalent to a delta opiate receptor. The auto-radiographic distribution of Type 2 opiate binding sites is diffuse over most areas of rat brain. Thus, Type 2 opiate binding sites are different from Type 1 opiate receptors which are very discretely distributed in rat brain. Our results suggest that Type 2 opiate receptors, unlike Type 1 opiate receptors, are receptors locked in a delta-like ligand selectivity conformation.     

Accumulation of mu-type opiate receptor ligands during the culture of phytohemagglutinin-stimulated lymphocytes

Substances displacing labelled ligands from opiate receptors of the rat brain membrane fractions were found in the extracts of human blood lymphocytes. Pronase treatment has shown that at least part of the opiate receptor ligands present in lymphocyte extracts are of peptide nature. Opiate mu-receptor ligands content was identical in the extracts of the total population, T-lymphocyte and B-cell-enriched population. However, opiate delta-receptor ligands concentration in T-cell population is 15 times lower than in B-cell-enriched population. Opiate receptor ligands content is not changed during cultivation of nonstimulated lymphocytes. However, phytohemagglutinin activation results in a threefold increase of opiate mu-receptor ligands content in lymphocytes. The results obtained suggest that synthesis and processing of opiate mu-receptor ligands precursor occur in stimulated T-lymphocytes.     

Studies on the effects of glucose in vitro and of the glycaemic state in vivo on the binding characteristics of mu, delta and kappa opiate receptors in mouse brain.

The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.     

Competitive inhibition of stereospecific opiate binding by local anesthetics in mouse brain

Cationic local anesthetics inhibit competitively the stereospecific binding of naltrexone and etorphine on the mouse brain opiate receptor. In contrast, the inhibition produced by benzocaine, a non-cationic local anesthetic, is non-competitive. It is suggested that the cationic group of local anesthetics interacts with a specific anionic binding site on the opiate receptor and that there are certain structural similarities between the receptors for both types of drugs. It is evident from these studies that several drugs can unspecifically modify the pharmacologic effects of opiates and that they could be useful tools to further characterize the opiate receptor.     

Synthesis and receptor binding characteristics of [D-Ala2, cysteamine 5] enkephalin, a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.     

Solubilization in good yield of active opiate binding sites from mammalian brain

Active opiate binding sites have been solubilized from mammalian brain cell membranes. The presence of 0.5-0.1 M NaCl during treatment of membranes from rat brain, human frontal cortex, and bovine corpus striatum with glycodeoxycholate or digitonin resulted in the extraction of active opiate binding sites in yields ranging up to 43%. The criteria for solubility of the sites were their inability to sediment at 10(5) x g after 2 hr and their apparent molecular weight of 3- 4 x 10(5) as determined by gel filtration. The receptors in solution resemble the membrane-bound sites with respect to saturability, stereo-specificity, sensitivity to heat and reagents, and high affinity for opioid ligands. The interaction of solubilized sites with immobilized lectins was used to demonstrate the glycoprotein nature of the opiate receptor. Soluble receptors from all species studied were retained by wheat germ agglutinin(WGA)-agarose and could be specifically eluted with N-acetylglucosamine. No retention of solubilized material was observed with eight other lectins examined, including horseshoe crab lectin, a sialic acid specific agglutinin. The receptor protein eluted from WGA columns was enriched 25-50-fold over the crude soluble fraction.     

Characterization of a photoalkylated psoralen receptor in HeLa cells

Psoralens in combination with ultraviolet light are potent modulators of epidermal cell growth and differentiation. Responsive cell types contain specific, saturable, high-affinity binding sites for the psoralens. These binding sites become covalently modified by the psoralen molecule following ultraviolet light exposure. In the present studies the psoralen receptor, labeled with [3H]8-methoxypsoralen, was visualized in the cytoplasmic and plasma membrane fractions of HeLa cells following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor had an apparent molecular mass of approximately 22,000 daltons and was shown to be sensitive to protease, but not nuclease treatment. The radiolabeled receptor could not be visualized in nuclear extracts of cells. Covalent binding of the radioligand to the receptor protein was inhibited by excess unlabeled 8-methoxypsoralen, indicating that covalent psoralen-receptor binding was saturable. In addition, the covalently modified receptor was found to persist in cells for over 5 h. The presence of a cellular protein that exhibits specific affinity for the psoralens and becomes photoalkylated by these compounds, together with previous data showing that the psoralens have direct effects on the cell surface membranes, supports our model that some of the biological effects of photoactivated psoralens are receptor-mediated.     

Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.     

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.     

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.