Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC.

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.

A new surfactant series, the N-alkylamino-1-deoxylactitols: application for extraction of 'op' opiate receptors from frog brain.

A new series of surfactants, the N-alkylamino-1-deoxylactitols, was prepared and employed to extract 'op' opiate receptors from frog brain. These surfactants are both cheap and convenient to prepare. Receptors were reproducibly extracted in a good yield using N-nonylamino-1-deoxylactitol. This derivative, which was not denaturing during the extraction process, could thus be used instead of the more costly digitonin, whose rather variable purity affects yield.

Inactivation and solubilization of opiate receptors by phospholipases A2.

(1) As previously shown, stereospecific binding of opiates to membrane bound receptors is inhibited by treatment with small amounts of phospholipase A2 from Vipera russelli. This effect is quantified and compared with the enzymes from the venoms of Naja Naja siamensis, Apis Mellifica and from porcine pancreas. All enzymes are equally effective. The inhibition is due to partial phospholipid hydrolysis leading to inactivation of membrane-bound receptor. (2) Bee venom phospholipase A2 together with the synergistically acting peptide, melittin, causes receptor solubilization up to 80% of preformed receptor-ligand complex can be solubilized in this manner. (3) Lysophosphatidylcholine, a product of phospholipid hydrolysis, solubilizes performed receptor-ligand complex to a similar extent. Several other detergents were tested for their ability to solubilize receptor-ligand complex. Digitonin appears to be most effective in solubilizing such a complex.

Immunohistochemical analysis of opioid receptors in peripheral tissues.

Immunohistochemical staining is widely used to identify opioid receptors in specific cell types or anatomical structures throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditions in mice. Moreover, we consider the current debate on the specificity of antibodies.

[(35)S]GTPγS autoradiography for studies of opioid receptor functionality.

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR) that are Gi-coupled which make them suitable for studying the receptor functionality. The [(35)S]GTPγS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the α-subunit of the G protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.

Analysis of potassium and calcium imaging to assay the function of opioid receptors.

As the activation of opioid receptors leads to the modulation of potassium and calcium channels, the ion imaging represents an attractive method to analyze the function of the receptors. Here, we describe the imaging of potassium using the FluxOR™ potassium ion channel assay, and of calcium using Fura-2 acetoxymethyl ester. Specifically, we (1) characterize the activation of the G-protein-coupled inwardly rectifying potassium 2 channel by agonists of µ- and δ-opioid receptors with the aid of the FluxOR™ assay in cultured mouse dorsal root ganglion neurons, and (2) describe calcium imaging protocols to measure capsaicin-induced transient receptor potential vanilloid 1 channel activity during opioid withdrawal in transfected human embryonic kidney 293 cells.

Electrophysiological patch clamp assay to monitor the action of opioid receptors.

The patch clamp is a valuable electrophysiological technique, which allows the study of single or multiple ion channels in cells, and it is particularly useful in testing the excitable cells such as neurons. Activation of neuronal opioid receptors results in the modulation of various ion channels, which enables to examine the receptors' action with the patch clamp. In this chapter, we analyze the activation of the G-protein-coupled inwardly rectifying potassium channel 2 by opioids, and the capsaicin-induced transient receptor potential vanilloid 1 channel currents during opioid withdrawal, using the whole cell patch clamp in transfected human embryonic kidney 293 cells as well as in mouse and rat primary dorsal root ganglion neurons.

Opioid receptors: methods for detection and their modes of actions in the eye.

This study provides evidence for the presence of opioid-receptors in the retina, optic nerve, and optic nerve head astrocytes. These receptors were measured by more than one technique including Western blotting, immunohistochemistry, and functional assays such as scotopic electroretinogram (ERG) and Pattern ERG. I also have provided evidence in recently published work from my laboratory that opioid receptors, more specifically δ-opioid receptors, play crucial roles in retina neuroprotection against ischemic and glaucomatous injuries. This chapter provides detailed procedures to measure opioid receptor activation and their roles in retina neuroprotection using functional assays such as scotopic ERG and pattern ERG.

Affinity purification of the opioid receptor in NG 108-15 cells using an avidin-biotin system with a novel elution method.

Affinity purification of the opioid receptor in NG 108-15 cells was carried out using an affinity resin based on the avidin-biotin interactions, but a new elution method was employed with a radioligand of sub-micromolar concentration. A synthesized biotinyl derivative of leucine-enkephalin has a high affinity for the delta-receptor, but the affinity was lowered 10-fold in the presence of avidin. The new elution method is based on this affinity decrease and resulted in a 100-fold purification over the initial crude materials in the single step. SDS-polyacrylamide gel electrophoresis of the purified receptor revealed three polypeptides of 58, 65 and 71 kDa as possible components of the delta-receptor.

Separation of kappa-opioid receptor subtype from frog brain.

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Immunoaffinity-purified opiate receptor specifically binds the delta-class opiate receptor ligand, cis-(+)-3-methylfentanylisothiocyanate, SUPERFIT.

Using an antibody generated against the opiate receptor on NG108-15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108-15 cells.

Solubilization in high yield of opioid receptors retaining high-affinity delta, mu and kappa binding sites.

The binding sites for opiates (agonist and antagonist) and opioid peptides can be solubilized from rat brain membranes with digitonin in the presence of Mg2+ (10 mM). High affinity and high capacity binding to the soluble delta, mu, and kappa receptors is obtainable when the membranes are treated in Mg2+ (30 degrees C, 60 min) prior to solubilization. The yields of solubilized binding sites extracted with digitonin, 40-90%, are higher than those obtained from Mg2+-pretreated membranes with other detergents commonly used for receptor solubilization. The stability of the digitonin-soluble opioid receptor at room temperature makes it useful for purification and characterization.

Biological activity of mu opioid receptor.

The purification of the mu-opioid receptor has proved to be more difficult than most other receptors for at least two reasons. First, they are easily inactivated by most detergents and the second problem is the heterogeneous existence of the opioid receptor. Despite these problems, this laboratory, and several others, have recently reported purification of the mu-opioid receptor. The 58,000 molecular weight of the purified mu-opioid receptor agrees closely with the value reported by other investigators. In addition, it was found that this purified mu-opioid receptor was associated with a phosphatase activity. While the relevance of this phosphatase activity to opioid receptor action remains to be determined, it is interesting to note that this activity was stimulated by Mn++ and Mg+ ions, both of which also increase opioid binding.

Hybromet: a ligand for purifying opioid receptors.

Condensation of the Grignard reagent derive from 2-[4-(allyloxy)phenyl]ethyl bromide (4b) with 7 alpha-acetyl-6,14-endo-ethenotetrahydrothebaine (5) furnished the (R) tertiary carbinol, 7, which upon methoxymercuration followed by treatment with the KBr gave the bromomercurio compound 10 (Hybromet). The corresponding N-cyclopropylmethyl analogue, 11, was prepared also. The bromomercurio compound, 1, and the mercaptobenzothiazole derivative, 3, gave allyl phenyl ether when treated with BAL at room temperature. Similar treatment of 10 with BAL gave 7 in high yield. Binding studies using rat brain homogenates indicated that 7, 13, and 14 have moderately high affinities for mu rather than delta binding sites. Although much weaker, 10 showed preferential mu binding also. These results along with the fact that 10 reacted smoothly with sulfhydryl groups suggest that Hybromet would be a suitable ligand for use in affinity chromatography.

Characterization of a polyclonal antibody to the mu opioid receptor.

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the mu receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [3H]etorphine (universal opioid) and [3H]dihydromorphine (mu opioid) from rat membranes, but was ineffective against the binding of [3H]ethylketocyclazocine (kappa [3H]D-Ala2,D-Leu5-enkephalin (delta opioid) or [3H]phencyclidine (phencyclidine/sigma receptor ligand). The antibody was able to precipitate the Mr 94,000 component of the 125I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma X Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.

Hydrodynamic parameters of opioid receptors from frog brain.

Active opioid receptors were solubilized from frog (Rana esculenta) brain membranes using 1% digitonin. The solubilized preparation was sedimented in sucrose density gradient and applied to Sepharose-6B column. In the ultracentrifugation experiments, two distinct molecular forms of the opioid receptors were observed with apparent s20, w values of 15.7 and 10.8 S. The estimated molecular weights were 470 and 180 kD. The Stokes radii of the two separate forms were determined by gel filtration and found to be 71 and 42 A. The corresponding molecular weights were 500 and 140 kD indicating a good correlation with data obtained from the sedimentation experiments.

Mu-receptor specificity of the opioid peptide irreversible reagent, [3H]DALECK.

A novel affinity reagent DALECK, i.e. D-Ala2-Leu5-enkephalin with a C-terminal chloromethyl ketone group, was previously synthesized in normal and in tritiated form and shown to react irreversibly at opioid receptors, with some evidence for selectivity for the mu subtype. DALECK tritiated in its phenolic group has been synthesized at 13-fold higher specific radioactivity than in the previous study. In the irreversible reaction of this product at pH 8.1 with rat brain membranes it was confirmed that only one polypeptide there is labelled, of apparent Mr 58,000. Competition between this reaction and ligands highly selective for the mu, delta or kappa binding sites yielded curves demonstrating the very high selectivity of the DALECK irreversible reaction for the mu site. The results provide evidence that the mu opioid receptor protein contains only one type of binding subunit, whose apparent Mr is 58,000, this size being dependent upon the conditions used in the gel electrophoresis and being higher when stringent conditions which would reduce all internal disulphide bonds are applied.

Solubilization of two molecular forms of the frog brain opioid receptor.

In equilibrium binding studies, using 3H-etorphine and 3H-diprenorphine, digitonin extracts of frog brain membranes are found to contain two classes of sites, one of which is seen only in the presence of Na+ ions. Centrifugation of the extracts in sucrose gradients separates two macromolecular components (10S and 12S) which display specific opiate binding activity. The 12S component appears to carry the site that binds opiates in the absence of Na+ ions while the 10S component would carry the other site, i.e. the one which is seen only in the presence of Na+ ions in equilibrium binding studies. Preliminary evidence is also given that in extracts of frog brain membranes which have been pre-incubated with 120 mM NaCl, the balance of the two components is shifted in favor of the slower sedimenting (10S) one. These results are discussed in terms of the regulation of the state of equilibrium between an agonist (12S) and an antagonist (10S) form of the opioid receptor.

Some properties of brain opioid receptors in membrane bound and solubilized state.

In brain membrane preparations, 3H-naloxone binding to bullfrog brain gave the highest values versus other species such as carp, chicken, rat and mouse. Scatchard analysis revealed that the Bmax value of bullfrog brain membranes was three fold greater than that of rat brain membranes. Opioid receptors were solubilized from bullfrog brain membranes using digitonin or 3-(cholamidopropyl)-dimethylammonio-1-propane sulfonate (CHAPS) as the detergent. The properties of solubilized opioid receptors were essentially the same as membrane bound receptors with regard to sensitivity to various treatments. The order of potency for a series of agonists in displacing 3H-naloxone binding in both membrane and solubilized preparations was bremazocine greater than pentazocine greater than morphine greater than delta-receptor peptide. Partial purification of opioid receptors was performed using a fast protein liquid chromatography system. Specific binding activity of 3H-naloxone was increased 10-15 fold within 20 min using the Mono Q column.

Solubilization and characterization of kappa opioid binding sites from guinea pig cerebellum.

Solubilization of opioid binding sites from guinea pig cerebellum by digitonin, in the absence and presence of NaCl, resulted in very similar yields (25-30%) of [3H]bremazocine binding. Saturation curves of [3H]bremazocine binding give linear Scatchard plots for both soluble and membrane-bound binding sites yielding similar Kd's and Bmax's. Soluble kappa sites seem to resemble closely their membrane-bound counterparts and retain high affinity and selectivity for various kappa opioid ligands. The apparent molecular weight of soluble kappa sites is ca. 4 X 10(5). Results from this study, along with our previous findings with toad and guinea pig brain, indicate that kappa sites (unlike mu and delta) can be solubilized in good yield by digitonin even in the absence of NaCl. This supports the hypothesis that kappa sites may represent molecular species different from those of mu and delta sites.