Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis.

BACKGROUND: Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. RESULTS: Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the mu-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. CONCLUSION: A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids.

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).

Ligand-regulated internalization of the opioid receptor-like 1: a confocal study.

To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTPgamma(35)S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (>75%) with the periplasmic membrane. In the presence of 0.1 microm noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 m sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe(1)psi(CH(2)-NH)Gly(2)]-noc-(1-13)NH(2)) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH(2)) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.

Utilizing functional genomics to identify new pain treatments : the example of nociceptin.

Nociceptin/orphanin FQ (noc/oFQ) is the first novel bioactive substance to have been discovered by the implementation of a functional genomics/reverse pharmacology approach. The neuropeptide was indeed identified in brain extracts as the natural ligand of a previously cloned orphan G protein-coupled receptor, the opioid receptor-like 1 (ORL1) receptor. Since its discovery in 1995, noc/oFQ has been the subject of intensive study to establish its role in normal brain function and its possible involvement in neurophysiopathology. Although the neuropeptide, an inhibitor of neuronal activity, has been found to have a wide spectrum of pharmacological effects in vivo, none has been as intensively investigated as its action on nociception and nociceptive processing. There is now substantial evidence that noc/oFQ has a modulatory role in nociception. However, dependent on the dose and site of injection, and possibly the animal's genetic background and even psychological status, the peptide has been variously reported to cause allodynia, hyperalgesia, analgesia, and even pain, in rodents. Overall, noc/oFQ tends to facilitate pain when administered supraspinally, and to inhibit it when administered spinally. These opposing effects beg the obvious, yet still unanswered, question as to what would be the net effect on nociception of an ORL1 receptor ligand, agonist or antagonist, able to target supraspinal and spinal sites simultaneously. Owing to the research effort of several drug companies, such ligands, i.e. nonpeptidic, brain-penetrating agonists and antagonists, have recently been produced whose systematic screening in animal models of acute and inflammatory pain may help validate the ORL1 receptor as the target for novel, non-opioid analgesics.

Nociceptin antagonism: probing the receptor by N-acetyl oligopeptides.

In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the kappa-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [(35)S]GTPgammaS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K(e)) of 2.44 nM, and no agonist effect at 10 microM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the delta-opioid receptor agonist [D-Ala(2),D-Leu(5)]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [(3)H]nociceptin-NH(2) binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [(35)S]GTPgammaS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.

Description of the low-affinity interaction between nociceptin and the second extracellular loop of its receptor by fluorescence and NMR spectroscopies.

The second extracellular loop (ECL2) of the Noc receptor has been proposed to be involved in ligand binding and selectivity. The interaction of Noc with a constrained cyclic synthetic peptide, mimicking the ECL2, has been studied using fluorescence and NMR spectroscopies. Selective binding was shown with a dissociation constant of approximately 10 microM (observed with the constrained cyclic loop and not with the open chain), and residues involved in ligand binding and selectivity have been identified. This bimolecular complex is stabilized by (i) ionic interactions between the two Noc basic motives and the ECL2 acidic residues; (ii) hydrophobic contacts involving Noc FGGF N-terminal sequence and an ECL2 tryptophane residue. Our data confirm that Noc receptor's ECL2 contributes actively to ligand binding and selectivity by providing the peptidic ligand with a low affinity-binding site.

Effects of clonidine on diuretic response in ascitic patients with cirrhosis and activation of sympathetic nervous system.

The effects of the addition of clonidine to diuretics on the mobilization of ascites in the short term (diuretic response and requirement of diuretics) and the long term (readmissions for tense ascites and requirement of diuretics) were examined in patients with cirrhosis and with increased sympathetic nervous system (SNS) activity. We also studied neurohormonal, hemodynamic effects and side effects of clonidine and diuretics. Patients were randomized to receive placebo (group 1, n = 32) or clonidine (0.075 mg) twice daily (group 2, n = 32) for 3 months. After 8 days and for 10 days duration, spironolactone (200 mg/day) was added in both groups. After this period, the dosages of diuretics were individually increased until diuretic response. Responding patients were discharged and followed at the outpatient clinic. During the first hospitalization, the time needed for diuretic response was shorter in group 2 than in group 1. The mean requirement for diuretics was significantly higher in group 1 than in group 2, and the diuretic complications (hyperkalemia and renal impairment) were significantly lower in group 2. Clonidine induced a permanent decrease in SNS activity and delayed decrease in renin/aldosterone levels. During the follow-up, the time to the first readmission for tense ascites was shorter in group 1 than in group 2. Readmissions related to tense ascites or diuretic complications were significantly lower in group 2. The mean requirement for diuretics was significantly higher in group 1 than in group 2. In conclusion, the additional administration of clonidine to diuretics induced an earlier diuretic response associated with fewer diuretic requirements and complications.

Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role.

Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2.

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-L-Phe (Bpa)(2)]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa(2)]-HP1 binds the receptor with high affinity (K; approximately 3nM) and is as potent as HP1 in stimulating GTPgammaS binding (50-60% of nociceptin maximal effect). UV irradiation at 365nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa(2)]-HP1 results in the irreversible labelling of a glycoprotein of M(r) approximately 66kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.

Agonist-independent localization of the NOP receptor in detergent-resistant membrane rafts.

A lipid rafts/detergent-resistant membrane (DRM) fraction was prepared from recombinant HEK293 cells stably expressing the human NOP receptor fused to the green fluorescent protein EGFP (hNOPr-EGFP), and probed for the presence and functionality of the fusion protein. Fluorescence detection as well as immunoblotting with an anti-GFP antibody revealed that most of the fusion protein was recovered in the DRM fraction, wherein it mediated efficiently NOP-induced stimulation of GTPgamma(35)S binding. Recovery of hNOPr-EGFP in the DRM fraction was not affected had the cells been acutely or chronically exposed to NOP prior to detergent treatment. Therefore, in HEK cells, the NOP receptor localizes constitutively to DRMs wherein it retains ability to couple with hetero-trimeric G protein.