Biomedical subjects

H Nagase

Publications and source records attributed to H Nagase.

At least 127 records · Page 7Linked to original sources

PubMed1999

Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate.

The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.

Cell Line↗

PubMed1999

Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma.

Although matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components in vitro, the role of MMPs in the in vivo accumulation of the cells to the site of inflammation in bronchial asthma is still obscure. In this study, we investigated the role of MMPs in the pathogenesis of bronchial asthma, using a murine model of allergic asthma. In this model, we observed the increase of the release of MMP-2 and MMP-9 in bronchoalveolar lavage fluids after Ag inhalation in the mice sensitized with OVA, which was accompanied by the infiltration of lymphocytes and eosinophils. Administration of tissue inhibitor of metalloproteinase-2 to airways inhibited the Ag-induced infiltration of lymphocytes and eosinophils to airway wall and lumen, reduced Ag-induced airway hyperresponsiveness, and increased the numbers of eosinophils and lymphocytes in peripheral blood. The inhibition of cellular infiltration to airway lumen was observed also with tissue inhibitor of metalloproteinase-1 and a synthetic matrix metalloproteinase inhibitor. These data suggest that MMPs, especially MMP-2 and MMP-9, are crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness, which are pathophysiologic features of bronchial asthma, and further raise the possibility of the inhibition of MMPs as a therapeutic strategy of bronchial asthma.

Administration, Inhalation↗

PubMed1999

Algogenic mediator-induced nociceptive response in diabetic mice.

The duration of the somatostatin-, bradykinin- or prostaglandin F2alpha-induced nociceptive response was significantly less in diabetic mice than in non-diabetic mice. Subcutaneous injection of 7-benzylidenenaltrexone (0.1, 0.3 and 1 mg/kg), an antagonist of delta1-opioid receptors, had no significant effect on either somatostatin-, bradykinin- or prostaglandin F2alpha-induced nociceptive responses in non-diabetic mice. 7-Benzylidenenaltrexone (0.1 and 0.3 mg/kg, s.c.) also had no significant effect on somatostatin- or prostaglandin F2alpha-induced nociceptive responses in diabetic mice. However, the bradykinin-induced nociceptive response in diabetic mice was dose-dependently and significantly increased when 7-benzylidenenaltrexone (0.1, 0.3 and 1 mg/kg, s.c.) was injected 10 min before the injection of bradykinin. These results suggest that a spinal delta1-opioid receptor-mediated endogenous antinociceptive system may inhibit the bradykinin-mediated nociceptive responses in the second phase of the formalin-induced nociceptive response in diabetic mice.

Animals↗

PubMed1999

Different roles of mu-, delta- and kappa-opioid receptors in ethanol-associated place preference in rats exposed to conditioned fear stress.

The present study was designed to investigate the role of the endogenous opioid system in the development of ethanol-induced place preference in rats exposed to conditioned fear stress (exposure to an environment paired previously with electric foot shock), using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) with conditioned fear stress induced significant place preference. Naloxone (1 and 3 mg/kg, s.c.), a non-selective opioid receptor antagonist, significantly attenuated this ethanol-induced place preference. Moreover, the selective mu-opioid receptor antagonist beta-funaltrexamine (3 and 10 mg/kg, i.p.) and the selective delta-opioid receptor antagonist naltrindole (1 and 3 mg/kg, s.c.) significantly attenuated ethanol-induced place preference. In contrast, the selective kappa-opioid receptor antagonist nor-binaltorphimine (3 mg/kg, i.p.) significantly enhanced ethanol-induced place preference. Furthermore, 75 mg/kg ethanol (which tended to produce place preference) combined with the mu-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or the selective delta-opioid receptor agonist 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha- octahydroquinolino [2,3,3,-g] isoquinoline (TAN-67; 20 mg/kg, s.c.), at doses which alone did not produce place preference, produced significant place preference. However, co-administration of the selective kappa-opioid receptor agonist trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenacetamide methanesulfonate (U50,488H; 0.3 and 1 mg/kg, s.c.) with ethanol (300 mg/kg, i.p.) dose dependently attenuated ethanol-induced place preference. Moreover, conditioned fear stress shifted the response curve for the aversive effect of U50,488H to the left. These results suggest that mu- and delta-opioid receptors may play critical roles in the rewarding mechanism of ethanol, and that kappa-opioid receptors may modulate the development of the rewarding effect of ethanol under psychological stress.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1999

Digestion of C1q collagen-like domain with MMPs-1,-2,-3, and -9 further defines the sequence involved in the stimulation of neutrophil superoxide production.

C1q, a subunit of the first component (C1) of the classical complement pathway, binds to neutrophils via its collagen-like region (C1q-CLR) stimulating superoxide production. We previously identified a region of C1q-CLR, defined by fragments generated by trypsin and endoLys-C digestion, that was required for triggering this respiratory burst. To further localize that critical site, purified human C1q was digested with pepsin to generate C1q-CLR, and subsequently cleaved with the matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9. Digestion of C1q-CLR with any of these MMPs did not alter the circular dichroism spectra, demonstrating that the fragments generated had maintained the secondary structure observed in the native molecule. All fragments retained the ability to trigger superoxide production by neutrophils. Analysis of the amino acid sequences of the purified cleavage products (none of which are identical to the published cleavage site specificities for these enzymes) demonstrated that it is the C-chain, but not the A-chain of C1q, that is critical for stimulating this activity, and thus may be a target for future therapeutic intervention.

Amino Acid Sequence↗

PubMed1999

Frequency Measurement of Pure Rotational Transitions of H217O and H218O from 0.5 to 5 THz.

Frequencies of pure rotational transitions of isotopic species of water, H217O, and H218O were measured in the 1-5 THz region with a high-precision far-infrared spectrometer using a tunable radiation source. Measured frequencies of more than 100 spectral lines for each species provide an excellent frequency standard for the far-infrared region. Molecular parameters based on a Watson-type Hamiltonian have been obtained to reproduce the observed frequencies. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Structural properties of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Amino Acid Sequence↗

PubMed1999

Pharmacological profile of MS-377, a novel antipsychotic agent with selective affinity for sigma receptors.

RATIONALE: MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]me thyl-2- pyrrolidinone L-tartrate) was discovered as a new chemical entity with affinity for sigma receptors and without affinities for dopamine receptors. OBJECTIVE: In the present study, we examined the antipsychotic profile of MS-377 in several in vitro and in vivo experiments. METHODS: As in vitro assays, radioligand binding assays for sigma 1, sigma 2, D2 and 5-HT2 receptors were performed. As in vivo animal models, the effects of MS-377 on several behavioral models induced by phencyclidine (PCP), (+)-N-allylnormetazocine (NANM), apomorphine (Apo) and 5-hydroxytryptamine (5-HTP) were evaluated. All assay systems were conducted using the clinically active antipsychotic agents as reference standards. RESULTS: MS-377 displaced ligand bound to sigma 1 receptors in vitro. However, no such displacement was observed at sigma 2 or 5-HT2 receptors in vitro, or at D2 receptors either in vitro or in vivo. In behavioral studies, MS-377 inhibited both NANM- and PCP-induced head-weaving at low doses in mice and rats, whereas antipsychotic agents used in the present study were only effective against NANM-induced head-weaving behavior in mice. In addition, MS-377 antagonized Apo-induced climbing behavior and 5-HTP-induced head-twitching behavior in mice. MS-377 was inactive in models of extrapyramidal side effect (EPS) liability such as prevention of Apo-induced stereotypy and induction of catalepsy in rats. CONCLUSION: The present study demonstrated that MS-377 had not only anti-PCP activity but also anti-dopaminergic and anti-serotonergic activities in vivo, without acting directly through D2 or 5-HT2 receptors. Therefore, MS-377 could be a novel antipsychotic agent with clinical efficacy for overall symptoms of schizophrenia including its negative symptoms and without EPS liability.

Amisulpride↗

PubMed1999

Natural killer (NK) cell activity and NK cell subsets in workers with a tendency of burnout.

The involvement of cellular immunity in the burnout syndrome remains to be elucidated. We assessed three components of burnout of the Maslach Burnout Inventory: emotional exhaustion; depersonalization (DP); and personal accomplishment, as well as natural killer cell activity (NKCA) and NK cell subsets in 42 male workers. Workers with a higher DP score showed a lower NKCA and a lower proportionality of CD57+CD16+ to total lymphocytes. There were no differences in any of the health behaviors (e.g., smoking, alcohol, or obesity) between workers showing higher burnout and those showing lower burnout. A stepwise multiple regressions analysis demonstrated that NKCA was closely correlated with DP, independent of other variables, including a stress index. These results suggest that the relationship between reduced cellular immunity and DP is not due to traditional work stress or health behavioral problems. Further studies on DP as a psychosomatic disorder as well as an occupational health problem should be performed in the future.

Adult↗

PubMed1999

Role of the kappa-opioid system in the attenuation of the morphine-induced place preference under chronic pain.

We previously reported that the morphine-induced place preference was attenuated under inflammation produced by the unilateral injection of 2.5 % formalin (50 microl) into the hind paw of rats. In the present study, to elucidate the mechanism of this attenuation, the effects of pretreatment with delta- and kappa-opioid receptor antagonists, naltrindole (NTI) and nor-binaltorphimine (nor-BNI), on the development of the morphine-induced place preference under inflammation were examined in rats. Nor-BNI, but not NTI, eliminated the suppression of the morphine-induced place preference in inflamed groups. These results suggest that endogenous kappa-opioid systems may be activated in the presence of chronic inflammatory nociception; as a result, the development of morphine's rewarding effect may be suppressed under inflammation.

Animals↗

PubMed1999

Roles of 5-HT3 and opioid receptors in the ethanol-induced place preference in rats exposed to conditioned fear stress.

The effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (beta-endorphin and enkephalins), was investigated using the conditioned place preference paradigm. In addition, we also examined the effect of ondansetron on the ethanol-induced place preference enhanced by the administration of mu- and delta-opioid receptor agonists (exogenous opioids). The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference in rats exposed to conditioned fear stress. Pretreatment with ondansetron (0.01 and 0.1 mg/kg, s.c.) effectively attenuated this ethanol-induced place preference. When the mu-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or the selective delta-opioid receptor agonist 2-methyl-4a(alpha)-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a(alpha)-octah ydroquinolino [2,3,3-g] isoquinoline (TAN-67; 20 mg/kg, s.c.) was administered in combination with 75 mg/kg ethanol (which tended to produce a place preference), the ethanol-induced place preference was significantly enhanced. The selective mu-opioid receptor antagonist beta-funaltrexamine at a dose of 10 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Ondansetron (0.1 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Furthermore, the selective delta-opioid receptor antagonist naltrindole at a dose of 3 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. Ondansetron (0.1 mg/kg, s.c.) slightly, but significantly, attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding mechanism of ethanol under psychological stress, and may play an important role in the rewarding effect of ethanol through the activation of mu- and delta-opioid receptors.

Animals↗

PubMed1999

Potent antinociceptive effects of TRK-820, a novel kappa-opioid receptor agonist.

TRK-820, a new type of 4,5-epoxymorphinan derivative, was investigated in vivo for antinociceptive activities and its selectivity on various opioid receptors in mice. TRK-820 given s.c. or p.o. was found to be 351- and 796-fold more potent than U50,488H with acetic acid-induced abdominal constriction test. The duration of the antinociceptive effect produced by TRK-820 was longer than that produced by mu-opioid receptor agonist morphine or other kappa-opioid receptor agonists. In addition, with four other antinociceptive assays, low temperature hot plate (51 degrees C), thermal tail flick, mechanical tail pressure and tail pinch tests, TRK-820 was also found to be 68- to 328-fold more potent than U-50488H, and 41- to 349-fold more potent than morphine in producing antinociception, as comparing the weight of the different compound. However, TRK-820 was less active in inhibiting the high temperature (55 degrees C) hot plate response. The antinociceptive effects produced by TRK-820 were inhibited by nor-BNI, but not by naloxone or naltrindole (NTI) with the abdominal constriction test, indicating that the antinociception is selectively mediated by the stimulation of kappa-, but not mu- or delta-opioid receptors. Co-administration of TRK-820 with morphine slightly enhanced the antinociception induced by morphine in the mouse hot plate test. On the other hand, pentazocine significantly reduced the morphine-induced antinociception. TRK-820 produced sedation at doses, which are much higher than the doses for producing antinociception. These results indicate that the potent antinociception induced by TRK-820 is mediated via the stimulation of kappa-, but not mu- or delta-opiod receptors.

Administration, Oral↗

PubMed1999

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain.

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.

Analgesics, Opioid↗

PubMed1999

The mu-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in mu-opioid receptor knockout mice.

There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Analgesics, Opioid↗

PubMed1999

Catharanthine oxidation in flavin mononucleotide-mediated catharanthine-vindoline coupling reaction for synthesis of dimeric indole alkaloids under near-ultraviolet light.

A reaction model for the flavin mononucleotide-mediated coupling of catharanthine (C) and vindoline (V) under near-ultraviolet light was established based on the results of experiments on the effects of various physical and chemical factors on the disappearance of C and V, and the synthesis of the product, a dihydropyridinium intermediate (IM). The following events were deduced to occur. (i) C is oxidized in the presence of FMN and oxygen under near-ultraviolet light (C(OX1)). (ii) C(OX1) then couples with V to form IM. Degradation of C occurs simultaneously with its specific oxidation, as a result of which a part of the C is converted into the product (C*) which is incapable of coupling with V. When a reaction in which C(OX1) is further oxidized to another form (C(OX2)) is added and the counterpart for the coupling with V is changed to C(OX2) from C(OX1), the reaction model more appropriately describes the exponential increase in the product, IM, that occurs in the early stage of the coupling reaction. (iii) The degradation rate of IM is much greater after C has been consumed than before its disappearance. Using this reaction model, the coupling reactions under basal and optimized conditions were simulated and the results showed a good fit with the experimental values under both conditions. The kinetic study suggests that manganese ion (Mn2+) stimulates the binding of C(OX2) with V in addition to suppressing the degradation of C, resulting in an increased yield of IM. Mn2+ thus appears to be an important factor in the coupling reaction, in which it plays two different roles.

Journal Article↗

PubMed1999

A retrospective study of ocular toxocariasis in Japan: correlation with antibody prevalence and ophthalmological findings of patients with uveitis.

To classify the clinical characteristic of ocular toxocariasis in Japan, the prevalence of antibodies to Toxocara antigens was examined in patients with uveitis of unknown aetiology. From 1982 to 1993, serum specimens of 383 cases and intraocular fluid samples of 22 cases were serologically screened for Toxocara infection with five immunodiagnostic tests. Fifty-five sera and 11 intravitreous fluid samples were estimated to have significantly high antibody levels against larval excretory-secretory (ES) antigens of T. canis. Eight cases were positive in both serum and vitreous fluid, and three were positive only in the vitreous fluid. Among the 58 antibody positive samples, 20 cases were omitted due to a lack of detailed description of ocular findings. The remaining 38 cases are described in this study. Of these 38 cases, 34 (89%) were older than 20 years of age. Ocular lesions were located in the posterior fundus in 11 cases, in the peripheral fundus in 18 cases, and in both areas in seven cases. Of the eight cases in which papillary oedema or redness was observed, chorioretinal lesions were also present in seven of them. Tractional retinal detachment was present in five cases. These observations suggest that ocular toxocariasis in Japan has a different clinical profile compared with those in the other countries, and indicate a need for revised classification of ocular toxocariasis.

Adolescent↗

PubMed1999

Eosinophil chemotaxis by chemokines: a study by a simple photometric assay.

BACKGROUND: The effects of a panel of 15 chemokines on eosinophil chemotaxis were studied by a new photometric assay which is both less tedious and less laborious than the conventional manual counting methods. Approximately 40 chemokines have been identified to date, but there is little information on the eosinophil migration-inducing ability of chemokines other than CC chemokine receptor (CCR) 3 ligands. METHODS: Eosinophil migration was measured by the Boyden chamber technique with a 96-well multiwell chamber and polycarbonate membrane filter. Eosinophil migration was assessed by determination of the eosinophil peroxidase (EPO) activity, and photometric measurement was performed with a microtiter plate reader. RESULTS: The assay was sensitive enough to detect 200 eosinophils, and the time required was within 4 h. CCR3 ligands, i.e., regulated on activation normal T-cell expressed and secreted (RANTES), eotaxin, eotaxin-2, and monocyte chemoattractant protein (MCP)-3, induced significant migration, while other chemokines showed no significant migration-inducing ability. Although the chemotaxis induction by these chemokines was efficiently inhibited by anti-CCR3 mAb, anti-CCR1 mAb failed to show any inhibitory effects. CONCLUSIONS: The photometric assay is suitable for analyzing a large number of samples. CCR3 ligands are the most important chemokines inducing eosinophil chemotaxis; thus, CCR3 represents a possible therapeutic target for the treatment of allergic diseases.

Antibodies, Monoclonal↗

PubMed1999

Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗