Molecular modelling of the ORL1 receptor and its complex with nociceptin

The opioid receptor like (ORL1) receptor is a G-protein coupled receptor superfamily, and regulates a plethora of neurophysiological functions. The structural requirements for receptor activation by its endogenous agonist, nociceptin (FGGFTGARKSARKLANQ), differ markedly from those of the kappa-opioid receptor and its putative peptide agonist, dynorphin A (YGGFLRRIRPKLKWDNQ). In order to probe the functional architecture of the ORL1 receptor, a molecular model of the receptor has been built, including the TM domain and the extra- and intracellular loops. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterized. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by TM helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the selective preference of the ORL1 receptor for nociceptin over dynorphin A, conferred by residue positions 5 and 6 (TG versus LR), and the favourable interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. The functional roles of the EL2 loop and the conserved N-terminal tetrapeptide opioid 'message' binding site are discussed in the context of the different structural requirements of the ORL1 and kappa-opioid receptors for activation.     

The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans

1. The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated. 2. CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan. 3. CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets. 4. These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists. 5. The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide. 6. Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.     

Tissue fibronectin is an endogenous ligand for galectin-1

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.     

Guanylin, an endogenous ligand for C-type guanylate cyclase, is produced by goblet cells in the rat intestine

BACKGROUND & AIMS: Guanylin activates an intestinal guanylate cyclase (GCC) and stimulates electrolyte movement across the gut epithelium. Cells expressing guanylin messenger RNA have been localized to the epithelial cell layer of the intestine; however, the identity of the guanylin-producing cells has not been determined. The aim of this study was to identify cells that express guanylin in the rat intestine. METHODS: Antibodies were raised against defined proguanylin epitopes, evaluated by Western blotting, and used for immunoperoxidase histochemistry. RESULTS: Guanylin-like immunoreactivity was localized to a subset of goblet cells. In the small intestine, most, perhaps all, goblet cells in the villi were immunopositive, as were some goblet cells in upper crypts; however, goblet cells deep within crypts were unlabeled. In the colon, goblet cells clustered in the necks and around the openings of crypts were immunopositive, whereas (as in the small intestine) goblet cells in deeper crypt regions were unlabeled. In some animals, immunoreactive columnar epithelial cells were also observed in the colon (although such cells were not apparent in the small intestine). Relative labeling of columnar cells varied from animal to animal. CONCLUSIONS: Guanylin is expressed in mature goblet cells. If secreted in conjunction with mucin, it could play a role in the hydration of mucus.     

Different domains of the ORL1 and kappa-opioid receptors are involved in recognition of nociceptin and dynorphin A

In order to gain further insight into the functional architecture of structurally related G protein-coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and mu-, delta- and kappa-opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1-kappa-opioid (O-K) hybrid construct has retained high affinity for non-type-selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and mu- and/or delta-opioid receptor-selective enkephalins analogs, thus behaving like a 'universal' opioid receptor. Most significantly however, whilst the ORL1 and kappa-opioid receptors display high binding preference (KD 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O-K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (KD < 1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and kappa-opioid receptors are involved in recognition of their endogenous peptide ligands.     

The effect of electrical stimulation on colonic transit following spinal cord injury in cats

The distribution of type I collagen, the major component of human dermis, was characterized by immunohistochemistry in skin lesions of chromoblastomycosis, a chronic cutaneous mycosis, before and after a specific antifungal treatment with terbinafine to study the changes induced in the lesions by the treatment. Newly synthesized type I collagen was studied with an antibody directed against the aminoterminal propeptide of the molecule (PINP), whereas mature, cross-linked type I collagen was detected with an antibody against the carboxyterminal telopeptide of type I collagen (ICTP). The isopeptide N epsilon gamma-glutamyl lysine (N epsilon gamma GL), synthesized by transglutaminase and able to cross-link several components of the extracellular matrix, has also been investigated with two monoclonal antibodies to determine if it is involved in the stabilisation of the fibrotic cutaneous lesions. The degradative process involved in the remodelling has also been assessed by immunohistochemistry with anti-metalloproteinase (MMP-1 and MMP-9) and anti-tissue inhibitor (TIMP-1) antibodies. All tissue macrophages stained for CD68 and MMP-9, but not for MMP-1, while the polymorphonuclear neutrophils had an elastase and a weak MMP-9 phenotype. The fibroblasts of fibrotic areas stained constantly for N epsilon gamma GL and PINP. The immunostaining of extracellular matrix for ICTP and N epsilon gamma GL, and the number of PINP-positive fibroblasts, decreased significantly after one year of antifungal treatment. Terbinafine treatment decreases the synthesis of type I collagen and leads to a partial reversal of the cutaneous fibrotic lesions, independently of the cure of the fungal infection.     

The effect of cisapride on delayed colonic transit time in patients with idiopathic Parkinson's disease

Disorders of autonomic regulation are common in patients with Parkinson's disease (PD). Patients most frequently complain of dysphagia and therapy resistant constipation, as far as the gastrointestinal tract is concerned. These symptoms have to be attributed to a neuronal degeneration. In a pilot study we therefore investigated the effect of stimulation of the myenteric plexus by cisapride. 11 women and 13 men were examined, the average age was 67.3 years, the Webster rating 17 points. In 2 out of 24 patients, colonic transit was prolonged up to the limit, both with and without therapy. The other 22 patients showed an acceleration in transit on response to cisapride. On average the colonic transit of 130 hours was reduced to 79 hours. This objective improvement was associated with a subjective improvement. Central side effects or a worsening of Parkinsonian symptoms were not found. We conclude that cisapride is effective in the treatment of constipation in idiopathic PD.     

Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10(-10) M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.     

Simplified method for the measurement of segmental colonic transit time

Segmental colonic transit has been measured in 101 patients. Two MBq of 111Indium absorbed on resin pellets and encapsulated in an enteric coated capsule was given at 7 00 am. Hourly images during the first day, and three images during each subsequent day were acquired for up to three days. Using all scan and patient data the scans were categorised in one of the five patterns of colonic transit: normal, rapid, right delay, left delay, or generalised delay. The geometric centres and per cent activity at each time point was compared between the five groups of colonic transit patients to find the best time for imaging and so to distinguish the five groups. During the first day, early images did not help in diagnosis of patterns of transit, however, in the later images (six hours onwards after the ingestion of the activity) the rapid transit groups could be identified. Images at 27 and 51 hours were both required to distinguish all five groups of patients from each other. Only in the 'normal' transit patients was there some excretion of the activity during the course of the second day, otherwise there was no difference in the images taken in the course of a day (second or third day). A simplified protocol requires a minimum of three images to distinguish all five patterns of colonic transit. The activity should be ingested in the morning (7 00 am) and the first image taken at the end of the working day (8-10 hours after ingestion), the second image on the morning of the second day, and the third image during the course of the third day. This simple protocol would provide all the clinically relevant information necessary for correct classification of the colonic transit.     

Structure-activity study of the nociceptin(1-13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist

In the present study, the minimal fragment sequence required to fully activate the nociceptin (NC) receptor, namely NC(1-13)-NH2, was used as template for the design of a series of new compounds. Changes were made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe, which has been shown to be essential for receptor occupation and activation. The new compounds were tested for their ability to inhibit the electrically evoked contraction of the mouse vas deferens, a pharmacological preparation sensitive to NC. Results obtained indicate that (a) the replacement of Gly2 or Gly3 with an aromatic residue (Phe) of L or D chirality eliminates the ability of the peptide to occupy the NC receptor; (b) the distance between Phe1 and Phe4 of NC appears to be critical, since any alteration of it leads to a marked decrease or a total elimination of biological activity; and (c) the insertion of a pseudopeptide bond between Phe1 and Gly2 maintains affinity but eliminates the ability of the peptide to activate the NC receptor and leads to antagonism. The peptide [Phe1psi(CH2-NH)Gly2]-NC(1-13)-NH2 acts as a selective NC receptor antagonist and is inactive on opioid receptors. The results summarized in this paper confirm and extend our previous findings by showing that the structural requirements for NC binding to its receptor are clearly different from those of opioids; in addition, this structure-activity study has led to the identification of the first NC receptor selective antagonist.     

Delta9-tetrahydrocannabinol, but not the endogenous cannabinoid receptor ligand anandamide, produces conditioned place avoidance

Although exogenous cannabinoid ligands such as delta9-tetrahydrocannabinol (THC) have been implicated in reward-related learning and aversion, the hedonic effects of the endogenous cannabinoid agonist anandamide (arachidonylethanolamide) have never been assessed. Thus, the effects of anandamide were tested in a place conditioning task. Male Wistar rats received THC (0.0-8.0 mg/kg) or anandamide (0.0-16.0 mg/kg) during conditioning sessions. The half-life of anandamide was increased by pretreatment with the protease inhibitor phenylmethylsulfonyl fluoride (2.0 mg/kg). A significant place aversion was found at the 1.0 and 1.5 mg/kg doses of THC. No significant place conditioning effects were found with anandamide. Locomotor activity during conditioning was significantly decreased by the 1.0, 1.5, 2.0 and 4.0 mg/kg doses of THC as well as the 8.0 and 16.0 mg/kg doses of anandamide. These results fail to implicate the endogenous cannabinoid anandamide in reward-related learning or aversion.     

The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.     

The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats

1. We have investigated the effect of nociceptin on the micturition reflex evoked by distension or topical application of capsaicin on the urinary bladder of urethane-anaesthetized rats. 2. Nociceptin produced a dose-dependent (3-100 nmol kg(-1) i.v.) transient suppression of the distension-evoked micturition reflex: its effect was not modified by guanethidine (68 micromol kg(-1) s.c.) nor by bilateral cervical vagotomy, alone or in combination, and by naloxone (1.2 micromol kg(-1) i.v.). 3. Nociceptin (100 nmol/kg i.v.) slightly (about 30%) inhibited the contractions of the rat bladder produced by pre- or postganglionic electrical stimulation of the pelvic nerve. 4. Nociceptin almost totally abolished the reflex component of the response to topical capsaicin (1 microg in 50 microl). 5. In the rat isolated bladder, submaximal contractions produced by electrical field stimulation were slightly reduced (25+/-4% inhibition) by 1 microM nociceptin. Nociceptin did not affect the contraction of the rat bladder induced by acetylcholine (10 microM) or ATP (1 mM). 6. These findings indicate that nociceptin exerts a naloxone-resistant suppression of the volume-evoked micturition reflex which involves inhibition of transmitter release from postganglionic bladder nerves. An inhibitory effect on bladder afferent nerves is also suggested.     

Evidence for an endogenous cAMP-adenosine pathway in the rat kidney

In the rat kidney, exogenous adenosine-3'-5'-monophosphate (cAMP) is converted to adenosine via the metabolism of cAMP to adenosine-5'-monophosphate by phosphodiesterase and adenosine-5'-monophosphate to adenosine by 5'-nucleotidase. Our purpose was to investigate whether in the rat kidney adenosine is synthesized from endogenous cAMP via the same pathway. Rat kidneys were perfused with Tyrode's solution, and stabilized for 3 hr to minimize basal renal purine secretion. In control experiments (n = 6), the renal venous secretion rate of adenosine, inosine, hypoxanthine and Sigmapurines (adenosine + inosine + hypoxanthine) did not change over the two 10-min experimental periods. In contrast, the beta adrenoceptor agonist (+/-)-isoproterenol (1 and 10 microM added to the perfusate) caused a significant (1-factor analysis of variance with repeated measures; n = 31) increase in the renal venous secretion of adenosine (P <.0001), inosine (P <.0007), hypoxanthine (P <.0007) and Sigmapurines (P <.0001) as measured by high-performance liquid chromatography with ultraviolet detection. The Sigmapurines was the most discriminating index of isoproterenol-induced changes in purine release, and the renal venous secretion of Sigmapurines was significantly (2-factor analysis of variance with repeated measures) attenuated by inhibition of beta adrenoceptors with propranolol (.1 microM, n = 6; P <.05), phosphodiesterase with 3-isobutyl-1-methylxanthine (1 mM, n = 5; P <.002) and 5'-nucleotidase with alpha, beta-methyleneadenosine-5'-diphosphate (0.1 mM, n = 5; P <.03). Our data indicate that activation of beta adrenoceptors increases purine biosynthesis in the rat kidney via a mechanism that involves phosphodiesterase and 5'-nucleotidase. These results support the existence of an endogenous cAMP-adenosine pathway in the rat kidney.     

Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin

L-selectin participates in the initial attachment of leukocytes to the vascular endothelium. On lymphocytes, it mediates binding to high endothelial venules of lymph nodes. As a selectin it functions as a calcium-dependent lectin recognizing carbohydrate-bearing ligands on endothelial cells. Two lymph node ligands for L-selectin have been identified as sulphated glycoproteins of M(r) approximately 50K and approximately 90K, called Sgp50 and Sgp90 (ref. 10). The recently cloned Sgp50 (ref. 12), now designated GlyCAM-1, is a high endothelial venule-associated, mucin-like glycoprotein containing predominantly O-linked carbohydrate chains. Sialylation of GlyCAM-1 is necessary for its ligand activity and a role for fucosylation is suspected. We have used chlorate as a metabolic inhibitor of sulphation, and report here that GlyCAM-1 has an additional requirement for sulphate.     

EMD 57445, the selective sigma receptor ligand, has no effect on the 5-hydroxytryptamine system

The behavioral and biochemical effects of EMD 57445, a selective sigma receptor ligand with potential antipsychotic activity, on the 5-hydroxytryptamine (5-HT) system were studied in rats and mice. The drug influence was investigated in three behavioral tests: 8-OH-DPAT (5-HT1A agonist)-induced behavioral syndrome in rats, m-chlorophenylpiperazine (m-CPP, 5-HT1B agonist)-induced hypothermia in mice and L-5-hydroxytryptophan (L-5-HTP)-induced head twitches (5-HT2A stimulation) in rats. EMD 57445 did not show any activity in all three behavioral models. In biochemical studies, no changes in the 5-HT and 5-HIAA levels in rat brain cortex, nucleus accumbens, striatum, hypothalamus and hippocampus were found. The results indicate that EMD 57445 does not interact with 5-HT (5-HT1A, 5-HT1B, 5-HT2A) receptor subpopulations and does not affect 5-HT metabolism.     

Effect of the high-affinity estrogen receptor ligand ICI 182,780 on the rat tibia

We examined the specificity of the steroidal antiestrogen ICI 182,780 (ICI) on bone and reproductive tissues in adult and growing female rats. Using a 1.5-mg/kg dose (s.c.), we evaluated the effects of ICI on the bone, body weight, uterine weight, serum cholesterol, and serum estradiol in either adult and/or growing rats. ICI increased serum estradiol cholesterol in ovary-intact rats, had no effect on uterine weight in ovariectomized rats, and resulted in uterine atrophy in ovary-intact animals comparable with ovariectomy. In contrast, ICI had no effect on body weight. In bone, ICI significantly increased the rate of periosteal bone formation in long bones of growing and mature female rats. In contrast, ICI had no effect on longitudinal bone growth in rapidly growing rats. When ICI was administered to mature rats with or without ovaries, two-factor ANOVA revealed significant interaction (P < or = 0.05) between ovariectomy and ICI treatment for cancellous bone area and labeled bone perimeter. ICI increased skeletal indices of bone turnover in the cancellous bone of ovary-intact rats but reduced these indices of bone turnover in the cancellous bone of ovariectomized rats. The increase in bone turnover was associated with a reduction in cancellous bone area in the ovary-intact rats. A reduction in bone turnover was similarly associated with an increase in bone area in the ICI-treated ovariectomized rats. In summary, ICI exhibited complete estrogen antagonism in cortical and cancellous bone, partial agonism in cancellous bone, and no activity on tibial longitudinal growth rate of growing ovary-intact rats. The effects in adult rats were influenced by circulating levels of estradiol. ICI had no activity on body weight and complete antagonism on uterine weight. These results demonstrate that a ligand with high binding affinity to the estrogen receptor(s) can elicit an array of estrogen-mediated regulation of bone metabolism.