The Concise Guide to PHARMACOLOGY 2013/14: overview.

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Contribution of melanin-concentrating hormone (MCH1) receptor to thermoregulation and sleep stabilization: evidence from MCH1 (-/-) mice.

Recent studies have explored the implication of melanin-concentrating hormone (MCH) in the process of vigilance states. The current experiments were carried out in mice lacking the MCH(1) receptor (-/-) and wild-type (WT) littermates, to assess the role of MCH(1) receptor in the regulation of sleep architecture, body temperature (BT) and locomotor activity (LMA) under normal condition and following a 1h restraint stress at lights onset. Under baseline conditions, MCH(1) (-/-) mice exhibited consistent changes in waking and sleeping time across the 24-h recording period. We found an increase in the amount of wakefulness (MCH(1) (-/-) 680.1 ± 15.3 min vs. WT, 601.9 ± 18.1, p<0.05) at the expense of total duration of non rapid eye movement (NREM) sleep (MCH(1) (-/-) 664.1 ± 13.9 min vs. WT 750.1 ± 18.5, p<0.05). Additionally, MCH(1) (-/-) mice had a higher mean basal body temperature (MCH(1) (-/-), 36.6 ± 0.1°C vs. WT, 36.0 ± 0.1°C, p<0.05), particularly during the light-resting period. Restraint stress resulted in an immediate increase in wakefulness with a concomitant reduction in NREM sleep and REM sleep in both genotypes, followed by a homeostatic rebound sleep. A concomitant long lasting increase in BT, independently of the behavioural state accompanied those changes in both genotypes. The elevated basal body temperature and reduction in NREM sleep time resulting from shorter NREM episode durations observed in MCH(1) (-/-) suggests that central MCH(1) receptor has a role in thermoregulation and presumably stabilization of NREM sleep.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

The highly selective CRF(2) receptor antagonist K41498 binds to presynaptic CRF(2) receptors in rat brain.

1. Novel analogues of antisauvagine-30 (aSvg-30), a selective antagonist for CRF(2) receptors, have been synthesized and characterized in vitro and in vivo. 2. The analogues were tested for their ability to compete for [(125)I-Tyr(0)]Svg binding and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF(1) (hCRF(1)), hCRF(2alpha) and hCRF(2beta) receptor. One analogue [D-Phe(11), His(12), Nle(17)]Svg(11-40), named K41498, showed high affinity binding to hCRF(2alpha) (K(i)=0.66+/-0.03 nM) and hCRF(2beta) (K(i)=0.62+/-0.01 nM) but not the hCRF(1) receptor (k(i)=425+50 nM) and decreased Svg-stimulated cAMP accumulation in hCRF(2) expressing cells. In conscious Wistar-Kyoto rats, K41498 (1.84 microg, i.v.) antagonized the hypotensive response to systemic urocortin (1.4 microg, i.v.), but did not block the pressor response to centrally administered urocortin (2.35 microg, i.c.v.). 3. K41498 was subsequently radio-iodinated, and in autoradiographic studies, specific (sensitive to rat urocortin, astressin and aSvg30, but insensitive to antalarmin) binding of (125)I-K41498 (100 pM) was detected in the heart and in selected brain regions including the nucleus tractus solitarius (NTS), spinal trigeminal nucleus, lateral septum and around the anterior and middle cerebral arteries. 4. Following unilateral nodose ganglionectomy, binding of (125)I-K41498 was reduced by 65% in the ipsilateral NTS, indicative of presynaptic CRF(2) receptors on vagal afferent terminals. 5. These data demonstrate that K41498 is a useful tool to study native CRF(2) receptors in the brain and periphery.

Design, synthesis and pharmacological characterization of new highly selective CRF(2) antagonists: development of 123I-K31440 as a potential SPECT ligand.

Novel analogs of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF(2)), have been synthesized and characterized in vitro and in vivo. The N-terminal amino acid D-phenylalanine in aSvg-30 was replaced by a D-tyrosine residue for specific radioactive labeling with 123I. Additionally, Met(17) of aSvg-30 was substituted by norleucine and the N-terminus of the peptide was acetylated to increase in vivo metabolic stability. The aSvg-30 analogs were tested for their ability to displace [125I-Tyr(0)]Svg in binding experiments and to inhibit Svg-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for the human CRF(1) (hCRF(1)), hCRF(2alpha) and hCRF(2beta) receptor. Ac-[D-Tyr(11), His(12), Nle(17)Svg(11-40), named K31440, showed high specific binding to hCRF(2alpha) (K(i) = 1.48 +/- 0.34 nM) and hCRF(2beta) (K(i) = 2.05 +/- 0.61 nM) but not the hCRF(1) receptor (K(i) = 288 +/- 13 nM) and decreased Svg-stimulated cAMP activity in hCRF(2)-expressing cells in a similar fashion as aSvg-30. In biodistribution studies specific uptake of 123I-K31440 was detected after 1 h in small intestine of BALB/c nude mice. These data demonstrate that 123I-K31440 may serve as a useful tool to detect native CRF(2) receptors and elucidate their role in gastrointestinal disorders and diseases such as irritable bowel syndrome or cancer.

Pharmacological characterization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: rapid and reversible desensitization of the ORL1 receptor in vitro and lack of tolerance in vivo.

The novel nonpeptide orphanin FQ/nociceptin (OFQ/N) ligand [(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] (Ro 64-6198) was characterized in vitro and in vivo for its agonistic potential. Ro 64-6198 was 130- to 3500-fold selective for the OFQ/N receptor (ORL1) compared with opiate receptors. In the cAMP inhibition assay, Ro 64-6198 was a full agonist at the ORL1 and a partial agonist at the mu opiate receptor. When human embryonic kidney 293 cells stably expressing the human ORL1 receptor were pre-exposed (30 min) to either OFQ/N or Ro 64-6198, the ability of both agonists to inhibit forskolin-mediated cAMP accumulation was strongly reduced, indicating a functional desensitization of the second messenger cascade. However, acidic washes of OFQ/N-exposed cells fully restored the sensitivity of the ORL1 receptor for agonists. In contrast, the cAMP response in Ro 64-6198-exposed cells remained impaired after acidic washes, suggesting sustained receptor internalization at 30 min. In agreement with this finding, the number of cell-surface ORL1 receptors was significantly reduced after Ro 64-6198 pre-exposure, and this effect could be blocked with high sucrose concentrations. When Ro 64-6198 was chronically administered to rats, no signs of tolerance to its anxiolytic-like effects were detected following 15 days of daily drug exposure. In agreement with the behavioral results, Ro 64-6198 was able to reduce brain ORL1 binding sites in both acutely and chronically treated rats. Full recovery of ORL1 binding sites was observed 24 h after Ro 64-6198 administration with a t1/2 of approximately 5.5 h. These data show that nonpeptide agonists at the ORL1 receptor have a good clinical potential as anxiolytics without causing tolerance.

Molecular biology of the CRH receptors-- in the mood.

Dysfunctioning of corticotropin-releasing hormone (CRH) and its receptors (CRH(1) and CRH(2)) has been linked to the development of stress-related disorders, such as mood and eating disorders. The molecular characterization of CRH(1) and CRH(2) receptors and their splice variants has generated detailed information on their pharmacology, tissue distribution and physiology. While mammalian CRH(1) receptors nonselectively bind CRH analogs, the ligand specificity of CRH(2) is narrower. CRH(1) receptors are predominantly expressed in the brain and pituitary, whereas CRH(2) receptor expression is limited to particular brain areas and to some peripheral organs. Molecular approaches to block CRH(1) receptor expression in the brain argue in favor of its involvement in the regulation of some aspects of the stress response. The CRH(2alpha) receptor may be more important for motivational types of behavior essential for survival, such as feeding and defense.(1)

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation.

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.

G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor type 1 (PAC(1)) signaling and desensitization were investigated in human retinoblastoma Y-79 cells. Concentration-dependent stimulation of cAMP accumulation was observed in Y-79 cells incubated for 30 min with PACAP38, PACAP27, or VIP (10(-12) to 10(-6) M). The following EC(50) values were calculated: PACAP38, 24+/-3 pM; PACAP27, 99+/-8 pM; and VIP, 29+/-3 nM. Homologous desensitization of PAC(1) receptors in Y-79 cells pretreated with 10 nM PACAP38 or PACAP27 for 60 min was characterized by a 30-50% reduction in PACAP-stimulated cAMP accumulation (p<0.0001) and a two- to fivefold rightward shift in EC(50) values (p<0.0001). PAC(1) receptor desensitization was not accompanied by a reduction in PAC(1) mRNA expression. We concluded that the desensitizing effect of PACAP38 was homologous because neither corticotropin-releasing factor- nor (-)-isoproterenol-stimulated cAMP accumulation was altered by PACAP38 preincubation. Pretreating Y-79 cells with the protein kinase A (PKA) inhibitor H89 failed to inhibit homologous PAC(1) receptor desensitization. Similarly, pretreating Y-79 cells with the protein kinase C (PKC) inhibitors staurosporine or bisindolylmaleimide failed to alter homologous PAC(1) receptor desensitization. Although activation of PKA by dibutyryl cAMP or forskolin did not desensitize PAC(1) receptors, direct activation of PKC by PMA heterologously desensitized PAC(1) receptors, reducing cAMP accumulation 34.2+/-2.2% (p<0.001). Using RT-PCR, mRNA levels for G-protein-coupled receptor kinase 3 (GRK3), but not GRK2, were found to increase 2.2- to 4.8-fold in Y-79 cells exposed to PACAP38 for 10 min to 24 h (p<0.001). PAC(1) receptor desensitization decreased 72.5+/-4.3% (p<0.001) in Y-79 cells transfected with a GRK3 antisense cDNA construct that also reduced GRK3 protein expression 48.5+/-7.9% (p<0.0005). These experiments demonstrate that GRK3 plays an important role in the homologous desensitization of retinoblastoma PAC(1) receptors, whereas PKC, but not PKA, contributes to the heterologous desensitization of retinoblastoma PAC(1) receptors.

Interaction of neuropeptide Y and corticotropin-releasing factor signaling pathways in AR-5 amygdalar cells.

Corticotropin-releasing factor (CRF) is a 41 amino acid neuropeptide which is involved in the stress response. CRF and neuropeptide Y (NPY) produce reciprocal effects on anxiety in the central nucleus of the amygdala. The molecular mechanisms of possible CRF-NPY interactions in regulating anxiety behavior is not known. In the central nervous system, the action of NPY leads to inhibition of cAMP production while CRF is known to stimulate levels of cAMP in the brain. Consequently, we hypothesized that NPY may antagonize anxiety-like behavior by counter-regulating CRF-stimulated cAMP accumulation and activation of the protein kinase A pathway. We have engineered an immortalized amygdalar cell line (AR-5 cells) which express via RT-PCR, the CRF(2alpha), Y(1) and Y(5) NPY receptor. In addition, in these cells CRF treatment results in significant concentration-dependent increases in cAMP production. Furthermore, incubation of 3 microM CRF with increasing concentrations of NPY was able to significantly inhibit the increases in cAMP compared to that observed with 3 microM CRF treatment alone. These findings suggest that CRF and NPY may counter-regulate each other in amygdalar neurons via reciprocal effects on the protein kinase A pathway.

125I-Antisauvagine-30: a novel and specific high-affinity radioligand for the characterization of corticotropin-releasing factor type 2 receptors.

Corticotropin-releasing factor (CRF) receptors type 1 (CRF(1)) and type 2 (CRF(2)) differ from each other in their pharmacological properties. The human and ovine CRF versions bind to CRF(1) receptors with significantly higher affinity than to CRF(2) receptors. Recently antisauvagine-30, an N-terminally truncated version of the CRF analog sauvagine, was characterized as a specific antagonist to mouse CRF(2B). We have synthesized the radiolabeled version (125)I-antisauvagine-30 and tested it for its affinity at human CRF(1) (hCRF(1)), hCRF(2A), Xenopus CRF(1) (xCRF(1)) and xCRF(2) receptors. In control binding studies (125)I-labeled hCRF, sauvagine and astressin were also bound to these receptors. (125)I-antisauvagine-30 exclusively bound to hCRF(2A) and xCRF(2) but not to hCRF(1) and xCRF(1) receptors. (125)I-antisauvagine-30 binding to hCRF(2A) and xCRF(2) receptors was saturable and of high affinity (hCRF(2A): K(d)=125 pM; xCRF(2): K(d)=1.1 nM). In displacement binding experiments using (125)I-antisauvagine-30 as radioligand several CRF analogs bound to hCRF(2A) and xCRF(2) receptors with similar rank orders as reported with other CRF radioligands. Finally, preliminary studies using (125)I-antisauvagine-30 binding to membrane homogenates prepared from different rat brain structures showed that the peptide bound specifically to brain areas expressing CRF(2) receptors. These data demonstrate that (125)I-antisauvagine-30 is the first high-affinity ligand to specifically label CRF(2) receptors.

Different binding modes of amphibian and human corticotropin-releasing factor type 1 and type 2 receptors: evidence for evolutionary differences.

The binding characteristics of corticotropin-releasing factor (CRF) type 1 (CRF(1)) and type 2 (CRF(2)) receptors from human (hCRF(1) and hCRF(2alpha)) and Xenopus (xCRF(1) and xCRF(2)) were compared using four different (125)I-labeled CRF analogs, the agonists (125)I-CRF and (125)I-sauvagine, and the antagonists (125)I-astressin ((125)I-AST) and (125)I-antisauvagine-30 ((125)I-aSVG). The hCRF(2alpha) and xCRF(2) receptors bound all four radioligands with different affinities, whereas hCRF(1) did not bind (125)I-aSVG, and xCRF(1) bound neither (125)I-sauvagine nor (125)I-aSVG. Competitive binding studies using unlabeled agonists and antagonists with hCRF(1) and hCRF(2alpha) receptors revealed that most agonists exhibited higher affinity in displacing agonist radioligands compared with displacement of antagonist radioligands. Exceptions were the agonists human and rat urocortin, which displayed high-affinity binding in the presence of either (125)I-labeled agonist or antagonist ligands. In contrast, the affinities of antagonists were independent of the nature of the radioligand. We also found that, in contrast to the mammalian CRF receptors, the affinity of ligand binding to xCRF(1) and xCRF(2) receptors strongly depended on the nature of the radioligand used for competition. For xCRF(1), competitors showed different rank order binding profiles with (125)I-CRF compared with (125)I-AST as the displaceable ligand. Similarly, binding of competitors to the xCRF(2) receptor showed markedly different profiles with (125)I-CRF as the competed ligand compared with the other radioligands. These data demonstrate that amphibian CRF receptors have distinctly different binding modes compared with their mammalian counterparts.

Synthesis of (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, a potent and selective orphanin FQ (OFQ) receptor agonist with anxiolytic-like properties.

The development of 8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3, 8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (micro, kappa, delta) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPgamma(35)S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.

Evidence for the abundant expression of arginine 185 containing human CRF(2alpha) receptors and the role of position 185 for receptor-ligand selectivity.

The abundance of a histidine residue at position 185 (His(185)) of the human corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2alpha)) was investigated. His(185) has only been reported in hCRF(2); CRF(2) proteins from other species and all CRF(1) receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) cDNAs from a variety of neuronal and peripheral tissues revealed the existence of receptor molecules encoding Arg(185) only. Sequence analysis of the hCRF(2) gene verified the existence of Arg(185) also on genomic level. Full-length cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185)) variants of hCRF(2alpha) were stably expressed in HEK293 cells and tested for ligand binding properties. In displacement studies R2H(185) and R2R(185) displayed a similar substrate specificity, human and rat urocortin, and the peptide antagonists astressin and alpha-helical CRF((9-41)) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. Significant differences were observed for sauvagine and urotensin I, which bound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2R(185). These data indicate that hCRF(2), like all vertebrate CRF(1) and CRF(2) proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role for the discrimination of some CRF peptide ligands.

A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat.

The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound [(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] has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). When given systemically, the compound has an efficacy and potency comparable to those of a benzodiazepine anxiolytic such as alprazolam or diazepam. However, this compound is differentiated from a classical benzodiazepine anxiolytic by a lack of efficient anti-panic-like activity, absence of anticonvulsant properties, and lack of effects on motor performance and cognitive function at anxiolytic doses (0.3 to 3 mg/kg i.p.). No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (>/=10 mg/kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ/nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ/nociceptin in adaptive behavioral fear responses to stress.

Functional characterization of corticotropin-releasing factor type 1 receptor endogenously expressed in human embryonic kidney 293 cells.

The endogenous expression in human embryonic kidney 293 (HEK293) cells of corticotropin-releasing factor (CRF) receptors was detected. High-affinity binding sites for human CRF (K(i)=3.6 nM), ovine CRF (K(i)=4.6 nM), rat urocortin (K(i)=2.2 nM), sauvagine (K(i)=2.4 nM) and astressin (K(i)=4.3 nM) with the pharmacological characteristics for CRF type 1 (CRF(1)) receptors and B(max) values of approximately 30 fmol/mg protein were determined. The four CRF receptor agonists nonselectively stimulated cAMP production in HEK293 cells at low agonist concentrations, whereas the antagonist astressin shifted the dose-response curve for ovine CRF significantly rightward. Transfection of the pcDNA3 vector into HEK293 cells strongly reduced the expression of the endogenous CRF receptor. Northern blot analysis revealed the expression of a CRF(1) transcript in human neuronal tissues, HEK293, human NTera-2 (NT2) carcinoma, Y-79 retinoblastoma and African green monkey kidney (COS-7) cells. Neither by Northern blot analysis nor by reverse transcriptase PCR (RT-PCR), the expression of CRF(2) could be detected. In cAMP stimulation experiments, functional CRF receptors were detected in these cell lines. These data show that HEK293 and other cell lines endogenously express CRF(1) receptors.

Rapid agonist-induced phosphorylation of the human CRF receptor, type 1: a potential mechanism for homologous desensitization.

Agonist-induced phosphorylation of the human corticotropin-releasing factor type 1 receptor (hCRF(1)-R) was investigated using an influenza hemagglutinin (HA) epitope-tagged receptor transiently expressed in COS-7 cells. The HA-hCRF(1)-R migrated as a broad band (M(r) 60,000-70,000) in SDS-PAGE and showed increased mobility (M(r) approximately 48,000) after enzymatic deglycosylation with peptide-N-glycosidase F, consistent with the predicted size (47 kDa) of the nonglycosylated HA-hCRF(1)-R protein. A marked increase in HA-hCRF(1)-R phosphorylation was observed in HA-hCRF(1)-R-expressing COS-7 cells exposed to 1 microM ovine CRF for 5 min, whereas activation of protein kinase A (PKA) by 50 microM forskolin, or of Ca(2+)/calmodulin (CaM)-dependent kinases by 10 microM ionomycin, had little effect. These findings are consistent with preliminary data suggesting that CRF(1)-R phosphorylation mediated by G protein receptor kinase 3 (GRK3), but not by PKA or CaM-dependent kinases, has an important role in the homologous desensitization of brain CRF(1)-Rs.

High-affinity, non-peptide agonists for the ORL1 (orphanin FQ/nociceptin) receptor.

The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1a, as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin) receptor led to the synthesis of a series of optimized ligands. These compounds exhibit high affinity for the human ORL1 receptor, exhibit moderate to good selectivity versus opioid receptors, and behave as full agonists in biochemical assays. In this paper we present the synthesis, structure-activity relationship (SAR), and biochemical characterization of substituted 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones culminating in the discovery of 8-(5-methyl-1,2,3,4-tetrahydro-naphthalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1p, and 8-acenaphten-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1q, two high-affinity, potent ORL1 receptor agonists with good to moderate selectivity versus the other opioid receptors.

N-terminal splice variants of the type I PACAP receptor: isolation, characterization and ligand binding/selectivity determinants.

Three full-length cDNAs encoding functional splice variants of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) were isolated from Y-79 retinoblastoma cells and human cerebellum. Although the third intracellular loops of the three splice variants were identical, their N-terminal extracellular domains differed. The first full-length PAC1 variant, PAC1normal (PAC1n), encoded the entire N-terminus, whereas the second variant named PAC1short (PAC1s) was deleted by 21 amino acids (residues 89-109). Finally, the third variant, named PAC1very short (PAC1vs), was deleted by 57 amino acids (residues 53-109). Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, it was established that all three variants were expressed in neuronal tissues. Binding- and cAMP studies using human embryonic kidney 293 (HEK293) cells stably transfected with PAC1n, PAC1s and PAC1vs showed significant differences in the affinities and selectivities towards PACAP38, PACAP27 and VIP. PAC1n bound PACAP38 and PACAP27 with affinities in the low nanomolar range whereas VIP was bound with up to 400-fold lower affinity. PAC1vs preferentially bound PACAP38 (Ki=121 nM) and PACAP27 (Ki=129 nM) over VIP (Ki>1000 nM) but with 100-fold lower affinity than PAC1n. Surprisingly, PAC1s unselectively bound all three ligands with high affinity. These data indicate that residues 53-88 within the N-terminal domain of the PAC1 are important for high affinity ligand binding, whereas residues 89-109 determine the receptor's ligand selectivity.

8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.