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N Adham

Publications and source records attributed to N Adham.

At least 19 recordsLinked to original sources
PubMed2001

Trace amines: identification of a family of mammalian G protein-coupled receptors.

Tyramine, beta-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some "trace amines" have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of two G protein-coupled receptors for neuropeptide FF.

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system.

Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide.

Amino Acid Sequence↗
PubMed1999

N-Methyl-5-tert-butyltryptamine: A novel, highly potent 5-HT1D receptor agonist.

It has been observed that reported 5-HT1D receptor agonists have at least one heteroatom (N, O, or S) on the 5-substituent of the indole. This has led to the hypothesis that a 5-substituent capable of participating in hydrogen bonding is critical for conveying high affinity. This article describes the synthesis and biological evaluation of a new series of 5-alkyltryptamine analogues, which does not have a heteroatom in the 5-substituent group. In contrast to the hypothesis, 5-alkyltryptamines all exhibit high binding affinities for the human 5-HT1D receptor. The size of the lipophilic alkyl group at the 5-position of the indole has significant impact on the 5-HT1D binding affinity. Compounds with a tert-butyl group at the 5-position such as 9d, 10, and 11 were identified. These analogues display high binding affinity (Ki < 1 nM) and moderate receptor selectivity in comparison with known antimigraine agents such as sumatriptan, naratriptan, rizatriptan, and VML-251.

Cell Line↗
PubMed1998

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation.

Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk-) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160-190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine >/= 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities (Ki values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies (Kb values) to block 5-HT-stimulated [cAMP]i were in close agreement with their corresponding Ki values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Galphas protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.

Adenylyl Cyclases↗
PubMed1997

5-HT1F receptor agonists inhibit neurogenic dural inflammation in guinea pigs.

The serotonin (5-HT) receptor subtype mediating inhibition of neurogenic dural inflammation in guinea pigs was investigated using a series of serotonin agonists with differing affinities for the 5-HT1B, 5-HT1D and 5-HT1F receptors. When agonist potencies for inhibiting neurogenic inflammation were compared with affinities for these receptor subtypes, a significant positive correlation was seen only with the 5-HT1F receptor. The potency of agonists in inhibiting adenylate cyclase in cells transfected with human 5-HT1F receptor was also highly correlated with their potency in the animal model of migraine. In situ hybridization demonstrated 5-HT1F receptor mRNA in guinea pig trigeminal ganglion neurons. These data suggest that the 5-HT1F receptor is a rational target for migraine therapeutics.

Animals↗
PubMed1997

Modeling the G-protein-coupled neuropeptide Y Y1 receptor agonist and antagonist binding sites.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.

Amino Acid Sequence↗
PubMed1996

[3H]5-hydroxytryptamine labels the agonist high affinity state of the cloned rat 5-HT4 receptor.

We have used the cloned rat 5-HT4 receptor, and determined that the single protein product produced is able to bind both [3H]5-HT and [3H]GR113808 ([1-[(2-methyl sulphonyl) amino] ethyl-4-piperidinyl] methyl-1-methyl-1H-indole-3-carboxylate) with high affinity. The affinities of agonists for the [3H]5-HT (agonist)-labelled receptor were significantly higher than for the [3H]GR113808 (antagonist)-labelled receptor. Furthermore, [3H]5-HT binding was reduced by addition of guanyl nucleotides. These results strongly support the hypothesis that the 5-HT4 receptor displays two interconvertible affinity states (high and low) for agonists, characteristic of many G protein coupled receptors. [3H]5-HT, at the concentration employed, therefore labels the agonist high affinity state of the 5-HT4 receptor in systems in which high densities of this receptor are found.

Affinity Labels↗
PubMed1995

The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants.

Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable.

Adenylyl Cyclases↗
PubMed1993

Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase.

An intron-containing gene encoding a novel human serotonin (5-HT) receptor was isolated from human genomic and cDNA libraries with probes directed to transmembrane regions of the adenylate cyclase stimulatory Drosophila serotonin receptor gene, 5-HTdrol. Membranes harvested from transiently transfected Cos-7 cells displayed high affinity (Kd = 8.5 nM), saturable (Bmax = 6.6 pmol/mg protein) [3H]5-HT binding. The rank order of potencies for serotonergic ligands to displace specific [3H]5-HT binding was: 5-carboxamido-tryptamine > methiothepin > metergoline > 5-HT > 8-hydroxy-2-(di-n-propylamino)tetralin > sumatriptan > ketanserin > zacopride. 5-HT produced a dose-dependent (EC50 = 992 nM) stimulation (approximately 20-fold) of cAMP accumulation in transiently transfected cells, and this response was antagonized by the nonselective 5-HT antagonist methiothepin. RNA for this gene was predominantly detected in the human brain and a subset of peripheral tissues including coronary artery and several tissues of the gastrointestinal tract. The molecular biological and pharmacological properties of this receptor suggest that it is the first member of a new serotonin receptor subfamily (5-HT7). The second messenger coupling, and tissue distribution indicate a possible identity to 5-HT receptors that mediate relaxant responses in certain isolated blood vessels.

Adenylyl Cyclases↗
PubMed1993

Cloning of another human serotonin receptor (5-HT1F): a fifth 5-HT1 receptor subtype coupled to the inhibition of adenylate cyclase.

An intronless gene encoding an additional human serotonin (5-HT) 5-HT1-like receptor subtype was isolated from a human genomic library with probes obtained from degenerate PCR primers used to amplify 5-HT-receptor-specific sequences. The highest degree of homology was found with the 5-HT1E subtype (70%) and the 5-HT1D alpha (63%) and 5-HT1D beta (60%) receptors. RNA for this gene was detected in the human brain but was not detected in kidney, liver, spleen, heart, pancreas, and testes. High-affinity (Kd = 9.2 nM) 3H-labeled 5-HT binding was detected. Competition studies revealed the following rank order of potencies for serotonergic ligands: 5-HT > sumatriptan >> 5-carboxyamidotryptamine > 8-hydroxy-2(di-1-propylamino)tetralin > spiperone. 5-HT produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation (EC50 = 7.9 nM) in transfected cells. These properties distinguish this receptor from any previously characterized and establish a fifth 5-HT1-like receptor subtype (5-HT1F) coupled to the inhibition of adenylate cyclase.

Adenylyl Cyclase Inhibitors↗
PubMed1993

Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways.

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)1 (serotonin1) receptor class that inhibits adenylyl cyclase, namely the human 5-HT1F receptor (Adham et al. 1993a). In the present study we have examined in greater detail the functional coupling of the 5-HT1F receptor in two different cell lines, NIH-3T3 and LM(tk-) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk-) cells, whereas the EC50 values were comparable. To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88 +/- 2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (Kb = 438 nM). We have also investigated activation of additional signal transduction pathways by the 5-HT1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca2+]i was observed even at very high agonist concentrations (100 microM). In contrast, in LM(tk-) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca2+]i. The 5-HT1F receptor failed to alter arachidonic acid release in either cell line. The maximal increase in IP accumulation in LM(tk-) cells was modest, averaging about 100% above basal. The increases of IP and [Ca2+]i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC50 = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 microM methiothepin. All three responses (cAMP, IP, and [Ca2+]i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of Gi/Go protein(s) in these signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1993

Receptor reserve masks partial agonist activity of drugs in a cloned rat 5-hydroxytryptamine1B receptor expression system.

The present study was undertaken to evaluate the behavior of the rat 5-hydroxytryptamine (5-HT)1B receptor stably expressed in a Y-1 cell line, using both radioligand binding ([125I]iodocyanopindolol) and functional assays (inhibition of forskolin-stimulated cAMP release). The measured EC50 values for agonists were lower than expected from the measured Ki values (e.g., 5-HT, EC50 = 0.49 +/- 0.043, nine experiments; Ki = 5.3 +/- 0.82, four experiments). Furthermore, beta-adrenergic antagonists such as propranolol and pindolol, which have been reported to be partial agonists or antagonists at the 5-HT1B receptors in other systems, were found to be full agonists. To investigate the relationship between receptor occupancy and inhibition of cAMP release (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ treatment shifted the dose-response curve for 5-HT to the right by 6-7-fold, accompanied by a reduction (30-50%) in maximal response. Analysis of the data by the method of Furchgott revealed a very steep hyperbolic relationship between receptor occupancy and response for 5-HT, with 92 +/- 1.4% (three experiments) receptor reserve at the 50% maximal response level. In contrast to its effect on 5-HT, EEDQ treatment did not significantly shift the dose-response curves for pindolol; rather, only the maximal responses were reduced and a linear relationship was found between receptor occupancy and response for this compound. According to classical receptor theory these data indicate that pindolol is a partial agonist, relative to 5-HT, but because of the high density of 5-HT1B receptors in this system the difference between the intrinsic activities of the two drugs is masked. Therefore, one has to be cautious when interpreting functional data in transfected systems that often display large receptor reserve.

Adrenergic beta-Antagonists↗
PubMed1992

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.

Amino Acid Sequence↗
PubMed1992

Acute desensitization of muscarinic receptors in the isolated guinea-pig ileal longitudinal muscle.

1. The effects of acute desensitization of muscarinic receptors mediating contractile responses of the guinea-pig ileal longitudinal muscle were studied in vitro, using similar conditions for both functional and radioligand binding studies. 2. The pA2 values for a number of muscarinic antagonists (pirenzepine, methoctramine, (+/-)para-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methyl piperidine-methiodide) indicated that the contractile response to carbachol was mediated through an M3 muscarinic receptor. In binding experiments the muscarinic receptor subtype population in ileal longitudinal muscle was found to be heterogeneous, consisting of approximately 77% M2 and 23% M3 receptors. 3. Pre-exposure of ileal longitudinal muscle to 10 microM carbachol for 30 min produced a reduction (28 +/- % of control maximum) in the maximum contractile response and a dextral shift in the concentration-effect curve to carbachol. Prior equilibration (60 min) with (+/-)p-F-HHSiD (1 microM), but not with methoctramine (1 microM) or pirenzepine (0.3 microM), prevented the desensitization. Desensitization under these conditions did not alter either the apparent affinity, the total number of binding sites or the relative, proportions of M2 and M3 muscarinic receptors, as determined in radioligand binding studies. Desensitization did not cause any meaningful change in either the apparent affinity of carbachol or the proportion of the high and low affinity binding sites. 4. It is concluded that desensitization of the contractile responses of the guinea-pig ileal longitudinal muscle is a result of M3 but not M2 muscarinic receptor desensitization. Acute desensitization, therefore, is not accompanied by meaningful changes in the total number of both M2 and M3 receptors or by alterations in the affinity of the receptor to ligands.

Animals↗
PubMed1992

The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5-hydroxytryptamine1D beta receptor.

The relationship between the serotonin 5-hydroxytryptamine1B (5-HT1B) and 5-HT1D receptors has been the topic of much investigation and speculation since their complementary species distribution was first appreciated. The cloning of genes encoding 5-HT1D receptors has provided tools to investigate this relationship directly. In this study, a rat gene has been cloned that encodes the rat 5-HT1B receptor. Evaluation of the structure of this gene shows that it is a member of the guanine nucleotide-binding protein-coupled receptor superfamily. Comparison of the amino acid sequence of the rat gene with the human 5-HT1D beta gene showed a 93% overall identity and a 96% identity in the transmembrane regions. Comparison of the two sequences revealed zero to two amino acid changes in each of these transmembrane regions, as well as a striking conservation in the connecting loops, indicative of the relationship expected for species homologues of the same gene. The rat gene was expressed transiently in COS-7 cells, and membranes derived from these cells were shown to bind [125I]iodocyanopindolol. The pharmacological profile of this binding site closely matched that of the native rat 5-HT1B receptor (r = 0.95) but not the 5-HT1D receptor (r = 0.07). The cloned rat 5-HT1B receptor was found to couple to the inhibition of adenylate cyclase activity, as expected for a 5-HT1B receptor. These data indicate that, although the 5-HT1B and 5-HT1D receptors are pharmacologically distinct, they are species variants of the same receptor gene, the 5-HT1D beta gene.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and characterization of a high affinity dopamine receptor (D1 beta) and its pseudogene.

We have cloned a novel human intronless gene encoding a G-protein-coupled receptor of the dopamine receptor family. Expression of this receptor in Cos-7 cells led to the high affinity binding of a number of dopamine D1 antagonists, with a binding profile similar to that of the previously described dopamine D1 receptor. In contrast, the agonist binding profile of this new receptor did not exactly match any previously defined dopamine D1 receptor and was notable for its unusually high affinity for dopamine. This new receptor caused a 13-fold increase in adenylylcyclase activity in transfected Cos-7 cells, following addition of dopamine. Messenger RNA encoding this new receptor appears to be widely distributed in the human brain, including cortical regions, choroid plexus, hippocampus, and brain stem. This new receptor appears to be identical to the recently described dopamine D5 receptor. A second closely related gene, GL39, was isolated and shown to represent a pseudogene, the first to be described in the G-protein-coupled receptor superfamily. This pseudogene exhibits 94% nucleotide sequence homology to the GL30 sequence and may have arisen from a gene duplication event followed by a mutation approximately 8 million years ago, prior to the emergence of man. This recently evolved pseudogene is transcribed in the human brain with a tissue distribution similar to that for its closely related functional gene.

Amino Acid Sequence↗