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.

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.

Analysis of the pharmacological and molecular heterogeneity of I(2)-imidazoline-binding proteins using monoamine oxidase-deficient mouse models.

The I(2) subgroup of imidazoline-binding sites was identified as monoamine oxidases (MAOs), but it is unclear whether there are I(2)-binding sites located on proteins distinct from MAOs. To address this issue, we characterized I(2)-binding proteins in liver and brain of wild-type and MAO A- and MAO B-deficient mice. I(2)-binding sites were identified using [(3)H]idazoxan and the photoaffinity adduct 2-[3-azido-4-[(125)I]iodophenoxyl]methylimidazoline ([(125)I]AZIPI). [(3)H]Idazoxan labeled binding sites with ligand recognition properties typical of I(2) sites in both brain and liver of wild-type mice. High-affinity, specific [(3)H]idazoxan binding were not altered in MAO A knockout (KO) mice. In contrast, [(3)H]idazoxan binding was completely abolished in both liver and brain of MAO B KO mice. In wild-type mice, [(125)I]AZIPI photolabeled three proteins with apparent molecular masses of approximately 28 (liver), approximately 61 (brain), and approximately 55 kDa (liver and brain). The photolabeling of each protein was blocked by the imidazoline cirazoline (10 microM). Photolabeling of the approximately 61- and approximately 55-kDa proteins was not observed in MAO A and B KO mice, respectively. In contrast, photolabeling of the liver approximately 28-kDa protein was still observed in MAO-deficient mice, indicating that this protein is unrelated to MAOs. These data indicate that I(2) imidazoline-binding sites identified by [(3)H]idazoxan reside solely on MAO B. The binding sites on MAO A and the liver approximately 28-kDa protein may represent additional subtypes of the family of the imidazoline-binding sites.

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.

Imidazoline-binding domains on monoamine oxidase B and subpopulations of enzyme.

A series of phenoxy-substituted methylimidazoline derivatives were synthesized and used to define the ligand recognition properties of the imidazoline-binding domain (IBD) on monoamine oxidase (MAO)-B and its role in substrate processing. The rank order of potency for selected compounds in competitive binding studies with the imidazoline [(3)H]idazoxan was different from that in enzyme activity assays, suggesting that the IBD and the site involved in enzyme inhibition are distinct. IC(50) values for inhibition of MAO-B activity by imidazoline/guanidinium ligands were one to two orders of magnitude greater than ligand concentrations that probably saturate the IBD, but were equal to the K(d) values of these ligands in competitive binding assays with the reversible MAO-B inhibitor [(3)H]Ro 19-6327. In addition, the degree of enzyme inhibition by these ligands was similar in platelet and liver, tissues exhibiting 10-fold differences in the amount of the IBD-accessible enzyme subpopulation. These data suggested that the inhibitory effect of these compounds on MAO-B activity involved a secondary interaction with the enzyme domain recognizing the inhibitor Ro 19-6327 and does not involve interaction with the IBD. Subsequent radioligand-binding studies indicated that human liver MAO-B actually existed as two distinct populations that differed in the accessibility of their IBD. The relatively small amounts of MAO-B possessing an accessible IBD ( approximately 5% in human liver) precludes determination of the functional consequences of ligand binding to the IBD. This subpopulation of MAO-B may be selectively regulated or generated in different individuals or tissues and targeted by pharmacologically active compounds in a cell type-specific manner.

Imidazoline binding domains on MAO-B. Localization and accessibility.

Various imidazoline and guanidinium derivatives elicit diverse cellular responses in both peripheral tissues and the central nervous system that are often difficult to attribute to known receptor signaling systems. Such molecules also exhibit high affinity for membrane proteins (imidazoline binding sites) that are distinct from receptors for known hormones and recognize endogenous bioactive substance(s) that mimic some of the effects of these compounds. These observations suggest a previously uncharacterized cell signaling system. However, limited information on the identity and functionality of this family of imidazoline binding sites has hampered the full understanding of this system. Unexpectedly and of particular significance, recent data indicate that two members of the family of imidazoline binding proteins are identical to the A and B isoforms of monoamine oxidase (MAO). The imidazoline binding domain on MAO is distinct from the enzyme active site that recognizes the mechanism-based inhibitors such as pargyline and deprenyl and is not equally accessible in all tissues.

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Localization of the imidazoline binding domain on monoamine oxidase B.

Monoamine oxidase B (MAO-B) was recently identified as a member of the family of imidazoline binding proteins. To localize the imidazoline binding domain on MAO-B, we labeled the domain with the imidazoline photoaffinity adduct [125I]2-(3-azido-4-iodophenoxy)methylimidazoline in rat and human liver and visualized labeled peptides by autoradiography/sodium dodecyl sulfate-polyacrylamide gel electrophoresis after CNBr cleavage of the labeled protein. Based on species-specific fragmentation patterns and immunoprecipitation of labeled peptides, the imidazoline binding domain was localized to residues K149 to M222 of human MAO-B. The imidazoline binding domain is encompassed within a region that influences substrate processing but is distinct from primary sites of interaction for the enzyme inhibitors pargyline and lazabemide (Ro 19-6327). Radioligand binding assays and photoaffinity labeling also indicated that the various classes of compounds did not cross-compete at the different enzyme domains. Identification of an imidazoline binding domain on MAO-B provides a new opportunity for the potential pharmacological development of imidazoline/guanidinium compounds and also presents additional avenues for structure/function analysis of the monoamine oxidase enzymes.

Relationship between imidazoline/guanidinium receptive sites and monoamine oxidase A and B.

Imidazoline binding sites or imidazoline/guanidinium receptive sites (IGRS) recognize bioactive endogenous substances and a variety of pharmacologically active compounds containing imidazoline or guanidinium moieties. The family of imidazoline binding proteins consists of multiple membrane-associated proteins that differ in their tissue/subcellular localization, M(r) and ligand recognition properties. Two of the imidazoline binding proteins are identical to the mitochondrial enzyme monoamine oxidase (MAO) A and B isoforms, which contain imidazoline binding domains distinct from the enzyme active site. The relationship between the imidazoline binding proteins and monoamine oxidases was further characterized in the present report using a covalent probe (2-[3-azido-4[125I]iodophenoxy] methyl imidazoline, [125I]-AZIPI) to label the imidazoline binding proteins in different species and following transient expression of MAO- A and -B in COS 7 cells. Species homologues of MAO-A and -B in rat and human differ in their apparent molecular weight by approximately 2000 Da. In rat and human liver [125I]-AZIPI identified peptides with apparent molecular weights similar to those of the species homologues of MAO. Peptides of M(r) approximately 63,000 (MAO-A) and approximately 59,000 (MAO-B) were also photolabeled in membranes prepared from COS-7 cells transfected with human cDNA clones encoding MAO-A or -B. Additional experiments indicate that the imidazoline binding domains on MAO-A and -B exhibit different ligand recognition properties. The covalent labeling of human liver MAO-B was more sensitive than that of placenta MAO-A to inhibition by the imidazoline 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224). These data indicate that the A and B isoforms of MAO possess imidazoline binding domains that differ in their ligand recognition properties. Allosteric regulation of the activity of MAO via the imidazoline binding domains may be of significance in various disease states associated with elevated enzyme expression or in which the enzyme is a therapeutic target.

Imidazoline/guanidinium binding domains on monoamine oxidases. Relationship to subtypes of imidazoline-binding proteins and tissue-specific interaction of imidazoline ligands with monoamine oxidase B.

Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins. To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[125I]iodophenoxy)methylimidazoline ([125I]A-ZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an M(r) = approximately 59,000 peptide in liver and an M(r) = approximately 63,000 peptide in placenta, consistent with the M(r) of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites. However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.

Structural and ligand recognition properties of imidazoline binding proteins in tissues of rat and rabbit.

Imidazoline/guanidinium receptive sites (IGRS) belong to a family of membrane proteins that selectively recognize certain pharmacologically active compounds with an imidazoline or a guanidinium moiety. The role of such proteins in the cellular responses elicited by these compounds is unclear, but two members of this protein family are identical to isoforms of monoamine oxidase, an enzyme involved in the metabolism of monamine neurotransmitters. To characterize the structural and ligand recognition properties of the imidazoline binding proteins, we used the photoaffinity adduct [125I]iodoazidophe-noxymethylimidazoline ([125I]AZIPI) to label their ligand binding subunits in selected target tissues (kidney, pancreatic B cells, liver, and salivary gland). Photoaffinity labeling of membrane preparations or subcellular particulate fractions from various rat, rabbit, or hamster tissues indicated two labeled peptides of M(r) approximately 55,000 and approximately 61,000, the relative tissue distribution of which mirrored the expression of the A or B isoforms of monoamine oxidase. The ligand binding subunit of imidazoline binding proteins was identified on two peptides of M(r) approximately 55,000 and approximately 61,000 in rat and rabbit kidney, rat liver, rabbit salivary gland, and the pancreatic B cell line RIN-5AH, whereas only an M(r) approximately 61,000 peptide was observed in rat salivary gland and the hamster pancreatic B cell line HIT-T15. Saturation labeling experiments indicated that [125I]AZIPI exhibited similar affinity (Kd approximately 2-3 nM) for both the M(r) approximately 55,000 and approximately 61,000 peptides. However, competitive inhibition of photolabeling indicated that the two peptides were distinguished by their affinity for the guanidinium guanabenz or their interaction with potassium. Although some types of imidazoline binding sites are located on the enzyme monoamine oxidase, the nonisoform selective enzyme inhibitor pargyline did not alter photoaffinity labeling of either the M(r) approximately 55,000 or approximately 61,000 peptide, indicating that imidazolines/guanidiniums and active site inhibitors of monoamine oxidase interact with different domains on the enzyme. In rat kidney and liver, an additional photolabeled peptide of M(r) approximately 25,000 was observed, and its ligand recognition profile was distinct from the M(r) approximately 55,000 and approximately 61,000 species. In contrast with the mitochondrial location of the larger peptides, subcellular fractionation of liver homogenates indicated that the M(r) approximately 25,000 localized to the plasma membrane.

Similar rates of phosphatidylinositol hydrolysis following activation of wild-type and truncated rat neurokinin-1 receptors.

The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.

Ligand binding kinetics of substance P and neurokinin A receptors stably expressed in Chinese hamster ovary cells and evidence for differential stimulation of inositol 1,4,5-trisphosphate and cyclic AMP second messenger responses.

Stably transfected Chinese hamster ovary cells expressing either the substance P receptor or neurokinin A receptor were constructed, isolated, and characterized. Equilibrium ligand binding studies performed on whole cells demonstrated that cell lines expressing either of these receptors contained a single class of high-affinity binding sites with an apparent KD of 0.16 nM for the substance P receptor and an apparent KD of 2.1 nM for the neurokinin A receptor. The higher affinity of substance P for its receptor was accounted for by both a greater association rate constant and a lesser dissociation rate constant. The time course and extent of ligand-stimulated inositol 1,4,5-trisphosphate mass increases in both cell lines were similar and displayed rapid and transient kinetics. Ligand-stimulated cyclic AMP accumulation was also apparent in the cell lines, although the time course and magnitude of the responses were substantially different, with the neurokinin A receptor mediating a greater and more prolonged response. These studies establish the presence of functional substance P receptors and neurokinin A receptors in the stably transfected cell lines and provide evidence for agonist-dependent differential stimulation of second messenger responses.

S-adenosylhomocysteine levels are not involved in the spasmolytic activity of 3-deazaadenosine in guinea-pig lung parenchyma.

The effects of two S-adenosylhomocysteine (SAH) hydrolase inhibitors, MDL 28,842 and 3-deazaadenosine (3-DAA), on the tissue levels of SAH, cAMP and cGMP and on the contractile responses to leukotriene D4 and histamine were compared in guinea-pig lung parenchyma. MDL 28,842 and 3-DAA (both in the presence of L-homocysteine) increased tissue levels of SAH to the same degree, indicating that both compounds had inhibited SAH hydrolase. However, only 3-DAA inhibited the contractile response of the lung parenchyma. The spasmolytic activity of 3-DAA was inhibited by co-incubation with MDL 28,842, suggesting that 3-DAA must be metabolized by SAH hydrolase, probably to 3-deazaadenosylhomocysteine, in order to exert its effect. 3-DAA did not elevate tissue levels of cAMP or cGMP. Although the mechanism by which 3-DAA exerts its spasmolytic effects is unknown, direct inhibition of methyltransferase is a plausible explanation. These data indicate that SHA levels do not regulate the contractile response of guinea-pig lung parenchyma.

MDL 28,753, an agonist of LTD4 but an antagonist of LTC4 in longitudinal muscle of guinea pig ileum.

This article describes the novel pharmacological activity of MDL 28,753, a compound designed as a potential receptor antagonist of the peptidoleukotrienes. In the isolated longitudinal muscle of the guinea pig ileum, MDL 28,753 was a full agonist at the leukotriene (LT) D4 receptor and had a pD2 of 8.9 compared with a pD2 of 9.2 for LTD4. The contraction induced by MDL 28,753 was antagonized in a competitive manner by known LTD4 receptor antagonists, FPL 55,712 (pA2 = 7.3) and ICI 198615, and the peptidoleukotriene receptor antagonist, MDL 43,291 (pA2 = 6.7). However, under conditions in which the LTD4 receptors, but not the LTC4 receptors, were selectively inhibited by ICI 198615, MDL 28,753 showed no agonist activity at the LTC4 receptor. Instead, it competitively antagonized the agonist activity of LTC4 (pA2 = 6.1) in the longitudinal muscle of the guinea pig. Interestingly, under the same conditions, LTD4 also antagonized the agonist activity of LTC4. The functional difference of activity at these receptors supplies additional evidence for multiple receptors for the peptidoleukotrienes in guinea pig tissues. The unexpected lack of agonist action and unexpected antagonist activity of LTD4 at the LTC4 receptor suggests that the third carboxyl group in LTC4 is a critical binding requirement for agonist activity at the LTC4 receptor in guinea pigs.

Conformationally restricted leukotriene receptor antagonists: [(octahydro-2-oxo-7-tetradecylidene-2H-1-benzopyran-8-yl)thio]ace tic acids.

[(Octahydro-2-oxo-7-tetradecylidene-2H-1-benzopyran-8-yl)thio]acet ic acid (MDL 43,291) is a novel leukotriene (LT) receptor antagonist. It is a competitive antagonist of LTD4 (pA2 = 6.7) and LTE4 (pA2 = 6.7) and an apparent noncompetitive inhibitor of LTC4 ('pseudo' pA2 = 6.8) in the longitudinal muscle of the guinea pig ileum. At concentrations that effectively antagonize peptidoleukotriene-induced contractions, MDL 43,291 does not antagonize histamine, carbachol or substance P. In vivo, 10-30 mg/kg MDL 43,291, given intravenously, effectively inhibits increases in insufflation pressure induced by 100 ng/kg i.v. LTD4. This compound is a prototype of a novel class of leukotriene receptor antagonists that may be useful in the treatment of bronchial asthma and related disorders.

Impact of atmospheric dispersion and transport of viral aerosols on the epidemiology of influenza.

Current theories of influenza viral epidemiology have not explained the persistence, seasonality, and explosive outbreaks of virus over large geographic areas. It is postulated in this paper that atmospheric dispersion and intercontinental scale transport of airborne aerosolized influenza virus may contribute to the spread, persistence, and ubiquity of the disease, the explosiveness of epidemics, and the prompt region-wide occurrence of outbreaks and that seasonal changes in circulation patterns and the dispersive character of the atmosphere may help to explain the regular annual cycle of influenza activity.