Kim S. Sugamori

A primordial dopamine D1-like adenylyl cyclase-linked receptor from Drosophila melanogaster displaying poor affinity for benzazepines

We report here the isolation from Drosophila melanogaster of a 2.0 kb cDNA clone encoding a 385 amino acid protein (dDA1) displaying, within putative transmembrane domains, highest amino acid sequence homology (49–53%) to members of the vertebrate dopamine D1‐like receptor family. When expressed in either Sf9 or COS‐7 cells, dDA1 did not bind the specific D1‐like receptor antagonist [3H]SCH‐23390 or numerous other dopaminergic, adrenergic or serotoninergic ligands with high affinity. However, like vertebrate dopamine D1‐like receptors, dDA1 stimulated the accumulation of cAMP in response to DA (EC50 ∼300 nM) and 6,7‐ADTN (EC50∼500 nM). The dopaminergic rank order of potency (DA > NE⪢5‐HT) and the lack of stimulation by other possible neurotransmitters (octopamine, tyramine, tryptamine) or DA metabolites (e.g. N‐acetyl dopamine) found in Drosophila suggests that this receptor functionally belongs to the dopamine D1‐like subfamily. Benzazepines, which characteristically bind to vertebrate dopamine D1‐like receptors with high affinity, were relatively poor in stimulating (SKF‐38393, SKF‐82526; EC50 > 10 μM) dDA1‐mediated accumulation of cAMP. Of the numerous compounds tested, a few dopaminergic antagonists inhibited DA‐stimulated production of cAMP in a concentration‐dependent manner, albeit with considerably reduced affinity, and with the rank order of potency: (+)‐butaclamol(K b∼125nM) > SCH‐23390(K b∼230nM) > α‐flupenthixol (K b ∼ 400 nM) > chlorpromazine ≥ spiperone (K b ∼ 680 nM) ≥ clozapine In situ hybridization revealed that dDA1 receptor mRNA is expressed as a maternal transcript, and at later blastoderm stages is restricted to apical regions of the cortical peripheral cytoplasm. The generation of inter‐species D1 receptor chimeras may help to identify those particular sequence‐specific motifs or amino acid residues confering high affinity benzazepine receptor interactions.

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

Early Emergence of Three Dopamine D1 Receptor Subtypes in Vertebrates MOLECULAR PHYLOGENETIC, PHARMACOLOGICAL, AND FUNCTIONAL CRITERIA DEFINING D1A, D1B, AND D1C R/ECEPTORS IN EUROPEAN EELANGUILLA ANGUILLA

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.

Pharmacogenetics of the Human Arylamine N-Acetyltransferases

This review briefly describes current understanding of one of the earliest discovered pharmacogenetic polymorphisms of drug biotransformation affecting acetylation of certain homo- and heterocyclic aromatic amines and hydrazines. This so-called acetylation polymorphism arises from allelic variation in one of the two known human arylamine N-acetyltransferase genes, namely NAT2, which results in production of NAT2 proteins with variable enzyme activity or stability. The NAT1 gene locus encodes a structurally related enzyme, NAT1, with catalytic specificity for arylamine acceptor substrates distinct from that exhibited by NAT2. NAT1 function is also genetically variable in human populations. Clinical and toxicological consequences of genetic variation in NAT1 and NAT2 activity are discussed.

Molecular and functional characterization of a partial cDNA encoding a novel chicken brain melatonin receptor.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domesticus) brain. Based on available deduced amino‐acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xenopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expression, a chimeric frog/chicken (flc)MLR was constructed in which the 5′ end of the cMLR, including the N‐terminus, TM1 and part of the first intracellular loop was substituted by fMLR sequence. [125I]Iodo‐ML bound with high affinity (K d of ∼35 pM) to COS‐7 cells transiently expressing the flcMLR in a saturable and guanine nucleotide‐sensitive manner with the following rank order of potency: 2‐iodo‐ML > ML > 6‐Cl‐ML > S20750 > 6‐OH‐ML > S20643 > S20753 > N‐acetyl‐5HT > > 5‐HT. Estimated K i values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6‐Cl‐ML and 6‐OH‐ML ∼10‐fold lower than mammalian receptors. Functionally, opposing interactions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1A‐receptor‐mediated cAMP accumulation in cells (HEK‐293) transiently co‐expressing these receptors. cMLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identification of those amino‐acid residues and structural motifs that regulate ML‐binding specificity and affinity.

In vivo and in vitro metabolism of arylamine procarcinogens in acetyltransferase-deficient mice.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of which are procarcinogenic agents. Interestingly, these enzymes are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play in either preventing or enhancing toxic responses. The ultimate direction may be substrate-specific and dependent on its tissue-specific metabolism by competing, but genetically variable, drug-metabolizing enzymes. To investigate the effect of N-acetylation on the metabolism of some classical procarcinogenic arylamines, we have used our double knockout Nat1/2(–/–) mouse model to test both in vitro activity and the in vivo clearance of some of these agents. As expected, N-acetylation activity was undetectable in tissue cytosol preparations from Nat1/2(–/–) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), whereas significant levels were measured in all wild-type tissue cytosols tested, indicating the widespread metabolism of these agents. Nat1/2(–/–) mice displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3- to 4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype, signifying a dependence on other drug-metabolizing enzymes. The elucidation of the role that N-acetylation plays in the clearance of procarcinogenic agents is the first step in attempting to correlate metabolism by NATs to toxic outcome prevention or augmentation.

Loss of the Mono-ADP-ribosyltransferase, Tiparp, Increases Sensitivity to Dioxin-induced Steatohepatitis and Lethality

Background: Tiparp is an aryl hydrocarbon receptor (AHR) repressor, but its role in dioxin toxicity is unknown. Results: Loss of Tiparp increases sensitivity to dioxin toxicity and lethality. Tiparp ADP-ribosylates AHR, which is reversed by the mono-ADP-ribosylase, MacroD1. Conclusion: We identify new roles for Tiparp, MacroD1, and ADP-ribosylation in AHR signaling and dioxin toxicity. Significance: These data reveal the importance of TIPARP in regulating AHR activity in mice. The aryl hydrocarbon receptor (AHR) mediates the toxic effects of the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD). Dioxin causes a range of toxic responses, including hepatic damage, steatohepatitis, and a lethal wasting syndrome; however, the mechanisms are still unknown. Here, we show that the loss of TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), an ADP-ribosyltransferase and AHR repressor, increases sensitivity to dioxin-induced toxicity, steatohepatitis, and lethality. Tiparp−/− mice given a single injection of 100 μg/kg dioxin did not survive beyond day 5; all Tiparp+/+ mice survived the 30-day treatment. Dioxin-treated Tiparp−/− mice exhibited increased liver steatosis and hepatotoxicity. Tiparp ADP-ribosylated AHR but not its dimerization partner, the AHR nuclear translocator, and the repressive effects of TIPARP on AHR were reversed by the macrodomain containing mono-ADP-ribosylase MACROD1 but not MACROD2. These results reveal previously unidentified roles for Tiparp, MacroD1, and ADP-ribosylation in AHR-mediated steatohepatitis and lethality in response to dioxin.

Dopamine D1B Receptor Chimeras Reveal Modulation of Partial Agonist Activity by Carboxyl-Terminal Tail Sequences

Abstract: NNC 01‐0012, a second‐generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01‐0012 acts as a full or poor antagonist at D1C and D1A receptor‐mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01‐0012, a series of receptor chimeras were constructed in which the divergent carboxyl‐terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl‐terminal‐tail at position Tyr345 with carboxyl‐terminal‐tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01‐0012 without affecting dopamine‐stimulated cyclic AMP accumulation. At vertebrate D1B/D1Cct‐tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01‐0012 (10 µM) was markedly enhanced (∼60% relative to 10 µM dopamine) with no concomitant alteration in the molecules ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF‐38393 and SCH‐23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl‐terminal tails with sequences encoded by the D1B receptor carboxyl‐terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well‐characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence‐specific motifs within the carboxyl‐terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1‐like receptors.

Effect of Arylamine Acetyltransferase Nat3 Gene Knockout on N-Acetylation in the Mouse

Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(–/–) knockout strain to further investigate the functional role of Nat3. Nat3(–/–) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(–/–) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(–/–) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(–/–) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(–/–) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(–/–) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.

Functional Differentiation of Multiple Dopamine D1-Like Receptors by NNC 01-0012

Abstract: Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype‐specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1‐like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS‐7 cells. Of all the compounds tested, only NNC 01‐0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH‐23390 binding with an estimated affinity (∼0.6 nM) 20‐fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01‐0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 µM)‐stimulated adenylyl cyclase activity. In contrast, NNC 01‐0012 (10 µM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by ∼33% relative to the full agonist dopamine (10 µM), an effect that was blocked by the selective D1 receptor antagonist NNC 22‐0010. These data clearly suggest that the benzazepine NNC 01‐0012, despite lacking the N‐methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01‐0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.