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H B Niznik

Publications and source records attributed to H B Niznik.

At least 19 recordsLinked to original sources
PubMed1998

High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol.

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.

Animals↗
PubMed1998

Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H] CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 microM), but not alphaPDD, reduced the Vmax (approximately 1 pmol/min/10(5) cells) for [3H]DA uptake by approximately 40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by approximately 30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (approximately 200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [3H] CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function.

Animals↗
PubMed1998

Functional differentiation of multiple dopamine D1-like receptors by NNC 01-0012.

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 (approximately 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 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) 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 approximately 33% relative to the full agonist dopamine (10 microM), 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.

Animals↗
PubMed1998

Dopamine D1B receptor chimeras reveal modulation of partial agonist activity by carboxyl-terminal tail sequences.

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 microM) was markedly enhanced (approximately 60% relative to 10 microM dopamine) with no concomitant alteration in the molecule's 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.

Animals↗
PubMed1997

Early emergence of three dopamine D1 receptor subtypes in vertebrates. Molecular phylogenetic, pharmacological, and functional criteria defining D1A, D1B, and D1C receptors in European eel Anguilla 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.

Adenylyl Cyclases↗
PubMed1997

Effects of intrastriatal infusion of D2 receptor antisense oligonucleotide on apomorphine-induced behaviors in the rat.

An antisense oligonucleotide strategy was employed to specifically deplete postsynaptic striatal D2 receptors in order to determine the possible role of presynaptic D2 autoreceptors in mediating behavioral responses induced by low doses of apomorphine. A phosphorothioate-modified antisense oligonucleotide complementary to the first 19 bases of the coding region of D2 receptor mRNA, a scrambled sequence comprising the same bases, or saline was infused bilaterally into the striatum of adult rats, twice daily for 2 days via indwelling cannulae. After an interval of 8-12 h, rats were habituated and challenged with high (300 micrograms/kg; subcutaneous) or low (50 micrograms/kg; s.c.) doses of apomorphine or its vehicle (0.1% ascorbic acid). Yawning, vacuous chewing mouth movements, hypoexploration, and penile grooming induced by low-dose apomorphine were unaffected by antisense infusion into the striatum, whereas stereotypic sniffing following high-dose apomorphine was markedly suppressed. Intrastriatal infusion of antisense resulted in significantly diminished [3H]-raclopride binding, while binding of [3H]-SCH 23390 (D1 receptors) and [3H]-WIN 35428 (dopamine transporter) was unchanged. D2 mRNA levels determined by quantitative in situ hybridization were normal in the striatum and the substantia nigra. Our results confirm that stereotypic sniffing is mediated via postsynaptic D2 receptors in the striatum, and favor the notion that behavioral responses induced by low doses of apomorphine are mediated by presynaptic D2 autoreceptors.

Animals↗
PubMed1996

NMDA and dopamine D2L receptor interaction in human neuroblastoma SH-SY5Y cells involves tyrosine kinase and phosphatase.

Blockade of N-methyl-D-aspartate (NMDA) receptors by the specific antagonists dizocilpine and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid on human neuroblastoma SH-SY5Y cells expressing human D21 receptors resulted in a significant increase in the density of D2L receptors. In order to understand the mechanism of dopamine D2L receptor induction following NMDA receptor blockade we used specific protein tyrosine kinase and phosphatase inhibitors to demonstrate their involvement in this interaction. The induction of dopamine D2L receptor was measured by radioreceptor binding assay. The density of the dopamine D2L receptor was increased to 109% by the inhibition of protein tyrosine kinase and prevented by the inhibition of phosphatase 1 or 2A. Inactivation of NMDA receptors might effect the phosphorylation-dephosphorylation states of the regulatory proteins and lead to the induction of the D2L receptor gene.

Cell Line↗
PubMed1996

The dopamine transporter carboxyl-terminal tail. Truncation/substitution mutants selectively confer high affinity dopamine uptake while attenuating recognition of the ligand binding domain.

In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted. Complete truncation of the carboxyl tail from Ser582 allowed for the expression of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax approximately 0.4 pmol/10(5) cells/min) high affinity (Km approximately 300 nM) component and one exhibiting low affinity (Km approximately 15 microM] and high capacity (Vmax approximately 5 pmol/10(5)cells/min) with a concomitant 40% decrease in overall apparent Vmax relative to wild type (WT) DAT. Truncation of the last 22 amino acids or substitution of the DAT-COOH tail with sequences encoding the intracellular carboxyl-terminal of either dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro597. The attenuation of WT transport activity, either by lowering levels of DAT expression or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 microM), did not affect the kinetics of [3H]dopamine transport. The estimated affinity of dopamine (Ki approximately 180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (approximately 2000 nM) and appears selective for the endogenous substrate, since the estimated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed significantly reduced high affinity [3H]CFT binding interactions with estimated Ki values for dopamine and numerous other substrates and inhibitors tested from 10-100-fold lower than that observed for WT DAT. Moreover, co-expression of truncated and/or substituted DATs with WT transporter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of uniphasic low affinity [3H]dopamine uptake kinetics (Km approximately 2000 nM) and high affinity substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were evident. These data clearly suggest the functional independence and differential regulation of the dopamine translocation process from the characteristics exhibited by its ligand binding domain. The lack of functional phenotypic expression of mutant DAT activities in cells co-expressing WT transporter is consistent with the contention that native DATs may exist as multisubunit complexes, the formation and maintenance of which is dependent upon sequences encoded within the carboxyl tail.

Animals↗
PubMed1996

A polymorphic dinucleotide repeat in the human dopamine D5 receptor gene promoter.

Previously, we have reported the cloning and characterization of the 5'-flanking region of the human dopamine D5 receptor encoding gene (D5) and that the major transactivation domain was 119-182 bp upstream of the transcriptional start site [Beischlag, T.V. et al., Biochemistry, 34 (1995) 5960-5970]. Within this region existed a small dinucleotide repeat termed (TC)13. In this report, we describe the screening of genomic DNAs from 18 unrelated individuals by single-strand conformation polymorphism (SSCP) analysis. SSCP analysis revealed the existence of two additional alleles, termed (TC)12 and (TC)14. Neither form significantly altered D5 promoter-mediated luciferase activity when compared to that of the wild-type control, suggesting that small differences in the number of dinucleotide repeats are not likely of any functional consequence for D5 transactivation.

Base Sequence↗
PubMed1996

Striatal dopamine, dopamine transporter, and vesicular monoamine transporter in chronic cocaine users.

Depletion of striatal dopamine (DA) has been hypothesized to explain some of the neurological and psychiatric complications of chronic use of cocaine, including increased risk for neuroleptic-precipitated movement disorders. We measured levels of DA, as well as two DA nerve terminal indices, namely, the DA transporter (DAT) and the vesicular monoamine transporter (VMAT2) in autopsied brain of 12 chronic cocaine users. Mean DA levels were normal in the putamen, the motor component of the striatum; however 4 of the 12 subjects had DA values below the lower limit of the control range. DA concentrations were significantly reduced in the caudate head (head, -33%; tail, -39%) with a trend for reduction in nucleus accumbens (-27%). Striatal DAT protein (-25 to -46%) and VMAT2 (-17 to -22%) were reduced, whereas DAT determined by [3H]WIN 35,428 binding was normal. In conclusion, our data suggest that chronic cocaine use is associated with modestly reduced levels of striatal DA and the DA transporter in some subjects and that these changes might contribute to the neurological and psychiatric effects of the drug.

Adult↗
PubMed1996

Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells.

To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH-SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by approximately 50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.

Base Sequence↗
PubMed1996

Differential changes in neurochemical markers of striatal dopamine nerve terminals in idiopathic Parkinson's disease.

To determine the extent that different dopamine (DA) neuronal markers provide similar estimates of striatal (caudate and putamen) DA nerve terminal loss in idiopathic Parkinson's disease (PD), we compared, in postmortem striatum of 12 patients with PD and 10 matched controls, levels of five different DA neuronal markers. These markers included DA itself, three different estimates of the density of the DA transporter (DAT) ([3H])GBR 12,935 and [3H]WIN 35,428 binding; DAT protein immunoreactivity), and one estimate of the vesicular monoamine transporter (VMAT2; [3H]DTBZ binding). Striatal levels of all examined DA markers in PD were significantly intercorrelated. However, the magnitude of loss relative to controls was unequal (DAT protein = DA > [3H]WIN 35,428 > [3H]DTBZ > [3H]GBR 12, 935), with the differences more marked in the severely affected putamen. The less severe reduction of binding of the DAT/VMAT2 radioligands relative to DA and DAT protein could be explained by differential regulation/degeneration of different DA nerve terminal components or lack of specificity of the radioligands for the DA neuron. These postmortem data may help in interpretation of in vivo neuroimaging studies in PD in which only one radioligand is routinely employed.

Aged↗
PubMed1995

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 (Kd of approximately 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 > S20642 > S20753 > N-acetyl-5HT >> 5-HT. Estimated Ki 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 approximately 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.

Adenylyl Cyclase Inhibitors↗
PubMed1995

The human dopamine D5 receptor gene: cloning and characterization of the 5'-flanking and promoter region.

Genomic and overlapping cDNA clones encompassing the entire 5'-untranslated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized intron (of either 179 or 155 bp). We have determined that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5' to the transcription initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Sp1 and Ap1. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5' of the transcription initiation site were able to stimulate transcription only in SK-N-SH cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained a positive modulator at 119-182 and a negative modulator 251-500 bases upstream from the site of transcription initiation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridization analysis of monkey and human brain using a 5' D5-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV, and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labeled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor-mediated events.

Base Sequence↗
PubMed1995

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 approximately 300 nM) and 6,7-ADTN (EC50 approximately 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 microM) 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(Kb approximately 125nM) > SCH-23390(Kb approximately 230nM) > alpha-flupenthixol (Kb approximately 400 nM) > chlorpromazine > or = spiperone (Kb approximately 680 nM) > or = 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 conferring high affinity benzaepine receptor interactions.

Adenylyl Cyclases↗