Cellular and molecular action of the putative GABA-mimetic, gabapentin.

Gabapentin was originally designed as an anti-convulsant gamma-aminobutyric acid (GABA) mimetic capable of crossing the blood-brain barrier. In the present review we show that although gabapentin is not a GABA mimetic, it has great utility as an add-on therapy for epilepsy and as a first-line treatment for neuropathic pain. We summarise the studies that have been performed which demonstrate that gabapentin appears to interact with a novel binding site expressed at high density within the central nervous system (CNS), namely the alpha2delta voltage-dependent calcium channel subunit. The review continues by examining the effects of gabapentin on calcium channel function and neurotransmitter release before, in the latter part of the review, summarising the more recently discovered actions of gabapentin in relation to intracellular signalling.

New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.

The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.

Syntheses and biological activities of chiral piperidines-tachykinin NK3 antagonists.

AIM: To develop nonpeptide tachykinin NK3 antagonists. METHODS: Five tachykinin NK3 antagonists were synthesized. Receptor binding assay and oral absorption study were made. RESULTS: The 4,4-disubstituted piperidine compounds (1b, 1c, and 1d) showed stronger activities (IC50 = 5.9, 6.2, and 11 nmol.L-1, respectively) than the monosubstituted ring compound 1e (IC50 = 17 nmol.L-1). 4-Phenyl (1b) and 4-phenylsulfonylmethyl (1c) compounds were more active than the 4-fluorobenzyl compound (1d). All antagonists were found to be orally absorbable, the T1/2 of 1b (6.4 h) was more than three-fold longer than that of 1a (1.9 h). CONCLUSION: Compound 1b had the best binding activity (IC50 = 5.9 nmol.L-1) and the best AUC (2081 micrograms.h.L-1).

Bacterial expression and characterization of human recombinant apolipoprotein(a) kringle IV type 9.

Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys4057 of kringle IV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusionprotein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single approximately 17. 3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted from dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys4057 --> Ser ([Ser4057]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB3732-3745 and apoB4319-4332) containing Cys3734 or Cys4326. Both of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoB-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala3734]apoB3732-3745 and [Ala4326]apoB4319-4332) had no effect in the gel-shift assay. Furthermore, [Ser4057]KIVt9 did not covalently interact with apoB3732-3745 or apoB4319-4332. These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies.

Cloning, expression and characterization of a human dopamine D4.2 receptor (CHO K1 cells) and various D4.2/D2L chimeras (COS-7 cells).

1. Using the gene splicing technique a synthetic human dopamine (DA) D4.2 gene was constructed and subsequently stably expressed in CHO K1 cells. 2. Binding of [3H]spiperone to membranes prepared from human DA D4.2 CHO K1 cells was saturable with a Kd of 93 +/- 0.51 pM and a Bmax of 768 +/- 22 fmol per mg protein. 3. Clozapine, apomorphine, and S(+)-NPA were more selective for D4.2 than for D2L receptors, with D2L/D4.2 ratios of 5.7, 7.1, and 19.6, respectively. 4. Functional studies indicated that DA D4.2 receptors expressed in CHO K1 cells inhibited forskolin stimulated cAMP levels showing coupling to G-proteins. 5. Two reciprocal human D2L and D4.2 chimeric receptors (D2L/D4.2 and D4.2/D2L) were constructed by exchanging the amino-terminal end to the third transmembrane (TM) of one receptor with the counter part of the other receptor and expressing them transiently into COS-7 cells. The chimeric D2L/D4.2 receptor displayed non-detectable specific binding of [3H] spiperone and other ligands. The chimeric D4.2/D2L receptor binding affinities of DA agonists were more affected than that of antagonists, suggesting that binding affinities of agonists are more sensitive to changes in receptor conformation than that of antagonists. 6. This study characterized the pharmacology of a novel synthesized DA D4.2 receptor that provides a useful model for screening of potential D4.2 receptor agonist and antagonist.

Inhibition of self-splicing group I intron RNA: high-throughput screening assays.

High-throughput screening assays have been developed to rapidly identify small molecule inhibitors targeting catalytic group I introns. Biochemical reactions catalyzed by a self-splicing group I intron derived from Pneumocystis carinii or from bacteriophage T4 have been investigated. In vitro biochemical assays amenable to high-throughput screening have been established. Small molecules that inhibit the functions of group I introns have been identified. These inhibitors should be useful in better understanding ribozyme catalysis or in therapeutic intervention of group I intron-containing microorganisms.

Structural motifs encoded by individual exons of the human neurokinin-1 receptor gene interact differentially with selective agonists and antagonists.

Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1-2, or exon 1-3 of the NK1 receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9, Met(O2)11] substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.

The unpredicted high affinities of a large number of naturally occurring tachykinins for chimeric NK1/NK3 receptors suggest a role for an inhibitory domain in determining receptor specificity.

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.

Transcriptional downregulation of stromelysin by tetracycline.

We investigated the role of tetracycline in the transcriptional regulation of matrix metalloproteinases. Using interleukin-1beta (IL-1) induced stromelysin as a model system, we describe the repression of the endogenous stromelysin RNA accumulation, as well as the transcriptional inhibition of various stromelysin promoter/chloramphenicol-acetyltransferase constructs in transient transfection assays. The inhibition occurred in a dose-dependent fashion, with an IC50 of about 1 microM. Our results suggest that the transcriptional inhibition by tetracycline is not due to a block of activity of the activating protein complex 1 (AP-1) but is mediated by sequences upstream of the AP-1 binding site.

Two classes of structurally different antagonists display similar species preference for the human tachykinin neurokinin3 receptor.

Two classes of structurally different tachykinin neurokinin3 (NK3) antagonists were used to evaluate species difference in antagonist binding between human and rat NK3 receptors. In competition binding experiments with [125I-MePhe7]NKB as radioligand, PD 154740, PD 157672, SR 48968, and SR 142801 displayed lower Ki values for the human NK3 receptor (40 +/- 4, 12 +/- 1,350 +/- 50, and 0.40 +/- 0.05 nM, respectively) than for the rat NK3 receptor (2450 +/- 130, 288 +/- 25, > 10,000, and 11.0 +/- 0.5 nM, respectively). Data from in vitro functional assay showed similar species preference as observed with the competition binding assay. It was shown previously that substitution of only two amino acid residues in the rat receptor to their human counterparts could change the species selectivity of SR 48968, a weak NK3 antagonist. In the double-substituted rat mutant, all three antagonists (PD 154740, PD 157672, and SR 142801) displayed Ki values (76 +/- 8, 16 +/- 2, and 0.50 +/- 0.05 nM, respectively) very similar to the Ki values for the wild-type human NK3 receptor. Thus, in addition to their previously reported effects on SR 48968, these two amino acid residues are responsible for the species selectivity of these three additional NK3 antagonists. Because PD 154740 and PD 157672 are very different structurally from SR 48968 and SR 142801, our results indicate that the two identified residues may be involved in adopting a receptor conformation that favors the binding of NK3 antagonists that display species preference for the human NK3 receptor.

Pharmacological differences between rat and human endothelin B receptors.

Cloned rat and human endothelin-B receptors (ETBR) were utilized to determine if there are significant pharmacological differences between highly homologous ETBR from different species. Recombinant rat and human ETBR were expressed in CHO-K1 cells, and radioligand binding studies were carried out with [125I]-ET-3 to determine the affinities of various ET receptor agonists and antagonists for rat and human ETBR. These receptors had similar affinities for a number of ETBR agonists (ET-1, ET-3, S6C, BQ 3020) and antagonists (Ro 47-0203, PD 142893). However, several peptide (PD 147452, PD 151583, BQ 788) and non-peptide (PD 156707, SB 209670) antagonists had different affinities for rat and human ETBR, with differences in Ki values between species ranging from 4.1- to 53.4-fold. The ETBR-selective agonist IRL 1620 also had a 5.7-fold higher affinity for rat ETBR than human ETBR. Thus despite their high degree of homology, rat and human ETBR show significant pharmacological differences with respect to both antagonist and agonist binding.

Characterisation of [125I][MePhe7]neurokinin B binding to tachykinin NK3 receptors: evidence for interspecies variance.

Human tachykinin NK3 receptors expressed in Chinese hamster ovary (CHO-K1) cells were characterised using the novel radioligand [125I]iodohistidyl,[MePhe7]neurokinin B ([125I][MePhe7]neurokinin B). [125I][MePhe7]neurokinin B was shown to label human NK3 binding sites with high affinity in a saturable and reversible manner. The rank order of affinity of a range of tachykinin ligands confirmed that the tachykinin receptor expressed was the NK3 receptor type. An interspecies comparison of NK3 binding sites revealed pharmacological differences between human, guinea pig and rat tachykinin NK3 receptors. The NK2 selective antagonist SR 48968, inhibited binding of [125I][MePhe7]neurokinin B to NK3 binding sites with Ki values of 287 nM and 205 nM in human and guinea pig respectively, but was > 30-fold less active in the rat.

Characterization of tachykinin mediated increases in [Ca2+]i in Chinese hamster ovary cells expressing human tachykinin NK3 receptors.

The nature of the senktide response of the human NK3 receptor expressed in Chinese hamster ovary cells was characterised using the Ca2+ sensitive dye Fura-2 and imaging methods. Application of the NK3 receptor agonist senktide caused an increase in [Ca2+]i in the cells. The profile for NK3 receptor agonists was that senktide was more potent than [beta-Ala8]neurokinin A-(4-10) which was more potent than [Sar9,Met(O2)11]substance P. SR 48968 was a poor antagonist of the senktide response in intact cells confirming the weak affinity of this agent for the NK3 receptor (IC50 of approximately 1 microM) shown in binding assays. The NK3 receptor mediated increase in intracellular Ca2+ was independent of [Ca2+]o, blocked by the microsomal Ca2+ ATPase inhibitor thapsigargin and the phospholipase C inhibitor U73122 but not by ryanodine. Thus the source of the Ca2+ was probably a ryanodine insensitive, inositol triphosphate sensitive intracellular store.

The non-peptide tachykinin NK2 receptor antagonist SR 48968 interacts with human, but not rat, cloned tachykinin NK3 receptors.

SR 48968, a non-peptide tachykinin NK2 receptor antagonist, has been shown to possess sub-micromolar affinity for NK3 receptors present in the guinea pig. In the present study, we have compared the binding affinities of SR 48968 to the cloned human and rat NK3 receptors expressed in CHO cells. Using [125I]-[MePhe7]-neurokinin B as the radioligand, SR48968 displayed an IC50 value of 350 nM for the human NK3 receptor as compared with a value of greater than 10 microM for the rat NK3 receptor. Exposure of cells transfected with human NK3 receptor cDNA to [Pro7]-neurokinin B increased inositol phospholipid turnover in a concentration-dependent manner and this response was blocked competitively by SR 48968. Our results demonstrate that SR 48968 is an antagonist at the human NK3 receptor and may be a useful tool for elucidating the species-dependent variations in the non-peptide antagonist binding site(s) on the NK3 receptor.

Identification of methionine134 and alanine146 in the second transmembrane segment of the human tachykinin NK3 receptor as reduces involved in species-selective binding to SR 48968.

We have shown that SR 48968 possess sub-micromolar affinity for the human tachyknin NK3 receptor; however, its affinity for the rat NK3 receptor is greater than 10 microM. To determine the functional domain(s) responsible for the species variation in binding affinities, we have constructed several human/rat chimeric NK3 receptors. Based on studies of these chimeric receptors, the species-specific binding sites for SR 48968 were localized to five residues in the 1st and 2nd transmembrane segments of the human NK3 receptor. We have individually mutated all five residues in the rat receptor to their corresponding residues in human. Only two single-substituted mutants (V121M and G133A) show a small increase in their binding affinities for SR 48968. However, a mutant containing both substitutions was shown to have the same affinity for SR 48968 as the wild type human NK3 receptor. It is concluded that collectively these two amino acid changes are responsible for the species difference in binding affinities for SR 48968.

Absence of functional inhibition of cloned human beta 2-adrenergic receptors by autoantibodies in asthmatic subjects.

Using guinea-pig lung membranes and cloned human beta 2-receptor adrenergic receptors the effects of whole serum, plasma, purified immunoglobulins and cellular activation products on beta 2-adrenergic receptor ligand binding and function were investigated. Sera from 24 non-asthmatic subjects and 115 asthmatics in different clinical categories were studied. There were no significant differences between antagonist ([125I] cyanopindolol) inhibition mediated by serum, plasma or by purified IgG when the asthmatics were compared with non-asthmatics. There was also no inhibition of 10(-6) M isoproterenol stimulated cAMP release from L cells expressing human beta 2-adrenergic receptors, by plasma, DEAE purified IgG fractions from asthmatics and non-asthmatics, or by products of activated platelets or lymphocytes. Since we have no evidence that immunoglobulins from asthmatic subjects exert functional inhibition of human beta 2-adrenergic receptors we conclude that autoantibodies to the beta 2-adrenergic receptors do not play an important functional role in the pathophysiology of asthma.

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.

Cloning, expression, and characterization of a gene encoding the human angiotensin II type 1A receptor.

The human angiotensin II (AII) type 1a receptor gene and its upstream control sequence has been cloned from a human leukocyte genomic library. The promoter element CAAT and TATA sequences were found at -602 and -538, respectively, upstream from the translational initiation site. The deduced protein sequence is homologous to rat and bovine AT1a receptors (94.7% and 95.3% identity). The expressed gene exhibited high-affinity AII and Dup753 binding and was functionally coupled to inositol phosphate turnover. Northern analysis of human tissues showed AT1 receptor mRNA expression in placenta, lung, heart, liver, and kidney. Using 5' untranslated and coding sequence as probes in a Southern blot analysis, it was established that another AT1 subtype exists in the human genome.

Substitution of lysine-181 to aspartic acid in the third transmembrane region of the endothelin (ET) type B receptor selectively reduces its high-affinity binding with ET-3 peptide.

In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.