Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Transient and long-term differential modulations of branched-chain α-keto acid decarboxylase activity in hypophysectomized rats

Hypophysectomy caused a marked but transient increase in branched-chain α-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain α-keto acids. The activities then fall to a new steady state in three weeks with α-ketoisovalerate decarboxylase activity within the normal range, α-keto-β-methylvalerate decarboxylase activity at twice normal, and α-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.     

A yeast-based model of α-synucleinopathy identifies compounds with therapeutic potential

We have developed a yeast-based model recapitulating neurotoxicity of α-synuclein fibrilization. This model recognized metal ions, known risk factors of α-synucleinopathy, as stimulators of α-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic α-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66–74 in α-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble α-synuclein, indicating that noxious α-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain α-synuclein toxicity. Subsequent structure–activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of α-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.     

The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase.

Alanine aminotransferase catalyzes exchange of the β-hydrogens of alanine with the solvent at a rate commensurate with the rate of exchange of the α-hydrogen. These methyl protons are lost sequentially and intermediates having protons on the α-carbon but deuterium on the β-carbon were detected by nuclear magnetic resonance. The overall rates of exchange of both α-hydrogen and β-hydrogen were less than the rate of transamination and did not vary from pH 6–8. The α-hydrogen of glutamate, on the other hand, was found to exchange at a greater rate than the overall transamination rate with ketoglutarate. However the β-hydrogens of glutamate are not removed during the enzymic reaction. It is concluded that a basic group on the enzyme removes the proton from the α-carbon of alanine at a rate at least as great as the rate of transamination. Because the proton is held on the enzyme, it appears to exchange more slowly in alanine. Labilization of the α-hydrogen of amino acids does not appear to be the ratelimiting reaction of alanine aminotransferase, but occurs at a rate comparable to that of the overall reaction.     

Studies on purified α-actinin : I. Effect of temperature and tropomyosin on the α-actinin/F-actin interaction

α-Actinin, purified by two passages through a DEAE-cellulose column, migrates either as a single band or as a single major band with a fainter trailing band during polyacrylamide gel electrophoresis at pH 8.3. In the presence of sodium dodecyl sulfate, purified α-actinin always migrates as a single electrophoretic zone during polyacrylamide gel electrophoresis. Temperature has large effects on the interaction of α-actinin with F-actin. At 0 °C, α-actinin causes large increases in F-actin viscosity, either in the presence or absence of tropomyosin. Quantitative binding studies show that α-actinin can displace tropomyosin from F-actin at 0 °C and that F-actin will quantitatively bind 45% of its weight of α-actinin either in the presence or absence of tropomyosin. This binding ratio corresponds to one α-actinin molecule to approximately 10 to 11 G-actin subunits and suggests that one molecule of α-actinin binds to each turn of the F-actin helix at 0 °C.     

A novel structural framework for α(1A/D)-adrenoceptor selective antagonists identified using subtype selective pharmacophores

In this study four and five-feature pharmacophores for selective antagonists at each of the three α(1)-adrenoceptor (AR) subtypes were used to identify novel α(1)-AR subtype selective compounds in the National Cancer Institute and Tripos LeadQuest databases. 12 compounds were selected, based on diversity of structure, predicted high affinity and selectivity at the α(1D)- subtype compared to α(1A)- and α(1B)-ARs. 9 out of 12 of the tested compounds displayed affinity at the α(1A) and α(1D) -AR subtypes and 6 displayed affinity at all three α(1)-AR subtypes, no α(1B)-AR selective compounds were identified. 8 of the 9 compounds with α(1)-AR affinity were antagonists and one compound displayed partial agonist characteristics. This virtual screening has successfully identified an α(1A/D)-AR selective antagonist, with low μM affinity with a novel structural scaffold of a an isoquinoline fused three-ring system and good lead-like qualities ideal for further drug development.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Evaluation of Hydrophobicity Versus Chaperonelike Activity of Bovine αA- and αB-Crystallin

Calf lens αA-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than αB-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to αA-crystallin than to αB-crystallin at room temperature. Bis-ANS binding to both αA- and αB-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of αA-crystallin is lower than that of αB-crystallin whereas at higher temperatures, αA-crystallin shows significantly higher protection against aggregation of substrate proteins compared to αB-crystallin. Therefore, calf lens αA-crystallin is more hydrophobic than αB-crystallin and chaperone-like activity of α-crystallin subunits is not quantitatively related to their hydrophobicity.     

Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues

α-Conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide α-conotoxin RgIA (α-RgIA) is a member of the α-4,3 family of α-conotoxins and selectively blocks the α9α10 nAChR subtype, in contrast to another well-characterized member of this family, α-conotoxin ImI (α-ImI), which is a potent inhibitor of the α7 and α3β2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of α-RgIA, and have modified its C-terminus. The effects of these changes on activity against α9α10 and α7 nAChRs were measured; the solution structures of α-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for α-RgIA [R9A]. The structures for α-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of α-ImI reveals a common two-loop backbone architecture within the α-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the α9α10 and the α7 subtypes. In loop 2, α-RgIA [Y10W] had activity near that of wild-type α-RgIA, with high potency for α9α10 and low potency for α7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the α9α10 subtype, probably because it is larger and more solvent accessible than Ala9 in α-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the α9α10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.     

Starch conversion by soluble and immobilized α-amylase

B. subtilis α-amylase was immobilized on cyanogen bromide activated carboxymethyl cellulose. The conversion of wheat starchwas carried out at 72°C in a stirred tank by soluble and immobilized α-amylase. The initial reaction rate with immobilized α-amylase was lower than with the soluble enzyme, but after 1 hr immobilized α-amylase produced a higher quantity of reducing sugars than the soluble enzyme. The action pattern of immobilized α-amylase was different from that of the soluble enzyme: immobilized α-amylase produced relatively more glucose and maltose, except at the beginning of conversion. Immobilized α- readily hydrolyze G₆. The starch conversion by immobilized α-amylase was not diffusion controlled at a stirring rate of 100-300 rpm.     

The calcium channel α2/δ1 subunit interacts with ATP5b in the plasma membrane of developing muscle cells

The α2/δ1 and α(1)1.1 subunits are present at a 1:1 ratio in the dihydropyridine receptor (DHPR) from adult skeletal muscle. In contrast, during early myotube development α2/δ1 is present at higher levels than α(1)1.1 and localizes at the ends of the cells, suggesting that α2/δ1 may have a role independent from DHPRs. We sought to identify binding partners of α2/δ1 at a period when levels of α(1)1.1 are low. Analysis of protein complexes in their native configuration established that α2/δ1 may be associating with ATP5b, a subunit of a mitochondrial ATP synthase complex. This interaction was confirmed with fluorescence resonance energy transfer and coimmunoprecipitation. The association of α2/δ1 and ATP5b occurs in intracellular membranes and at the plasma membrane, where they form a functional signaling complex capable of accelerating the rate of decline of calcium transients. The acceleration of decay was more evident when myotubes were stimulated with a train of pulses. Our data indicate that the α2/δ1 subunit is not only part of the DHPR but that it may interact with other cellular components in developing myotubes, such as the ATP5b in its atypical localization in the plasma membrane.     

Uptake of alpha-ketoisocaproic acid in lymphoblast line WI-L2.

The uptake of α-ketoisocaproate by the cultured human lymphoblast line WI-L2 appears to be mediated by a transport system which has an apparent Km of 125 μM. The rate of uptake of α-ketoisocaproate decreases with increasing pH values, i.e., pH 6 > 7 > 8 and is stimulated by sodium at all pH values. Closely related branched chain α-ketoacids, α-keto-β-methylvaleric and α-ketoisovaleric exhibited the greatest inhibition of α-ketoisocaproate transport. Straight chain α-keto acids inhibited α-ketoisocaproic acid uptake to a lesser degree as did the α-hydroxy analogs of the branched chain α-keto acids. Inhibitors of the general anion transport system of erythrocytes, 1-anilino-8-napthalene sulfonic acid and 4-acetamido-4-isothiocyanostilbene-2-1′-disulfonic acid did not affect α-ketoisocaproate transport. A reduced sulfhydryl group is critical for α-ketoisocaproate acid uptake; transport is partially or completely inhibited by sulfhydryl reagents such as dithio-bis-nitrobenzoate, iodoacetamide, and p-chloromercuribenzoate. Inhibition by the sulfhydryl reagents is reversed with β-mercaptoethanol or partially with dithiothreitol.     

Organization and expression of a-tubulin genes in Drosophila melanogaster: One member of the a-tubulin multigene family is transcribed in both oogenesis and later embryonic development

Genomic clones containing α-tubulin sequences were isolated from a library of Drosophila melanogaster DNA and identified by a hybridization-selection and in vitro-translation procedure. The in vitro translation products were identical to the two electrophoretic variants of α-tubulin present in Drosophila embryos. They co-assembled with an embryonic tubulin fraction to form microtubules in vitro and generated the same partial proteolytic fragments as Drosophila α-tubulins. Hybridization in situ to polytene chromosomes revealed that the α-tubulin sequences constitute a multigene family localized on the right arm of chromosome 3 at sites 84 B3–6, 84 D4–8 and 85 E6–10. Clones hybridizing to these sites corresponded to the three major α-tubulin sequences in genomic DNA. The α-tubulin sequences at 84 B3–6 were present twice per haploid genome, embedded in a large duplicated DNA segment. The sequences of the three genomic α-tubulin genes showed considerable divergence, making it possible to conclude that both of the α-tubulin variants present in embryos are encoded by the genes at 84 B3–6. Furthermore, the abundance of this α-tubulin messenger RNA changes with the requirements for microtubule synthesis during embryo development. The genes at 84 B3–6 encoded both the stored maternal mRNA of the oocyte and the major mRNA transcribed during embryonic development.     

Glycolytic enzyme–tubulin interactions: Role of tubulin carboxy terminals

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of α-or β-tubulin by cleaving a peptide fragment from the C-terminals. Generation of α′β′-tubulin, which is cleaved at both the α- and β-subunit terminals, and αβ′-tubulin, which is cleaved at the β′-subunit C-terminal, have already been reported. In this work an isotype, α′β-tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of α led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3-phosphate dehydrogenase. When the α-subunit C-terminal was intact, inhibition was greatest. These results imply that the C-terminal of the tubulin α-subunit is subunit is responsible for interactions with glycolytic enzymes.     

Loss of density-dependent growth inhibition and dissociation of α-catenin from E-cadherin

Normal human breast epithelial (HBE) cells at early (9th) passage ceased growth and formed a monolayer when they reached confluence. Immunostaining and Western blotting revealed that α- and β-catenins colocalized and coprecipitated with E-cadherin, suggesting a complex formation of E-cadherin with α- and β-catenins in early passage cells. In contrast, HBE cells at late (12–13th) passage did not cease growth after confluence but stratified. The late passage cells exhibited enhanced colony forming ability in soft agar compared with early passage cells, however, they had a definite proliferating lifespan and were primarily diploid. In late passage cells grown as multilayers, α-catenin was expressed but did not colocalize or coprecipitate with E-cadherin, suggesting its dissociation from E-cadherin. Coimmunoprecipitation of α-actinin with α-catenin suggested an indirect link between the E-cadherin-β-catenin complex and α-actinin via α-catenin in early, but not in late passage cells. β-Catenin in late passage cells was tyrosine phosphorylated and was not dephosphorylated following the addition of inhibitors of tyrosine kinases. Inhibition of dephosphorylation of β-catenin in early passage cells by vanadate, an inhibitor of protein tyrosine phosphatases, caused overgrowth of cells beyond the saturation density and loss of α-catenin from the E-cadherin-β-catenin complex. The results suggest that E-cadherin requires its association with α-actinin-associated α-catenin to maintain epithelial monolayers and accomplish the density-dependent inhibition of growth. In addition, association between E-cadherin and α-catenin is suggested to be prevented by the presence of tyrosine phosphorylated β-catenin which associates with E-cadherin. J. Cell. Physiol. 173:54–63, 1997. © 1997 Wiley-Liss, Inc.     

Effect of anesthetics on the stability of oxyhemoglobin S

The conversion of the serine-195 in α-chymotrypsin to dehydroalanine results in two conformational substates that differ in their extinction coefficients at 240nm. The active site methionine-192 in the substate with lower absorption at 240nm is alkylated by α-bromo-4-nitroacetophenone at a rate of 7.0×10^?4sec^?1, similar to that found for α-chymotrypsin; the substate with higher absorption at 240nm reacts 14 times slower. These two substates are not separated by an affinity resin containing lima bean trypsin inhibitor. These data infer that the serine-195 plays a role in the stabilization of the active site conformation in α-chymotrypsin.     

Agouti Protein Inhibits the Production of Eumelanin and Phaeomelanin in the Presence and Absence of α-Melanocyte Stimulating Hormone

Melanocytes synthesise two types of melanin: the brown-black eumelanin and the red-yellow phaeomelanin. In mice, the relative proportions of these two melanins are regulated by α-MSH, which preferentially increases the synthesis of eumelanin and by the Agouti protein (AP), the expression of which correlates with the growth of yellow phaeomelanin-containing hair. It has been proposed that AP acts by antagonizing the action of α-MSH at the MCI receptor, although it has been suggested that it may also act independently of α-MSH. In the present study we show that AP inhibits melanogenesis in B16F1 melanoma cells in the presence and absence of α-MSH and also causes dose-related decreases in the synthesis of both eumelanin and phaeomelanin. In the presence of α-MSH AP had a greater effect on eumelanin production and this is consistent with an antagonistic action at the MCI receptor. In the absence of α-MSH however, AP produced similar reductions in the synthesis of both melanins. These changes were not seen in B16G4F cells which lack the MCI receptor, suggesting that even in the absence of α-MSH AP acts at the MCI receptor. How this action is mediated at the intracellular level is not yet clear, although it appears to be associated with a decrease in tyrosinase activity.     

A mutational analysis of active site residues in trans-3-chloroacrylic acid dehalogenase

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.     

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

Synthesis and evaluation of C2 functionalized analogs of the α-tubulin-binding natural product pironetin

Pironetin is an α-tubulin-binding natural product with potent antiproliferative activity against several cancer cell lines that inhibits cell division by forming a covalent adduct with α-tubulin via a Michael addition into the natural product’s α,β-unsaturated lactone. We designed and prepared analogs carrying electron-withdrawing groups at the α-position (C2) of the α,β-unsaturated lactone with the goal to generate potent and selective binding analogs. We prepared derivatives containing halogens, a phenyl, and a methyl group at the C2 position to evaluate the structure-activity relationship at this position. Testing of the analogs in ovarian cancer cell lines demonstrated 100–1000-fold decreased antiproliferative activity.