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.     

Intestinal absorption and metabolism of a soluble flavonoid, αG-rutin,in portal cannulated rats

A highly soluble quercetin glycoside, αG-rutin, is a glucose adduct of insoluble rutin, and intestinal absorption and metabolism of αG-rutin has not been known. We investigated the intestinal absorption and metabolism of αG-rutin by using portal and duodenal cannulated rats and the isolated rat intestinal mucosa. After a duodenal instillation of αG-rutin (150 μmol), intact αG-rutin, rutin and quercetin were appeared in the portal blood and these concentrations were similarly increased at 15 min. Portal quercetin reached a peak value at 60 min, and the value was higher than those of αG-rutin and rutin at that time. Quercetin-conjugates were also increased 30 min after the instillation. The remaining of αG-rutin metabolites, mainly rutin, in the intestine were 58% of instilled αG-rutin after 150 min. In the experiment by using the isolated mucosa of the jejunum, ileum and cecum, αG-rutin and rutin, but not quercetin, appeared in the serosal sides of all segments, and they were increased linearly from 10 to 100 mmol/l of mucosal αG-rutin. We also showed portal injected αG-rutin was very rapidly cleared from the blood, and appeared a large amount of conjugates. In conclusion, a soluble flavonoid-glycoside, αG-rutin, was absorbed as glycosides into the portal blood. A part of αG-rutin was hydrolyzed to rutin, but not to aglycone, through the intestine.     

Primary structure of rabbit lens α-crystallins

The primary structure and posttranslational modifications of rabbit lens α-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the αA- and αB-crystallins. The previously determined primary structure of αA-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit αB-crystallin was determined to be the same as the bovine αB-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit αB-crystallin showed evidence of phosphorylation at the same three sites as bovine αB-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.     

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.     

Studies on α-amylase production by Bacillus licheniformis MIR-61

An acid α-amylase hyperproducing strain, designated as MIR-61, was isolated in a screening procedure from South American soil samples. MIR-61, a 60°C thermoresistant strain, was identified using 98 biochemical and morphological tests and characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of B. licheniformis MIR-61 showed extracellular α-amylase and α-glucosidase activities during the exponential growth phase. The production of α-amylase was studied at free and constant pH values at 37 and 45°C. Maximum α-amylase activity (4,767 kU/dm³ in a liquid medium) was detected at 45°C at a constant pH (7.0) in the late exponential phase. The α-amylase production by B. licheniformis MIR-61 is 10 to 300 times higher than the enzyme production reported in strains of the same species. Optimum α-amylase activity was found at 50 to 67°C in an acid pH range from 5.5 to 6.0. These properties would allow its use in starch industry processes.     

α-Tocopheryl phosphate: uptake, hydrolysis, and antioxidant action in cultured cells and mouse

α-Tocopheryl phosphate (α-TP), a water-soluble analogue of α-tocopherol, is found in humans, animals, and plants. α-TP is resistant to both acid and alkaline hydrolysis and may exert its own function in this form in vivo. In this study, the uptake, hydrolysis, and antioxidant action of α-TP were measured using α-TP with a deuterated methyl group, CD(3), at position 5 of the chroman ring (α-TP(CD3)). The hydrolysis of α-TP(CD3) was followed by measuring α-tocopherol containing the CD(3) group, α-T(CD3), in comparison to unlabeled α-tocopherol, α-T(CH3). α-TP(CD3) was incubated with cultured cells, and the intracellular α-T(CD3) formed was measured with HPLC-ECD and GC-MS. α-TP(CD3) was also administered to mice for 4 weeks by mixing in the diet, and α-T(CD3) was measured in plasma, liver, brain, heart, and testis to compare with endogenous unlabeled α-T(CH3). It was found that α-TP(CD3) was taken in and hydrolyzed readily to α-T(CD3) in cultured cells and in mice. The hydrolysis of α-TP(CD3) in cell culture medium was not observed. α-TP protected primary cortical neuronal cells from glutamate-induced cytotoxicity, and α-TP given to mice reduced the levels of lipid peroxidation products in plasma and liver. These results suggest that α-TP is readily hydrolyzed in vivo to α-T, which acts as an antioxidant, and that α-TP may be used as a water-soluble α-T precursor in intravenous fluids, in eye drops, or as a dietary supplement.     

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.     

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.     

Yolk sac: site of developmental microheterogeneity of mouse alpha-fetoprotein.

α-Fetoprotein was observed to be synthesized in mouse fetal liver and yolk sac by incorporation of radioactive leucine into appropriate tissue cultures. Cultured fetal liver during early (Day 13.5) and late (Day 16.5) development secreted predominantly the maximally sialylated Fp5. In contrast, the yolk sac secreted a developmentally changing array of α-fetoprotein: Day 11.5 yolk sac secreted predominantly the unsialylated Fp1, at Day 13.5, the yolk sac secreted all five electrophoretic forms of α-fetoprotein, and by Day 16.5, only Fp5 was predominantly secreted, as in the fetal liver. To ascertain whether the ³H-labeled proteins that appeared in the regions of α-fetoprotein on polyacrylamide gels represented α-fetoprotein, immunoprecipitations with anti-α-fetoprotein were carried out. After the immunoprecipitations, radioactivity in the regions of marker α-fetoprotein on polyacrylamide gels was decreased to background levels. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates was performed, radioactivity peaks comigrated with marker α-fetoprotein. The undersialylated α-fetoprotein forms do not appear to arise by loss of sialic acid following secretion as determined by mixing experiments of yolk sac and fetal liver in culture. The contribution of α-fetoprotein synthesized and secreted by fetal liver and yolk sac at Days 13.5 and 16.5 of development was compared. Day 13.5 yolk sac incorporated 6.7 times as much radioactivity into secreted α-fetoprotein as did fetal liver at this time. These results suggest that during early development, the yolk sac is primarily responsible for the synthesis and secretion of the undersialylated forms of α-fetoprotein. In addition to the microheterogeneity of α-fetoprotein attributed to the number of sialic acid residues attached to the glycoprotein, there appeared to be other changes in α-fetoprotein: Fp5 synthesized from fetal liver migrated slightly faster on polyacrylamide gels than that from yolk sac.     

Gas chromatographic–mass spectrometric screening for organic acidemias using dried urine filter paper: determination of α-ketoacids

There are several organic acid disorders that require information on α-ketoacids, such as maple syrup urine disease or α-ketoadipic acidemia. The recovery, stability and diagnostic availability of α-ketoacids in dried urine filter paper analyzed by GC–MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of α-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain α-ketoacids, α-ketoadipate and α-ketoglutarate was stable for at least 28 days, although some α-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.     

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.     

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.     

Immunohistochemical analysis of two stem cell markers of α-smooth muscle actin and STRO-1 during wound healing of human dental pulp

Recent studies have employed two markers, alpha-smooth muscle actin (α-SMA) and STRO-1, to detect cells with mesenchymal stem cell properties in dental pulp. The present study aimed to explore the expression profile of α-SMA and STRO-1 in intact dental pulp as well as during wound healing in adult dental pulp tissue. Healthy pulps were mechanically exposed and capped with the clinically used materials MTA (ProRoot White MTA) or Ca(OH)(2) to induce a mineralized barrier at the exposed surface. After 7-42?days, the teeth were extracted and processed for immunohistochemical analysis using antibodies against α-SMA, STRO-1 and nestin (a neurogenic cytoskeletal protein expressed in odontoblasts). In normal pulp, α-SMA was detected in vascular smooth muscle cells and pericytes. Double immunofluorescent staining with STRO-1 and α-SMA showed that STRO-1 was localized in vascular smooth muscle cells, pericytes and endothelial cells, in addition to nerve fibers. During the process of dental pulp healing, numerous α-SMA-positive cells emerged at the wound margin at 14?days, and the initially formed mineralized barrier was lined with α-SMA-positive cells similar in appearance to reparative odontoblasts, some of which co-expressed nestin. STRO-1 was abundant in nerve fibers. In the advanced stage of mineralized barrier formation at 42?days, cells lining the barrier were stained with nestin, and no staining of α-SMA was detected in those cells. These observations indicate that α-SMA-positive cells temporarily appear along the wound margin during the earlier phase of mineralized barrier formation and STRO-1 is confined in vascular and neuronal elements.     

Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.     

Identification of glycated and acetylated lysine residues in human α2-antiplasmin

BackgroundThe post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.AimWe sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.MethodsWe analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.ResultsEleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.ConclusionHuman α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.     

Oxidation of O-Alkyl-O-(subst. phenyl) phenylphosphonothioates

It has been found that exocellular α-amylase could be fixed to mycelia of Aspergillus oryzae at an acidic pH region and fixed α-amylase was released reversely at an alkaline pH region. The fixation has more remarkably been observed in mycelia obtained from a phosphate deficient medium where endocellular accumulation of a-amylase occurs more easily than in an ordinary mycelia which secrete a large amount of α-amylase into medium. Bound form of α-amylase was more resistant to low pH and less active than the free form. The results appear to support the previous suggestion that a large quantity of endocellular α-amylase might be located on mycelial surface of the mold.     

Up-regulated α-actin expression is associated with cell adhesion ability in 3-D cultured myocytes subjected to mechanical stimulation

This study was aimed to investigate the alteration of α-actin in three-dimensionally (3-D) cultured myocytes under cyclic tensile stress loading. Myocytes were collected from neonatal SD rat’s lateral pterygoid muscle for primary cell culture. The third-passage cells were implanted and 3-D cultured in poly lactic-co-glycolic acid (PLGA) scaffold, and then subjected to cyclic tensile stress (0.5 Hz, 2,000 μstrain) for 0, 2, 4, 8, 12, and 24 h through a four-point bending strain system. The α-actin mRNA was investigated by semi-quantitative RT–PCR. The α-actin protein expression was examined by immunofluorescent cytochemistry, laser confocal scanning microscopy (LCSM), and image analysis technology. The dynamic adhesion of myocytes to PLGA scaffolds was investigated by fluorescence microscope and the viability of the myocytes was measured by MTT assay. After mechanical loading, the α-actin mRNA increased at 2 h and then declined. The α-actin protein expression kept increased until peaked at 12 h, but declined at 24 h. The time course changing of α-actin protein expression parallelled with that of cell adhesion ability. It is concluded that α-actin expression is probably associated with cell adhesion ability in myocytes subjected to mechanical stimulation.     

猪血浆α_2巨球蛋白部分性质的研究

对猪血浆α２巨球蛋白性质研究表明其大部分理化性质与人α２巨球蛋白十分相似。而且猪α２巨球蛋白也存在有活性的天然的Ｓｌｏｗ－Ｆｏｒｍ和构象发生变化并不可逆失活的Ｆａｓｔ－Ｆｏｒｍ。猪α２巨球蛋白经测定其ｐＩ为４．５５,５．１。紫外吸收光谱在２７６ｍｍ处有最大吸收,Ｅ＝９．１４。其糖含量为９．３％。氨其酸组成分析表明酸性氨基酸含量较高。圆二色谱分析说明分子中含有较多的β折叠。它还具有连接锌离子的性质。