The Effect of Amiloride and Sodium Chloride on Rat Renal and Hepatic 11β-Hydroxysteroid Dehydrogenase Activities

Abstract: The ability of glucocorticoid hormones to interact with glucocorticoid or mineralocorticoid receptors is modulated by 11β-hydroxysteroid dehydrogenases, interconverting active 11β-hydroxyglucocorticoids to inactive 11-ketones. This is, amongst others, important in maintaining a normal salt-water homeostasis. In this study, we determined the effect of treating rats for 4 days with the potassium sparing diuretic amiloride (5 mg/kg subcutaneously) or with 3 % NaCl in drinking water on renal and hepatic microsomal oxidative and reductive 11 β-hydroxysteroid dehydrogenase activities and immunoreactive 11 β-hydroxysteroid dehydrogenase 1 protein. Treatment with amiloride resulted in a 1.5-fold rise of microsomal corticosterone 11 β-oxidation rates in kidney (using NAD and NADP as cofactors) and in liver (for NADP only), but had no effect on microsomal 11-dehydrocorticosterone reduction. Renal 11 β-hydroxysteroid dehydrogenase 1 immunoreactive protein was increased 1.6-fold by amiloride. NaCl treatment appeared to have no effect.     

Reduction of Zearalenone to Zearalenol in Female Rat Liver by 3α-Hydroxysteroid Dehydrogenase

Abstract: The distribution of the zearalenone reducing activity was investigated in liver fractions obtained by differential centrifugation of liver homogenate from adult female Sprague Dawley rats. The zearalenone reducing enzyme was identified as 3α-hydroxysteroid dehydrogenase. At least two multiple forms occur of the enzyme with different subcellular locations and pH-optima. The activity was localized in the microsomes with NADH as coenzyme and in both microsomes and cytosol with NADPH.     

A Novel 11β-Hydroxysteroid Dehydrogenase Inhibitor Contained in Saiboku-To,a Herbal Remedy for Steroid-dependent Bronchial Asthma

Abstract— To identify the inhibitor of prednisolone metabolism contained in Saiboku-To, we conducted in-vitro experiments of 11β-hydroxysteroid dehydrogenase (11β-HSD), using rat liver homogenate and Cortisol as a typical substrate. We studied the effects often herbal constituents on 11β-HSD. Five herbal extracts showed inhibitory activity with Glycyrrhiza glabra > Perillae frutescens< Zizyphus vulgaris > Magnolia officinalis > Scutellaria baicalensis. This suggests that unknown 11β-HSD inhibitors are contained in four herbs other than G. glabra which contains a known inhibitor, glycyrrhizin (and glycyrrhetinic acid). Seven chemical constituents which have been identified as the major urinary products of Saiboku-To in healthy and asthmatic subjects were studied; magnolol derived from M. officinalis showed the most potent inhibition of the enzyme (IC50, 1·8 × 10^?4 m ). Although this activity was less than that of glycyrrhizin, the inhibition mechanism (non-competitive) was different from a known competitive mechanism. These results suggest that magnolol might contribute to the inhibitory effects of Saiboku-To on prednisolone metabolism through inhibition of 11β-HSD.     

Analysis of γβ, βγ, γγ, ββ multiple turns in proteins

Abstract: The number of γ‐turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic γ‐turns and 57% inverse γ‐turns are associated as multiple turns with either another γ‐turn or a β‐turn. We refer to these as multiple turns of the (γβ)_1,2,3 or (βγ)_1,2,3 type, depending upon whether the γ‐turn is before or after the β‐turn along the protein chain, respectively. However, for multiple turns involving only γ‐turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) β‐turns. Fifty‐eight per cent β‐turns are associated as multiple turns with another β‐turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual γ‐ or β‐turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side‐chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins ( http://www.cdfd.org.in/dsmp.html ).     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

Synthesis of New 2-{[(Phenoxy or Phenyl)acetyl]amino}benzoic Acid Derivatives as 3α-Hydroxysteroid Dehydrogenase Inhibitors and Potential Antiinflammatory Agents

A number of 2-{[(phenoxy or phenyl)acetyl]amino}benzoic acid derivatives were prepared in about 50% yield from (phenoxy or phenyl)acetyl chloride and anthranilic acid derivatives. All the compounds were tested as in vitro inhibitors of 3α-hydroxysteroid dehydrogenase, since enzyme inhibition predicts potential antiinflammatory activity in vivo The most active compounds 3 1, m, s are about 3.5 times more active than acetylsalicylic acid (ASA). Activity is influenced by electronic as well as steric effects.     

Automated synthesis of 11C‐β‐hydroxybutyrate by enzymatic conversion of 11C‐acetoacetate using β‐hydroxybutyrate dehydrogenase

1‐[¹¹C]‐β‐hydroxybutyrate was produced by conversion from 1‐[¹¹C]‐acetoacetate using (D)‐β‐hydroxybutyrate dehydrogenase in the presence of nicotinamide adenine dinucleotide with purification by ion exchange column chromatography. Radiochemical yield at the end of the synthesis was 10% for a total synthesis time of 36 min. High‐performance liquid chromatography analysis showed ≤4% impurities, principally unconverted acetoacetate. Residual tetrahydrofuran (34±11 ppm) and ethanol (77±30 ppm) were well under the tolerable limits for human studies. Copyright © 2008 John Wiley & Sons, Ltd.     

Peripheral Metabolism of β-Carboline-Carboxylic Acid Esters

Abstract: Esters of β-carboline-3-carboxylic acid have recently been identified as potent inhibitors of brain benzodiazepine receptors in vitro. Ethyl β-carboline-3-carboxylate (β-CCE), however, is a rather weak inhibitor in vivo of benzodiazepine receptors in mice. The ED50-value was 91 mg/kg intraperitoneally 35 min. after administration (ED50 is that dose which inhibits by 50% the specific binding of ³H-flunitrazepam intravenously). ED50 for β-CCE was 2–20 fold lower in mice pretreated with organophosphorus esterase inhibitors, concomitantly with the observation of strong inhibition of liver and kidney hydrolyzing activity, using ³H-propyl β-carboline-3-carboxylate as substrate. The rat brain contains only approximately 0.1% of the hydrolyzing activity as compared to the liver. It is concluded that some esters of β-carboline-3-carboxylate exhibit only weak effects on benzodiazepine receptors in living animals due to hydrolysis outside the brain.     

Effects of Dietary β‐Cyclodextrin in Hypercholesterolaemic Rats

Abstract: β‐Cyclodextrin is a compound that forms inclusion complexes with a variety of molecules, specially bile acids and sterols. This study examines the effects of β‐cyclodextrin on cholesterol and bile acid metabolism in hypercholesterolaemic rats. Male Wistar rats were divided into 4 groups that received during 7 weeks: control diet, 2% cholesterol diet (A), A+2.5% β‐cyclodextrin (B) and A+5% β‐cyclodextrin (C). The cholesterol‐rich diet induced hepatomegaly and fatty liver and significantly reduced cholesterol, bile acid and phospholipid secretion. Addition of β‐cyclodextrin normalised biliary lipid secretion. Moreover, when compared to A, β‐cyclodextrin significantly lowered plasma phospholipid concentration (B: ?21%; C: ?29%) and the liver free/total cholesterol molar ratio (B: ?40%; C: ?38%), increased bile acid faecal output (B: +17%; C: +62%) and enhanced cholesterol 7α‐hydroxylase activity (B:+50%; C: +100%) and mRNA levels (B: +14%; C: +29%). 5% β‐cyclodextrin also reduced plasma triglycerides concentration (?38%). However, ALT and AST activities were significantly increased (B: +140% and +280%; C: +72% and +135%) and there was a high incidence of cell necrosis with portal inflammatory cell infiltration. Addition of β‐cyclodextrin to a cholesterol‐rich diet results in a triglyceride‐lowering action, enhancement of bile acid synthesis and excretion, and normalization of biliary lipid secretion, but produces a marked hepatotoxic effect.     

Effects of the Dopamine-β-hydroxylase Inhibitors FLA-57 and FLA-63 on Ethanol Metabolism and Aldehyde Dehydrogenase Activity in Rats

Abstract: In rats pretreated with the dopamine-β-hydroxylase (DBH)-inhibitors FLA-57 and FLA-63 (60 mg/kg, intraperitoneally, for 4 and 18 hrs), no effects on the blood acetaldehyde level after ethanol administration or on the activity of the low-K_m aldehyde dehydrogenase (ALDH) in the liver were found. FLA-63 but not FLA-57 decreased the rate of ethanol elimination. FLA-63 inhibited the low-K_m enzyme in vitro, but much less than the ALDH-inhibitors disulfiram and cyanamide. FLA-57 caused no inhibition in vitro. The results show that the previously observed suppression of ethanol intake in rats by FLA-57 and FLA-63 was not caused by an acetaldehyde-mediated aversion such as during the disulfiram-ethanol reaction.     

Aluminium β-Cyclodextrin Sulphate as a Stabilizer and Sustained-release Carrier for Basic Fibroblast Growth Factor

Abstract— The water-insoluble aluminium salt of β-cyclodextrin sulphate (Al · β-CyD-Sul) was used as a stabilizer and sustained-release carrier for recombinant human basic fibroblast growth factor (bFGF). An adsorbate of bFGF with Al · β-CyD-Sul was prepared by incubating the protein with a suspension of Al · β-CyD-Sul in water. The mitogenic activity of bFGF released from the adsorbate, as indicated by the proliferation of kidney cells of baby hamster (BHK-21), was almost comparable with that of the intact bFGF. Al-β-CyD-Sul significantly protected bFGF from proteolytic degradation by pepsin and α-chymotrypsin, compared with the water-soluble sodium salt. The in-vitro release of bFGF from the adsorbate was sustained in proportion to a rise in the ratio of Al · β-CyD-Sul to the protein in the adsorbate. Of the bFGF preparations evaluated, the adsorbate of bFGF with Al ·β-CyD-Sul, when given subcutaneously to the rat, showed the most prominent increase in the formation of granulation tissues, due to the stabilization and slow-release of the mitogen. The limited data presented here suggest that the adsorbate of bFGF with Al · β-CyD-Sul has a potent therapeutic efficacy for wound healing, and may be applicable to oral protein formulations for the treatment of intestinal mucosal erosions.     

Affinity Constants and β-Adrenoceptor Reserves for Isoprenaline on Cardiac Tissue from Normotensive and Hypertensive Rats

To determine whether there are differences in cardiac β-adrenoceptor responsiveness, isoprenaline affinity constants and fractional β-adrenoceptor occupancy—response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-β-(2-hydroxyl-3-α-naphthoxypropylamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible β-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD₂ values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline K_A values were 2–3 times 10^?6M, which is as expected for isoprenaline at β₁ or β₂-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline K_A values were 2–4 times 10^?8M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3–4% of the β-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25–35% and 55%, respectively, of the β-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller β-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline K_A values on the WKY right atrium. The isoprenaline K_A value on the SHR right atrium was 1–4 times 10^?8M. Because the isoprenaline K_A values for the left and right atria are markedly different from those previously reported for isoprenaline at β₁ or β₂-adrenoceptors, we suggest that atypical β-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower β-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.     

The Anaesthetic Potency of 3α-Hydroxy-5α-pregnan-20-one and 3α-Hydroxy-5β-pregnan-20-one Determined with an Intravenous EEG-Threshold Method in Male Rats

Abstract: Two progesterone metabolites 3α-hydroxy-5α-pregnan-20-one (5-α) and 3α-hydroxy-5β-pregnan-20-one (5-β) were investigated for anaesthetic potency in male rats with an EEG-threshold method. Dose rate curves were obtained by infusing 5-α and 5β intravenously with different rates until an EEG-criterion (a burst suppression of one sec. or more, the “silent second”) was seen in the EEG. The potency of the investigated drugs has been estimated by comparing threshold doses at optimal infusion rates. 5-α and 5-β were tested on both young (44 to 46 days) and adult (109 to 118 days) rats. The relation between age and the anaesthetic sensitivity of 5-β was tested by weekly threshold determinations. 5-α and 5-β infused separately with different infusion rates gave almost V-shaped dose rate curves. The optimal infusion rate was in all age groups 2 mg/kg/min. In young rats 5-α (6.7 mg/kg) was more potent than 5-β (8.9 mg/kg). In adult rats the sensitivity was increased but the relation in potency between 5-α (5.1 mg/kg) and 5-β (6.6 mg/kg) was unchanged. With 5-β the main change in this age-related increase in anaesthetic sensitivity was seen between 49 and 70 days of age. Both 5-α and 5-β exhibited an excitatory action seen as jerks during induction of the EEG-criterion.     

超声斑点追踪成像技术评价短期药物干预对高血压大鼠左心功能的影响

目的 应用超声斑点追踪成像技术(STI)观察短期药物干预对自发性高血压大鼠(SHR)左室节段心肌径向(RS)及圆周应变(CS)的变化,探寻较传统超声心动图更早评价左室收缩功能改变的方法 . 方法 以10只Wistar大鼠为正常对照组,将30只SHR随机分成对照组、硝苯地平组、三子养阴汤组各10只,分别测量左室EF,FS及左室短轴乳头肌水平RS和CS的收缩期峰值. 结果4组大鼠的EF、FS值无明显差异,模型对照组大鼠的PRS和PCS明显降低(P<0.05),与模型对照组比较,硝苯地平组PRS除了下壁与间隔外,其余4个节段明显升高(P<0.05),三子养阴汤组6个节段PCS降低,以前间隔、前壁、侧壁、后壁为著(P<0.05). 结论 STI能定量评价左室局部心肌的RS和CS,较传统超声更为敏感.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Metabolism of 4′-thio-β- -arabinofuranosylcytosine in CEM cells

Because of the excellent in vivo activity of 4′-thio-β- -arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of β- -arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5′-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5′-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5′-monophosphate of araC was a better substrate for deoxycytidine 5′-monophosphate deaminase than was the 5′-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.     

β-Adrenergic Responsiveness of the Gastrointestinal Tract in Diabetic Rats

Abstract: Recently, decreased gastrointestinal β-adrenergic responses in experimental diabetes have been demonstrated. Gastrointestinal responses to β-adrenoceptor agonists are impaired in both insulin-dependent and non-insulin-dependent diabetic rat. Insulin treatment improves the impaired gastrointestinal β-adrenergic responsiveness of diabetic rats. The improvement seen with insulin treatment on β-adrenergic responsiveness is closely related to protein biosynthesis. The decreased β-adrenergic responses in diabetic rat gastrointestinal tract seem to result from a decrease in the number of β-adrenoceptors. It is most likely that the decreased gastrointestinal β-adrenergic responsiveness is related to an impairment in the turnover of β-adrenoceptors as a consequence of diabetes and that insulin has a beneficial effect on the impaired receptor turnover.     

Selectivity of ABT-089 for α4β2* and α6β2* nicotinic acetylcholine receptors in brain

Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While α4β2 and α7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among α4β2*-nAChR, ABT-089 had partial agonist activity (7–23% of nicotine) and high selectivity for α4α5β2 nAChR as evidenced by loss of activity in thalamus of α5^−/− mice. ABT-089 stimulated [³H]-dopamine release (57%) exceeded the activity at α4β2* nAChR, that could be explained by the activity at α6β2* nAChR. The concentration–response relationship for ABT-089 stimulation of α6β2* nAChR was biphasic. EC₅₀ and efficacy values for ABT-089, respectively, were 28 μM and 98% at the less sensitive α6β2* nAChR and 0.11 μM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations ≤300 μM at α3β4-nAChR measured by [³H]-acetylcholine release from interpeduncular nucleus. Thus, ABT-089 is a β2* nAChR ligand with demonstrable agonist activity at α4β2* and α6β2* receptors. As one form of α6β2* nAChR is sensitive to sub-μM concentrations, we propose that this receptor in particular may contribute to the enhanced cognitive performance following low doses of ABT-089.     

Characterization of C-11 or I-123 Labelled β-CIT-FP and β-CIT-FE Metabolism Measured in Monkey and Human Plasma. Identification of Two Labelled Metabolites with HPLC

β-CIT-FP and β-CIT-FE which are novel fluoroalkyl derivatives of 2β-carbomethoxy-3β-(4-iodophenyl)tropane (β-CIT), have recently been labelled with¹¹C or¹²³I and used for imaging the dopamine transporter with positron emission tomography (PET) or single photon emission tomography (SPET). The metabolite pattern of these novel tracers was studied with HPLC in plasma in monkeys and in urine and plasma in humans. Four labelled metabolites were found of which two were identified, the¹²³I-labelled free acid and¹¹C- or¹²³I-labelled nor-β-CIT. The latter was detected only in small amounts (<4 per cent). Furthermore, a polar metabolite and an unknown lipophilic¹¹C- or¹²³I-labelled metabolite were found. The amount of this unidentified labelled metabolite of [¹¹C]β-CIT-FP or [¹¹C]β-CIT-FE in monkey plasma was 21±8 per cent and 16±9 per cent at 60 min after injection, respectively. In conclusion, four types of labelled metabolites were found after injection of¹¹C- or¹²³I-labelled β-CIT-FP and β-CIT-FE in monkeys and humans of which one may hamper the quantitation of the dopamine transporter by PET or SPET.     

Peracylated β-Cyclodextrins as Novel Sustained-release Carriers for a Water-soluble Drug,Molsidomine

Abstract— Peracylated β-cyclodextrins with different alkyl chains (acetyl-octanoyl) were prepared by acylating all hydroxyl groups of β-cyclodextrin (β-CyD), and their physical properties were evaluated. These hydrophobic β-CyDs decreased the release rate of molsidomine, a peripheral vasodilator, in proportion to the lengthening of alkyl chain and suppressed a peak plasma level of molsidomine following oral administration of peracylated β-CyD complexes to dogs. Among the peracylated β-CyDs tested, perbutanoyl-β-CyD maintained sufficient plasma drug levels for a long period of time, while other peracylated β-CyDs having shorter or longer chains were inappropriate to control the in-vivo release behaviour of molsidomine. The prominent retarding effect of perbutanoyl-β-CyD was ascribable to the appropriate mucoadhesive property and hydrophobicity, compared with other peracylated β-CyDs. The present results suggest that perbutanoyl-β-CyD is particularly useful in modifying the release rate of water-soluble drugs as a novel slow-release carrier.