Implications of QRS duration in dogs with pacing-induced heart failure

The objective of this study was to find out the implication of QRS duration in dogs with rapid pacing-induced heart failure. Sixteen Beagle dogs were implanted with transvenous cardiac pacemakers and underwent rapid right ventricular pacing for 3 weeks at 260 bpm to induce heart failure. Dogs were divided into two groups according to the QRS duration: 9 with normal QRS duration (<100 ms) and 7 with prolonged QRS duration (≥100 ms). Cardiac systolic function and size was analyzed by real time 3-dimensional echocardiography and left ventricular dyssynchrony was assessed by speckle tracking strain imaging. Congestive heart failure developed 3 weeks after rapid right ventricular pacing. Dogs with prolonged QRS duration showed more extensive radial strain and circumferential strain dyssynchrony than dogs with normal QRS duration. At the end of 4-week recovery, greater improvement of left ventricular ejection fraction and left ventricular end-systolic volume was detected in dogs with normal QRS duration. The findings suggested that left ventricular dyssynchrony, indicated by a prolonged QRS duration, predicted an unsatisfying recovery in dogs with rapid pacing-induced heart failure. QRS duration had the potential to be a prognostic indicator for dogs with heart failure.     

Tissue-specific regulation of Ca2+ channel protein expression by sex hormones

The L-type Ca²⁺ channel pore-forming α subunit, α_1C can be detected in brain and heart as two proteins with molecular masses of ∼240 kDa and ∼190 kDa known as α_1C-long and α_1C-short, respectively. In brain, the α_1C-short is thought to be the product of a ∼50 kDa C-terminus calpain-mediated proteolytic deletion. We now show that uterine smooth muscle also possesses α_1C-long and α_1C-short isoforms, and that the relative expression of these two forms is regulated by sex hormones in a tissue-specific manner. Protein expression of α_1C L-type Ca²⁺ channels was examined in uterine smooth muscle, brain and heart, comparing non-pregnant (NP) estrus vs. late-pregnant (21 days) rats. The two forms of α_1C were detected in all studied tissues. In late-pregnant uterus, α_1C-long doubled the expression of α_1C-short; in NP uterus the opposite occurred. However, these changes were restricted to the uterine muscle, with no changes in brain and heart. To investigate the mechanism of such regulation, ovariectomized rats were treated with sex hormones, progesterone (P4) and/or 17β-estradiol (estrogen, E2). P4 treatment, which yielded P4 plasma levels of 5±1 ng/ml and a high P4/E2 ratio (3±1.5×10³) similar to the ratio in late-pregnant uterus (1.5±0.3 ×10³), facilitated α_1C-long expression. In contrast, E2 or E2+P4 treatment that increased E2 plasma levels to 60±8 pg/ml and 75±24 pg/ml, produced low P4/E2 ratios of 0.03±0.006 and 0.2±0.1, respectively. These low P4/E2 ratios also found in NP rats at estrus (0.3±0.1) favored the expression of α_1C-short form in myometrium. Neither hormone treatment altered α_1C expression in brain or heart. Our results indicate that expression of α_1C isoforms depends on P4/E2 ratios. Plasma P4/E2 ratios <1×10³ favor the expression of the α_1C-short; whereas ratios >1×10³ facilitate the expression of the α_1C-long form. This regulation is tissue-specific for myometrium since it did not occur in heart and brain tissues.     

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50?kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO₂ hydration reaction: k_cat =?4.1?×?10⁵ s^?1 and k_cat/K_m of 3.6?×?10⁷ M^?1 × s^?1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a K_I of 380?nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO₂.     

Highly efficacious factor Xa inhibitors containing α-substituted phenylcycloalkyl P4 moieties

We previously disclosed a series of highly potent FXa inhibitors bearing α-substituted (CH₂NR¹R²) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the α-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K_i), human plasma anticoagulant activity (PT EC_2×) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC₅₀s = 29–81 nM).     

Cordil Reversibly Inhibits the Na,K-ATPase from Outside of the Cell Membrane. Role of K-dependent Dephosphorylation

Abstract

Cordil-LND796 is a new cardiotonic glycoside under development. In rat brain microsomes where three isoforms of the Na, K-ATPase with differential affinities for cardiac glycosides have been identified, Cordil had higher affinity for the α₃ (IC₅₀ = 0.02 μM) than for the α₂ (IC₅₀ = 0.6 μM) and the α₁ (IC₅₀ = 30 μM) isozymes. Cordil is potentially a selective inhibitor for both α₂ and α₃ Na, K-ATPase isoforms. Using inside out vesicles we have shown that Cordil binds to and inhibits Na, K-ATPase at an extracellular site. The dissociation kinetic rates (k_?1) from the ATPase and the phosphatase activity (K-dependent dephosphorylation) of the Na, K-ATPase were similar for Cordil. Despite these similarities to ouabain comparison of the kinetics of the Na, K-ATPase inhibition by ouabain and Cordil revealed marked differences in their association rates (k₊₁ = 0.7 1 mol¹ min^?1 and k₊₁ = 6 × 10_?3 1 mol_?1 min_?1 respectively) and their dissociation rates (k_?1 = 1.3 ± 0.2 × 10^?4 S^?1 and k_?1 = 69 ± 7 × 10^?4 s^?1 respectively). Both binding association and dissociation rates were enhanced for Cordil. These data are compatible with a stabilizing effect of Cordil on the E₂P conformational state of Na, K-ATPase.     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO₂ hydration reaction were measured. With a k_cat/K_m of 1.1?×?10⁸ M^?1 s^?1, and a k_cat of 1.3?×?10⁶ s^?1, clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K_Is in the range of 1.9–3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Pulse radiolytic study of α-tocopherol radical mechanisms in ethanolic solution

Pulse radiolytic studies of α-tocopherol (αTH) oxidation-reduction processes were carried out with low doses (5 Gy) of high-energy electrons in O₂₋, N₂₋, and air-saturated ethanolic solutions. Depending on the concentration of oxygen in solution, two different radicals, A· and B·, were observed. The first, A·, was obtained under N₂ and results from aTH reaction with solvated electron (k_aTH+csolv⁻ = 3.4 × 10⁸ mol⁻¹ liter s⁻¹) and with H₃C-ĊH-OH, (R·) (k_{aTH + R·} = 5 × 10⁵ mol⁻¹ liter s⁻¹). B·, observed under O₂, is produced by αTH reaction with RO₂ peroxyl radicals (k_aTH + RO₂^. = 9.5 × 10⁴ mol⁻¹ liter s⁻¹).     

The synthesis and hydrolysis of a series of deoxyfluoro-d-glucopyranosyl phosphates

The synthesis of all four deoxyfluoro-α-d-glucopyranosyl phosphates is described. Rate constants for their acid-catalyzed hydrolysis were determined, and fluorine substitution was shown to have a significant effect in lowering the rate, particularly when the substitution is adjacent to the anomeric center. Relative rate-constants measured in m HClO₄ at 25° are 60.30:1.00:7.05:3.97:16.5 for α-d-glucopyranosyl phosphate and the 2-, 3-, 4- and 6-deoxyfluoro derivatives, respectively. The hydrolysis of 2-deoxy-2-fluoro-α-d-glucopyranosyl phosphate was studied in more detail, and an activation entropy and enthalpy of 4.1 e.u. (m reactant) and 113.5 kJ.mol⁻¹, respectively, were determined for hydrolysis in m HClO₄ at 60° The pH dependence of its hydrolysis was investigated, and rate constants for hydrolysis of the monoanion (k_M = 1.88 × 10⁻⁶ s⁻¹) and neutral (k_N = 6.23 × 10⁻⁵ s⁻¹) species were thus extracted. Hydrolysis of the monoanion is not significantly affected by fluorine substitution, as expected. The ability or inability of several mechanistically distinct enzymes to utilize these fluorinated substrates is rationalized in the light of these findings.     

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H₂O = 7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k_s) for α-TocH in MeOH/H₂O solution with EYPC vesicles were apparently 3.45 × 10⁵ M⁻¹ s⁻¹, which was about 8 times higher than that (4.50 × 10⁴ M⁻¹ s⁻¹) in MeOH/H₂O solution without EYPC vesicles. The corrected k_s of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10 mM EYPC vesicles than in the bulk MeOH/H₂O solution, was 2.60 × 10³ M⁻¹ s⁻¹, which was one-seventeenth that in MeOH/H₂O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k_s of vitamin E in membranes increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H₂O solution among vitamin E analogues [k_s(vesicle)/k_s (MeOH/H₂O) = 7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k_s(micelle)/k_s(EtOH) = 100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH.     

Solution properties and chain flexibility of pullulan in aqueous solution

Molecular characteristics for pullulan, a polysaccharide produced by a fungus Aureobasidium pullulans, were measured by light scattering, viscometry, and gel-permeation chromatography. From the experimental data the Mark-Houwink-Sakurada viscosity equation in water at 25°C was determined for samples having the molecular weight M ranging from 48 × 10³ to 2.18 × 10⁶ g mol^?1 as [η] = (1.91 ± 0.02) × 10^?2M_w^0.67±0.01 (in cm³ g^?1); and as molecular weight decreased, the slope of the viscosity equation decreased, although the molecular weight values below 30 × 10³ g mol^?1 evaluated by gel-permeation chromatography were somewhat unreliable. The unperturbed dimensions 〈R²〉₀^1/2 of pullulan were estimated by determining the expansion factor α_s, from the theoretical combination of theories for the interpenetration function Ψ and those for α_s. The 〈R²〉₀/M value estimated from this procedure in 6.7 × 10^?17 cm² mol g^?1. We concluded that the polysaccharide chain that is linked by the α-1,6-glucosidic linkage behaves like a flexible chain in aqueous solution.     

Inhibition of NO production increases myocardial blood flow and oxygen consumption in congestive heart failure

Coronary blood flow (CBF) and myocardial oxygen consumption (MVO(2)) are reduced in dogs with pacing-induced congestive heart failure (CHF), which suggests that energy metabolism is downregulated. Because nitric oxide (NO) can inhibit mitochondrial respiration, we examined the effects of NO inhibition on CBF and MVO(2) in dogs with CHF. CBF and MVO(2) were measured at rest and during treadmill exercise in 10 dogs with CHF produced by rapid ventricular pacing before and after inhibition of NO production with N(G)-nitro-L-arginine (L-NNA, 10 mg/kg iv). The development of CHF was accompanied by decreases in aortic and left ventricular (LV) systolic pressure and an increase in LV end-diastolic pressure (25 +/- 2 mmHg). L-NNA increased MVO(2) at rest (from 3.07 +/- 0.61 to 4.15 +/- 0.80 ml/min) and during exercise; this was accompanied by an increase in CBF at rest (from 31 +/- 2 to 40 +/- 4 ml/min) and during exercise (both P < 0.05). Although L-NNA significantly increased LV systolic pressure, similar increases in pressure produced by phenylephrine did not increase MVO(2). The findings suggest that NO exerts tonic inhibition on respiration in the failing heart.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan from Ganoderma lucidum

Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and ¹³C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight M_w and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The M_w value (2.4×10⁴) of sulfated glucan S-GL-1 was much lower than that (44.5×10⁴) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C_∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10^-3M_w^1.06 (cm³ g^-1) and 16, respectively, in the M_w range from 1.1×10⁴ to 2.4×10⁴. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Biphasic nature in the autoxidation reaction of human oxyhemoglobin

In comparison with myoglobin molecule as a reference, we have studied the autoxidation rate of human oxyhemoglobin (HbO₂) as a function of its concentration in 0.1 M buffer at 35°C and in the presence of 1 mM EDTA. At pH 6.5, HbA showed a biphasic autoxidation reaction that can be described completely by a first-order rate equation containing two rate constants — k_f, for fast autoxidation of the α-chain, and k_s, for slow autoxidation of the β-chain, respectively. When tetrameric HbO₂ was dissociated into αβ-dimers by dilution, the value of k_f increased markedly to an extent comparable with the autoxidation rate of horse heart oxymyoglobin (MbO₂). The rate constant k_s, on the other hand, was found to remain at an almost constant value over the whole concentration range from 1.0 × 10⁻³ M to 3.2 × 10⁻⁶ M in heme. At pH 8.5 and pH 10.0, however, the autoxidation of HbO₂ was monophasic, and no enhancement in the rate was observed by diluting hemoglobin solutions. Taking into consideration the effects of 2,3-diphosphoglyceric acid and chloride anion on the autoxidation rate of HbO₂, we have characterized the differential susceptibility of the α- and β-chains to the autoxidation reaction in aqueous solution.     

Cloning,expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m of 3.5?×?10⁷?M^?1 ×s^?1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.     

Reversible H-Atom Abstraction from Alcohols by Thiyl Radicals: Determination of Absolute Rate Constants by Pulse Radiolysis

Hahn-Meitner-Institut Berlin, Bereich Strahlenchemie, Glienicker Str. 100, 1000 Berlin 39, Fed. Rep. Germany

Penicillamine thiyl radicals, PenS', are shown to abstract hydrogen atoms from 2-propanol and to establish an equilibrium

PenS' + (CH₁)₂ CHOH ? PenSH + (CH₃)₂ COH.

The rate constants for the forward and back reaction have been determined to (1.4 ± 0.3) × 10⁴ and (1.2 ± 0.3) × 10⁸ M^-1 s^-1, respectively, by means of pulse radiolysis. The data have been obtained from various independent methods which include direct measurements and competitive schemes involving irreversible interception of the alcohol radical by electron acceptors (e.g. CCl₄. PNAP) and/or the thiyl radical by antioxidants (e.g. α-tocopherol). The results demonstrate that the reaction of carbon-centered radicals with thiols, in radiation biology commonly known as “repair” reaction, may be reversed and thus imply the possibility of thiyl radical induced biological damage.