Propafenone for the prevention of atrial tachyarrhythmias after cardiac surgery: a randomized, double-blind placebo-controlled trial.

We studied the efficacy of propafenone in preventing atrial tachyarrhythmias after cardiac surgery, and the possible relationships between CYP2D6 polymorphism and the efficacy, pharmacokinetics, and tolerability of propafenone. One hundred and sixty patients were randomized (double blind) to receive propafenone (n= 78) or placebo (n= 82) for 1 week after cardiac surgery. The patients who were assigned to the propafenone group received 1 mg/kg infused in 1 h, followed by a continuous infusion at a rate of 4 mg/kg/24 h until the following morning, and subsequently 450 mg/day orally until the sixth postoperative day. Thirty-seven patients completed the trial in the propafenone group and 45 in the placebo group. The frequency of occurrence of atrial tachyarrhythmia was lower in the propafenone group than in the placebo group (29.7% vs. 53.3%, P< 0.05; relative risk, 0.56). Plasma propafenone concentrations were markedly influenced by CYP2D6 genotype-derived phenotype.

Effect of acute hyperhomocysteinemia on methylation potential of erythrocytes and on DNA methylation of lymphocytes in healthy male volunteers.

Homocysteine is a precursor of S-adenosylmethionine (AdoMet) and a metabolite of S-adenosylhomocysteine (AdoHcy). The ratio of AdoMet to AdoHcy, defined as the methylation potential (MP), indicates the flow of methyl groups within the cells. Chronic elevations of total homocysteine (tHcy) in plasma correlate with increased AdoHcy concentrations, decreased MP, and impaired DNA methylation. However, the influence of acute hyperhomocysteinemia on MP is unknown. We induced acute hyperhomocysteinemia in 14 healthy volunteers by oral administration of l-homocysteine (65.1 micromol/kg body wt) in an open, randomized, placebo-controlled two-period crossover study. The kinetics of tHcy in blood and urine, MP in blood, and global DNA methylation in lymphocytes were studied systematically during 48 h. Plasma tHcy concentrations reached a peak at 34 +/- 11 min after an oral load with l-homocysteine and decreased with a half-life of 257 +/- 41 min (means +/- SD). Only 2.3% of the homocysteine dose were recovered in urine. AdoHcy concentrations and MP in whole blood and erythrocytes were not affected by the oral homocysteine load. Furthermore, global DNA methylation in lymphocytes did not change under these conditions. We found no difference between the genotypes of 5,10-methylenetetrahydrofolate reductase in response to the homocysteine load. However, AdoMet content in erythrocytes was significantly higher in the C677T carriers (CT; n = 7) compared with the CC genotype (n = 7). Although chronic elevation of tHcy has been shown to affect MP and DNA methylation, acute elevation of plasma tHcy above 20 micromol/l for 8 h is not sufficient to change MP and to induce DNA hypomethylation in lymphocytes.

Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase.

BACKGROUND: Adenosine has been suggested to play an important role in the regulation of renal function. We developed a simple and sensitive binding assay for the detection of adenosine based on the displacement of [(3)H]adenosine from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. METHODS: SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive. This reduced enzyme retains its ability to bind adenosine with high affinity. To determine adenosine in urine or tissues, samples must be deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchloric acid). RESULTS: The reduced SAH hydrolase bound adenosine with a dissociation constant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher concentrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptable correlation with those by HPLC. CONCLUSIONS: The highly sensitive adenosine-binding protein assay is a simple test that allows detection of adenosine in samples with small volumes without purification, and is in this respect superior to HPLC.

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography.

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N6-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25-1.0 nmol/g wet weight for renal tissue and 0.25-0.5 microM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1 +/- 2.6% and 102.5 +/- 4.0% for renal tissue and urine, respectively (mean +/- S.D., n = 3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64 +/- 2.2, 0.67 +/- 0.18 and 46.2 +/- 1.9 nmol/g wet weight, respectively (mean +/- S.D., n = 6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low microM range and are negatively correlated with urine flow-rate.

ATP-Sensitive K+ channel modulator binding to sulfonylurea receptors SUR2A and SUR2B: opposite effects of MgADP.

KATP channels are heteromeric complexes of inwardly rectifying K+ channel subunits and sulfonylurea receptors (SURs). SUR2A and SUR2B, which differ within the carboxyl terminal exon 38, are characteristic for the cardiac and smooth muscle type channels, respectively. Here we compare binding of the tritiated KATP channel opener, [3H]P1075, to membranes from human embryonic kidney (HEK) cells transfected with murine SUR2A and 2B at 37 degrees C. Binding to both SURs required addition of Mg2+ and ATP in the low micromolar range. In the presence of MgATP, micromolar concentrations of MgADP, formed by the ATPase activity of the membrane preparation, increased binding to SUR2A but inhibited binding to SUR2B. Decreasing temperatures strongly reduced [3H]P1075 binding to SUR2A, whereas binding to SUR2B was increased in a bell-shaped manner. Kinetic experiments revealed a faster dissociation of the [3H]P1075-SUR2A complex, whereas the association rate constants for [3H]P1075 binding to SUR2A and 2B were similar. Openers inhibited [3H]P1075 binding to SUR2A with potencies approximately 4 times lower than to SUR2B; in contrast, glibenclamide inhibited [3H]P1075 binding to SUR2A approximately 8 times more potently than to SUR2B. The data suggest that SUR2A and 2B represent the opener receptors of cardiac and vascular smooth muscle KATP channels, respectively, and show that MgADP is an important modulator of opener binding to SUR. The different carboxyl termini of SUR2A and 2B lead to differences in the MgADP dependence and the thermodynamics of [3H]P1075 binding, as well as in the affinities for openers and glibenclamide, underlining the importance of this part of the molecule for KATP channel modulator binding.

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney.

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.

Evidence against a major role of adenosine in oxygen-dependent regulation of erythropoietin in rats.

This in vivo study investigated whether adenosine (ADO) plays a role in oxygen-dependent production of erythropoietin (EPO). Exposure of rats to 0.075% carbon monoxide (CO) for four hours was used as a stimulus for EPO production. To inhibit potential effects of ADO, rats were treated with the non-specific ADO antagonist theophylline, the selective ADO A1 receptor blockers DPCPX and KW-3902, the selective ADO A2 receptor blocker DMPX, and AOPCP, an inhibitor of 5'-ectonucleotidase, an ADO generating enzyme that is expressed on the surface of EPO producing cells. To stimulate ADO receptor activity, animals were treated with the selective ADO A1 and A2 receptor agonists CHA and CGS 21680, the ADO reuptake inhibitors dipyridamole and soluflazine and the ADO desaminase inhibitor EHNA. At doses known to interfere with ADO signal transmission in vivo, none of these substances either influenced EPO serum levels in normoxic rats or affected the approximately 30-fold rise in EPO serum levels and the increase in renal EPO mRNA after exposure to carbon monoxide. Continuous administration of theophylline to normoxic rats for seven days did not alter hematocrit, hemoglobin or EPO serum levels. Taken together, these experiments do not support the hypothesis that ADO plays an important role in the regulation of EPO production.

Erythropoietin production in healthy volunteers subjected to controlled haemorrhage: evidence against a major role for adenosine.

1. This study was carried out to assess the role of adenosine in the regulation of human erythropoietin (EPO) production. To this end we investigated in healthy volunteers whether the nonspecific adenosine antagonist theophylline increases and the adenosine uptake inhibitor dipyridamole decreases EPO production in response to an haemorrhage of 750 ml. 2. Healthy male nonsmokers received i.v. in a parallel, randomized, single-blind trial theophylline (loading dose 5 mg kg-1 over 20 min, followed by 0.5 mg kg-1 min-1), dipyridamole (0.21 mg kg-1 h-1) or placebo (0.9% NaCl) for 6 h following the phlebotomy. EPO concentrations were followed up to 72 h after phlebotomy. 3. Following blood loss EPO concentrations increased during all treatments. The AUCEPO (0,72 h) were not statistically significantly different (theophylline: 398 +/- 30, dipyridamole: 301 +/- 15, placebo: 332 +/- 57 [mu ml-1 h]). Creatinine clearance and urinary cAMP excretion were unaltered by any treatment. Urinary excretion of adenosine was significantly increased during infusion of dipyridamole. Plasma renin activity was significantly increased during theophylline infusion. 4. In our model of controlled, physiological stimulation of EPO production by haemorrhage, adenosine appears unlikely to play a major role as a mediator of renal EPO production.

Different pathomechanisms of altered biliary leukotriene C4 elimination in isolated perfused rat livers.

Hepatic retention of cysteinyl leukotrienes is a consequence of impaired bile secretion and may be involved in the pathogenesis of intrahepatic cholestasis. In order to assess the mechanisms of altered biliary leukotriene elimination, we studied the secretion and metabolic pattern of leukotriene C4 (LTC4) in bile early in the alterations of bile formation by xenobiotics. To this end, rats were pretreated with alpha-naphthylisothiocyanate (ANIT), ethionine (ETH), or estradiol valerate (EV) at doses which did not increase serum marker enzymes of cholestasis. Bile secretion was assessed in perfused livers isolated from the treated rats. In all models, the access of [14C]sucrose into bile was increased, indicating increased permeability of the bile tract. Biliary recovery of radioactivity infused as [3H]LTC4 was decreased by ANIT and ETH while 3H-efflux into the perfusate was increased concomitantly. The secretion rate of 3H-radioactivity into bile was correlated with that of [14C]taurocholate infused at the same time. After pretreatment with ANIT (but not in the other models) the venous efflux of [3H]LTC4-ANIT pretreatment was increased [14C]sucrose clearance into bile associated with greatly enhanced biliary access of [32P]phosphate. Thus, altered charge selectivity of the paracellular pathway appears to be a prerequisite for reflux of cholephilic anions. HPLC analysis of [3H]LTC4-derived radioactivity in bile revealed that in all models of altered bile secretion the relative amount of LTD4 in bile was elevated. These results demonstrate differential changes in hepatobiliary transport and metabolism of LTC4 in developing cholestasis. ANIT inhibits leukotriene secretion by increasing paracellular permeability with loss of charge selectivity. In contrast, ETH treatment inhibits transcellular transport while treatment with EV only results in enhanced LTC4 metabolism.

Some problems of cardiac energetics.

Special problems of the aerobic metabolism in the cardiac muscle cell as an energy producing and energy consuming system are discussed and demonstrated with some experimental results using superfused resting and working guinea-pig atria as an energetic model: 1. Influence on resting O2 uptake: a) Free fatty acids (FFA) increase the O2 uptake rate to approximately 20% compared with glucose oxidation. This can be explained as a compensating effect due to the 9.7% lower combustion value for 1 mol O2 of C16-FFA and the 10.7% lower P/O-ratio related to glucose oxidation. b) K+-depolarization increases the O2 uptake b.1. between 15 and 65 mmol/l KCl from 110 to 200% without activation of the actomyosin system. This effect is Ca++ dependent and is not observed in Ca++ free solution and can be inhibited completely by nifedipine. The enhanced O2 uptake rate due to K+-depolarization is not connected with an improved state of the energy quotient (ATP/ADP+AMP) indicating a lowering of the energy coupling. b.2. between 100 and 250 mmol/l KCl from 220 to approximately 350%, not influenced by nifedipine and connected with activation of the actomyosin system at low Na+ and/or high external Ca++ (contracture). 2. Stretching of resting atria up to 10 mN tension does not increase the O2 uptake rate. The 'Feng' effect could not be confirmed. 3. a) The 'Frank-Starling' effect can be observed between 2.5 and 10 mN preload with increase of contractile work/beat connected with an enhancement of O2 uptake rate to a lower percentage. At the maximum of the 'Frank-Starling' effect the highest efficiency of contractile work can be observed. With increasing beat rate this maximum is shifted to a lower preload. b) The auxotonic contractile work measured by a calibrated spring blade allows the calculation of the 'internal work' and the 'external contractile work'/beat in mm X mN. The total energy of the activated actomyosin system (total work) is stored by the displacement of the spring blade due to the constant of the spring blade, the preload (tension) and the afterload (force). The 'internal work' will be transformed into ATP dependent heat and force equal to the preload tension. The same ATP dependent energy from O2 uptake is transformed at the same beat rate and the same preload a) with the isometric type of auxotonic contraction into high force and very low internal and low external work.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of various precursors on the concentration of energy-rich phosphates and pyridine nucleotides in cardiac tissue and its possible meaning for anoxic survival.

A special "anoxia test" was developed with isolated guinea pig atria to test influences on the anoxic energy balance. Adenine, ribose, nicotinic acid or nicotinamide added as precursors to nutrition solutions inhibit the loss of cardiac adenine and pyridine nucleotides during anoxia and improve the energy balance under aerobic and anaerobic conditions in the myocardium.

Synthesis and degradation of NAD in guinea pig cardiac muscle: I. Dependence upon the extracellular concentration of nicotinamide and nicotinic acid.

The NAD concentration as well as the 14C-incorporation in NAD and the disappearance of 14C-NAD were studied in spontaneously beating atria of guinea pigs at high and low concentrations of the precursors nicotinamide or nicotinic acid. Atria were incubated in Krebs-Henseleit solution containing 15 mM glucose and the appropriate precursors at 30 degrees C. The control NAD concentration (33 nMol/100 mg w.w.) remained unchanged during a 24-h-incubation time. -20 mM 14C-nicotinamide increased the total NAD about three-fold (90 nMol/100 mg w.w.) after an incubation period of 24 h, with positive effects on the performance. The incorporation rate in 14C-NAD was calculated to be 43.7 nMol/100 mg w.w. . 24 h. The ADPR moiety for the NAD synthesis stemmed from an endogenous pool. Between 5 and 20 mM nicotinamide the increase in the NAD concentration followed an apparent Michaelis-Menten kinetics with a Km of 6.1 mM nicotinamide and a Vmax of 70.92 nMol NAD/100 mg w.w. . 24 h. This can be explained as a new synthesis of NAD by a high concentration of nictoinamide and also by a decreased degradation of NAD, due to inhibition of the glycohydrolase by the high concentration of nicotinamide. The ratio of incorporation and disappearance of 14C-NAD during the 8th and 16th h incubation period was 2:1. After pre-incubation with 20 mM nicotinamide for an 8-h period the NAD concentration decreased to normal values after incubation for 8 h in a nicotinamide free medium. -20 mM 14C-nicotinic acid did not change the total NAD level and no significant incorporation in 14C-NAD could be detected, whereas negative effects on the performance occurred. -10 muM 14C-nicotinamide showed a slight increase in the total NAD concentration (39.7 nMol/100 mg w.w.) and in the 14C-incorporation (4.8 nMol/100 mg w.w.) within 24 h. -10 muM 14C-nicotinic acid seemed here to be the better precursor in this concentration. The NAD concentration increased to 49.8 nMol/100 mg w.w. after a 16 h incubation period and the incorporation in 14C-NAD was 12.1 nMol/100 mg w.w. after an incubation time of 24 h. As consequences of the observed different influences of each precursor on NAD turnover and NAD concentration the pathways of NAD synthesis and degradation must be studied. The importance of an increased NAD level for the energy metabolism of the cardiac muscle under aerobic and anaerobic conditions is discussed.