Modulation of cross-bridge interaction by 2,3-butanedione monoxime in human ventricular myocardium

OBJECTIVES: 2,3-butanedione monoxime (BDM) has been suggested as an additive to cardioplegic solutions, because it may reduce the energy cost associated with force production in heart muscle. METHODS: The present study investigated the effect of BDM (10 mM) on developed tension (DT), Ca(2+)-dependent myosin ATPase activity (MYO) and tension cost (myosin ATPase activity/ tension ratio), characterizing the cross-bridge detachment rate, of skinned fiber preparations (1% Triton X, 20 h, 4 degrees C) of human left ventricular failing myocardium (dilative cardiomyopathy, heart transplants, n=6) at increasing concentrations of Ca2+ (0.01-32 microM). RESULTS: BDM decreased Ca2+ sensitivity of DT [EC50 Ca2+, control: 1.3+/-0.2 microM, + BDM (10 mM): 4.5+/-0.3 microM] and MYO [EC50 Ca2+, control: 0.9+/-0.2 microM, + BDM (10 mM): 3.1+/-0.3 mM]. In addition, BDM reduced maximal DT [control: 26.0+/-1.9 mN/mm2, + BDM (10 mM): 8.1+/-1.1 mN/mm2] and MYO [control: 124+/-21 RM ADP/s, + BDM (10 mM): 62 9 microM ADP/s]. However, the influence of BDM on maximal DT (-69%) was more pronounced than on maximal MYO (-50%). The myosin ATPase activity/tension relation was significantly higher in the presence of BDM. CONCLUSIONS: BDM exerts negative inotropic activity by reducing the number of force-generating cross-bridges, possibly by increasing the cross-bridge detachment rate as well as by reducing force generation per cross-bridge in human myocardium. As BDM reduces force generation more than ATPase activity, BDM may not necessarily reduce energy demand in human myocardium.     

Characteristic dysfunction of stunned myocardium induced by 2,3-butanedione monoxime without ischaemia

1. In the present study, we tested the hypothesis that, even in the absence of prior ischaemia, 2,3-butanedione monoxime (BDM), an inhibitor of contraction at the actin-myosin level, could produce the postischaemic dysfunction characteristic of stunned myocardium. 2,3-Butanedione monoxime was injected directly into the left anterior descending coronary artery (LAD) before and again after myocardial stunning produced by 15 min occlusion of the LAD followed by 30 min reperfusion. 2. Regional myocardial force, segment shortening and regional work were measured in both the LAD-perfused area and the area perfused by the circumflex coronary artery, which served as a control area. Regional dysfunction produced by BDM injection or ischaemia-reperfusion was assessed quantitatively by five parameters: end-diastolic length (EDL), shortening onset delay (delay), systolic bulge (bulge), end-shortening time delay (EST) and tail work ratio (TWR). 3. It was found that injection of BDM into the LAD caused dyskinesis similar to that caused by occlusion-reperfusion. Both displayed elevated EDL and marked increases in delay, bulge, EST and TWR; these parameters were significantly higher in the dyskinesis caused by BDM injection. Despite dysfunctional fibre shortening, intracoronary BDM injection did not reduce regional force. 4. Thus, BDM can elicit changes similar to those characteristic of postischaemic dysfunction. Because contractility was not impaired, dysfunction was apparently caused by disrupting the association between contractile force and muscle motion.     

Block of ATP-sensitive K+ channels in isolated mouse pancreatic beta-cells by 2,3-butanedione monoxime.

1. The patch-clamp technique has been used to examine the action of the chemical phosphatase 2,3-butanedione monoxime (BDM) on ATP-sensitive K+ channels (KATP-channels) from mouse isolated pancreatic beta-cells in the absence of ATP and Mg2+. 2. BDM reversibly inhibited whole-cell KATP-currents with a concentration for half maximal inhibition (K(i)) of 15 +/- 1 mM and a Hill coefficient (n) of 2.5 +/- 0.2 (n = 4). 3. In outside-out patches, external BDM reversibly reduced the activity of single KATP-channels with an affinity similar to that observed in whole-cell recordings (K(i) = 11 +/- 3 mM, n = 2.0 +/- 0.3, n = 7). In inside-out patches, internally applied BDM also reversibly blocked the activity of KATP-channels (K(i) = 31 +/- 2 mM, n = 2.2 +/- 0.4, n = 8). In both excised patch configurations, BDM decreased the mean open life-time and the burst duration, thereby producing a decrease in the channel open probability. The drug had no effect on the short intraburst closed times. 4. BDM had no effect on the single-channel current amplitude. 5. The results suggest that BDM blocks the KATP-channel directly, by mechanisms independent of channel dephosphorylation.     

Effect of 2,3-butanedione monoxime on force of contraction and protein phosphorylation in bovine smooth muscle

The aim of the study was to investigate the effects of the putative protein phosphatase (PP) activator 2,3-butanedione monoxime (BDM) in vascular smooth muscle. BDM concentration-dependently increased PP activity in homogenates of bovine coronary arteries and led to dephosphorylation of various smooth muscle proteins in ³²P-labelled bovine aortic smooth muscle cells. In isolated bovine coronary artery rings (CARs) the effects of 10 mmol/l BDM on force of contraction (FOC) under conditions of depolarization by 75 mmol/l KCl and PP inhibition by 100 μmol/l cantharidin were investigated. At the end of contraction experiments CARs were freeze-clamped and myosin light chain (MLC₂₀) phosphorylation was determined by two-dimensional gel electrophoresis. Pretreatment of CARs with BDM reduced KCl-induced FOC to 42 ± 4% vs. 118 ± 1% (no BDM) and cantharidin-induced FOC to 102 ± 2% vs. 120 ± 7% (no BDM) compared to a former KCl contraction (= 100%). Moreover, BDM increased the amount of unphosphorylated MLC₂₀ up to 56 ± 2% vs. 36 ± 5% (no BDM) and 28 ± 2% vs. 21 ± 1% (no BDM), respectively, demonstrating the central role of MLC₂₀ phosphorylation in initiating smooth muscle contraction. In KCl precontracted CARs BDM decreased FOC to 47 ± 4% vs. 100 ± 1% (no BDM) but did not affect MLC₂₀ phosphorylation, suggesting an uncoupling of force maintenance and MLC₂₀ phosphorylation. In contrast, BDM neither affected FOC nor MLC₂₀ phosphorylation in CARs precontracted with cantharidin. These results strengthen the hypothesis that PP activation by BDM only occurs on the holoenzyme level, e.g. by affecting regulatory subunits. Received: 15 July 1998 / Accepted: 22 March 1999     

Effects of 2,3-butanedione monoxime on induction of action potential bursts in central snail neurons: direct and indirect modulations of ionic currents

The effects of 2,3-butanedione monoxime (BDM) on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of giant African snail (Achatina fulica Ferussac). The effect of okadaic acid on the neuron was also tested. The RP4 neuron showed a spontaneous firing of action potential. Okadaic acid (1 micromol/l) did not alter the frequency of spontaneous action potential while BDM (3 mmol/l) reversibly elicited bursts of potential (BoP) of the RP4 neuron. The BoP elicited by BDM (3 mmol/l) were reversed 20 min after incubation with diazoxide (500 micromol/l) while the BoP were not altered in preparations treated with okadaic acid and BDM. The BDM-elicited BoP were not inhibited after administration with (a) hexamethonium (100 micromol/l), (b) atropine (1 mmol/l), (c) d-tubocurarine (100 micromol/l), (d) prazosin (100 micromol/l), (e) propranolol (100 micromol/l), (f) calcium-free solution, (g) high K(+) (12 mmol/l) or (h) with high Mg(2+) (30 mmol/l) solutions. The BDM-elicited BoP were inhibited by pretreatment with KT-5720 (10 micromol/l) or H89 (10 micromol/l), the protein kinase A inhibitors. However, the BoP were not affected after application of chelerythrine (10 micromol/l) or Ro 31-8220 (10 micromol/l), the protein kinase C inhibitors. Voltage-clamped studies revealed that BDM elicited a negative slope resistance (NSR) at membrane potentials between -50 and -10 mV. The NSR was not detectable at the same membrane potential in control RP4 neuron. It is suggested that the BoP elicited by BDM were not due to (1) the synaptic effects of neurotransmitters; (2) the activation of cholinergic, adrenergic receptors, or (3) phosphatase activity of the neuron. The BDM-elicited BoP were dependent on the protein kinase A related cAMP in the neuron and the delayed outward K(+) current may contribute to the BDM-elicited BoP.     

Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.     

The influence of repeated oral administration of dichlorvos on circulating esterases in buffalo calves (Bubalus bubalis)

Alterations in circulating esterases induced by dichlorvos and their reversibility were investigated in male buffalo calves. Changes in blood glucose and total plasma proteins were also monitored. Animals received 4 or 8 mg dichlorvos/kg/day po for 28 consecutive d. Dichlorvos caused dose- and time- dependent significant (P less than 0.01) inactivation of blood esterases. Maximal inhibition of plasma cholinesterase (87-94%) and serum carboxylesterase (51-67%) was observed on the 28th day. An increase in blood glucose and total plasma proteins occurred during the dosing period; these parameters returned to control values in surviving animals within 7 d after the final dose. Activities of esterases returned to 45-70% of normal the 14th d after cessation of dichlorvos dosing.     

Effect of diacetylmonoxime on blood enzymes in fenitrothion-dosed buffalo calves

The effect of iv diacetylmonoxime (DAM) alone or in combination with atropine was determined on blood enzymatic activities in fenitrothion-exposed buffalo calves. Fenitrothion given po at 435 mg/kg bw produced pronounced inhibition of blood acetylcholinesterase (AChE) and elevation in serum aspartate and alanine aminotransferases, acid and alkaline phosphatases, and lactate dehydrogenase within 30 min. The administration of DAM alone or in combination with atropine significantly reactivated AChE activity. The administration of DAM + atropine decreased serum aspartate and alanine aminotransferase enzymes within 1-3 d. The reversal effect of DAM + atropine on serum phosphatases and lactate dehydrogenase was greater than that of DAM or atropine alone.     

Pharmacokinetics of aditoprim and trimethoprim in buffalo calves

Pharmacokinetic parameters of two antifolates, trimethoprim and aditoprim, were studied in buffalo calves. The elimination half-life of aditoprim (6.14 h) was nearly twice as long as that of trimethoprim (3.08 h) and compares well with values observed in heifers. This longer half-life of aditoprim is a result of its much larger distribution volume (four to five times larger) because the clearance of aditoprim was about twice as high as that of trimethoprim. The longer half-life of aditoprim is expected to give a longer duration of in vivo bacteriostatic activity than that of trimethoprim.     

Influence of experimentally induced fever on the disposition of cefpirome in buffalo calves

The influence of Escherichia coli endotoxin-induced fever on the disposition of cefpirome was investigated in five male buffalo calves following a single intravenous dose of 10mgkg(-1). Blood samples were collected from 1min to 24h of drug administration. The drug concentration in plasma was estimated by microbiological assay using E. coli as a test organism. The disposition of cefpirome followed two-compartment open model and the drug was detected above the minimum inhibitory concentration in plasma up to 12h. The Vd(area) and AUC were 0.75±0.01Lkg(-1) and 35.1±0.46μgml(-1)h, respectively. The elimination half-life of 1.81±0.009h and Cl(B) of 0.29±0.004Lkg(-1)h(-1) reflected rapid elimination and body clearance of cefpirome in febrile buffalo calves. Based on the results, a satisfactory dosage regimen of cefpirome in febrile buffalo calves was calculated to be 6mgkg(-1) to be repeated at 8h intervals.     

Influence of experimentally induced fever on the disposition of cefpirome in buffalo calves

The influence of Escherichia coli endotoxin-induced fever on the disposition of cefpirome was investigated in five male buffalo calves following a single intravenous dose of 10 mg kg⁻¹. Blood samples were collected from 1 min to 24 h of drug administration. The drug concentration in plasma was estimated by microbiological assay using E. coli as a test organism. The disposition of cefpirome followed two-compartment open model and the drug was detected above the minimum inhibitory concentration in plasma up to 12 h. The Vd_area and AUC were 0.75 ± 0.01 L kg⁻¹ and 35.1 ± 0.46 μg ml⁻¹ h, respectively. The elimination half-life of 1.81 ± 0.009 h and Cl_B of 0.29 ± 0.004 L kg⁻¹ h⁻¹ reflected rapid elimination and body clearance of cefpirome in febrile buffalo calves. Based on the results, a satisfactory dosage regimen of cefpirome in febrile buffalo calves was calculated to be 6 mg kg⁻¹ to be repeated at 8 h intervals.     

Selenium toxicokinetics after oral and intravenous administration in buffalo calves

The blood levels, toxicokinetics and urinary excretion of selenium were investigated in healthy male buffalo calves after single oral and intravenous administration of selenourea at the dose rate of 0.75mg/kg (providing 0.48mg/kg selenium). The concentration of selenium in blood and urine was estimated spectrophotometrically. Following administration of the drug, the blood selenium disposition patterns exhibited two distinct peaks. The toxicokinetic parameters of selenium were determined by employing non-compartmental analysis. The values of AUC, t(1/2elm), Cl(B) and Vd(SS) were 18.46μgml(-1)h, 10.33h, 20.04mlkg(-1)h(-1)and 0.3lkg(-1), respectively, after oral administration and 23.97μgml(-1)h, 7.12h, 20.53mlkg(-1)h(-1) and 0.2lkg(-1), respectively, following intravenous injection of selenourea. The value of MRT was higher after oral dosing. The bioavailability of selenium, following oral administration of selenourea was 77%. Approximately, 22% of the total intravenous dose and 5.9% of total oral dose of selenium was excreted in urine within 24h of administration of selenourea. The data on blood Se levels may be of help in diagnosing the impeding selenium toxicosis and thus preventing mortality due to selenium toxicity.     

Effects of butanedione monoxime on neuromuscular transmission.

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.     

The influence of single topical application of dichlorvos on blood esterases and toxicity in male calves.

The effect of dichlorvos on inducing changes in blood esterase activities and systemic toxicity was investigated following single topical applications of 1, 3 or 6% dichlorvos concentrations to male calves. Dichlorvos at 1% concentration did not produce any signs of toxicity, whereas 3 and 6% concentrations induced mild to severe toxicity characteristic of anticholinesterase poisoning. Dichlorvos at all concentrations significantly inhibited erythrocyte cholinesterase (25-75%), plasma cholinesterase (30-85%), and serum carboxylesterase (15-51%) activities in male calves. The dose-dependent inhibition was maximum 12 h after insecticide exposure. The extent of inactivation of blood esterases was not correlated with the severity of toxicity. Inhibition of blood cholinesterases by the 6% dichlorvos was still present 21 d after the dichlorvos exposure.     

The influence of propranolol on catecholamine-induced changes in carbohydrate metabolism in the rabbit.

In vitro evidence has indicated that isoproterenol (ISO) is more potent than epinephrine (EPI) in releasing glucose from rabbit liver slices and that propranolol (PROP) is a competitive antagonist of ISO. In contrast, dose-response data from fasted rabbits have shown that EPI is more potent than ISO in increasing plasma glucose levels and in lowering hepatic glycogen levels. Pretreatment with PROP abolished ISO-induced hyperglycemia and changes in liver and muscle glycogen levels whereas only the muscle glycogen depleting effect of EPI was altered significantly. These results suggest that factors other than stimulation of hepatic beta-receptors must be involved in EPI-stimulated depletion of liver glycogen and hyperglycemia in the intact rabbit.     

The influence of methysergide on 5-hydroxytryptamine-induced changes in regional distribution of blood flow.

Systemic and regional haemodynamic variables were measured at the baseline and after saline or 5-HT infusions (5 microgram kg-1 min-1, i.v.) or methysergide injections (0.5 mg kg-1, i.v.). Cardiac output and its complete distribution were measured by the radioactive microsphere (15 micrometer diam) technique. Although 5-HT did not change the systemic variables, methysergide caused a moderate increase in systolic and mean blood pressure and heart rate. 5-HT caused a substantial increase in gastric and a moderate increase in cerebral and myocardial blood flow at the expense of that to the lungs (arteriovenous shunt + bronchial flows), kidneys and skin. While methysergide was able to reduce the vascular responses to 5-HT in stomach, skin, kidneys, heart, lungs and brain, the drug itself, like 5-HT, decreased the number of microspheres reaching the lungs. Since a large number of 15 micron microspheres can escape through the arteriovenous anastomoses to lodge in the lungs it seems likely that both 5-HT and methysergide can reduce the 'non-nutrient' flow through these anastomoses.     

Effects of diacetyl monoxime on neuromuscular transmission

The action of diacetyl monoxime on neuromuscular transmission has been studied in frogs, chickens, and cats, and in isolated rat phrenic nerve-diaphragm preparations. In frogs and chickens the oxime caused a flaccid paralysis; in chickens there was sometimes opisthotonos. In the indirectly stimulated rat diaphragm, diacetyl monoxime decreased the height of a single twitch, but a tetanus was well sustained. In cats, the twitch height of the indirectly excited gastrocnemius-soleus muscle was reduced by diacetyl monoxime more than was that of the tibialis anterior muscle, but in both muscles a tetanus was well maintained. Diacetyl monoxime reduced the response to direct stimulation of both the rat diaphragm and cat muscles. Diacetyl monoxime injected intra-arterially in the cat elicited a transient hypertension and a gasp. Diacetyl monoxime did not reverse the neuromuscular block caused by anticholinesterases either in isolated rat phrenic nerve-diaphragm preparations or in cats.     

Effect of temperature of thawing and diluent on the post--thaw physiological changes of buffalo frozen semen.

The effect of thawing was studied in buffalo semen diluted in three diluents (Tris egg-yolk, Egg-yolk citrate and Citric Acid whey) at three temperatures (5 degrees C, 35 degrees C and 75 degrees C) on motility, eosin staining, morphological and acrosomal changes, hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase activities. The motility and lack of staining of sperm by eosin were maximum on thawing at 35 degrees C and in tris egg-yolk diluent followed by egg-yolk citrate and citric acid whey. Hyaluronidase, glutamic oxalacetic transaminase and glutamic pyruvic transaminase increased significantly in the extra-cellular fluid on thawing of semen diluted with all the three diluents. The buffalo semen diluted in tris egg-yolk and thawed at 35 degrees C for 30 seconds gave the best results.