Single-Shot Diagnosis of Electron Energy Evolution via Streaked Betatron X Rays in a Curved Laser Wakefield Accelerator.

We report on an experimental observation of the streaking of betatron x rays in a curved laser wakefield accelerator. The streaking of the betatron x rays was realized by launching a laser pulse into a plasma with a transverse density gradient. By controlling the plasma density and the density gradient, we realized the steering of the laser driver, electron beam, and betatron x rays simultaneously. Moreover, we observed an energy-angle correlation of the streaked betatron x rays and utilized it in diagnosing the electron acceleration process in a single-shot mode. Our work could also find applications in advanced control of laser beam and particle propagation. More importantly, the angular streaked betatron x ray has an intrinsic spatiotemporal correlation, which makes it a promising tool for single-shot pump-probe applications.

Two-color high-harmonic generation from relativistic plasma mirrors.

High-intensity laser solid interactions are capable of generating attosecond light bursts via high-harmonic generation-most work focuses on single beam interactions. In this paper, we perform a numerical investigation on the role of wavelength and polarization in relativistic, high-harmonic generation from normal-incidence, two-beam interactions off plasma mirrors. We find that the two-beam harmonic-generation mechanism is a robust process described by a set of well-defined selection rules. We demonstrate that the emitted harmonics from normal-incidence interactions exhibit an intensity optimization when the incident fields are of equal intensity for two-color circularly polarized fields.

Homogeneous, Micron-Scale High-Energy-Density Matter Generated by Relativistic Laser-Solid Interactions.

Short-pulse, laser-solid interactions provide a unique platform for studying complex high-energy-density matter. We present the first demonstration of solid-density, micron-scale keV plasmas uniformly heated by a high-contrast, 400 nm wavelength laser at intensities up to 2×10^{21} W/cm^{2}. High-resolution spectral analysis of x-ray emission reveals uniform heating up to 3.0 keV over 1 µm depths. Particle-in-cell simulations indicate the production of a uniformly heated keV plasma to depths of 2 µm. The significant bulk heating and presence of highly ionized ions deep within the target are attributed to the few MeV hot electrons that become trapped and undergo refluxing within the target sheath fields. These conditions enabled the differentiation of atomic physics models of ionization potential depression in high-energy-density environments.

Millijoule few-cycle pulses from staged compression for strong and high field science.

Intense few-cycle laser pulses have a breadth of applications in high energy density science, including particle acceleration and x-ray generation. Multi-amplifier laser system pulses have durations of tens of femtoseconds or longer. To achieve high intensities at the single-cycle limit, a robust and efficient post-compression scheme is required. We demonstrate a staged compression technique using self-phase modulation in thin dielectric media, in which few-cycle pulses can be produced. The few-cycle pulse is then used to generate extreme ultravoilet light via high harmonic generation at strong field intensities and to generate MeV electron beams via laser solid interactions at relativistic intensities.

Polarization-Dependent Self-Injection by Above Threshold Ionization Heating in a Laser Wakefield Accelerator.

We report on the experimental observation of a decreased self-injection threshold by using laser pulses with circular polarization in laser wakefield acceleration experiments in a nonpreformed plasma, compared to the usually employed linear polarization. A significantly higher electron beam charge was also observed for circular polarization compared to linear polarization over a wide range of parameters. Theoretical analysis and quasi-3D particle-in-cell simulations reveal that the self-injection and hence the laser wakefield acceleration is polarization dependent and indicate a different injection mechanism for circularly polarized laser pulses, originating from larger momentum gain by electrons during above threshold ionization. This enables electrons to meet the trapping condition more easily, and the resulting higher plasma temperature was confirmed via spectroscopy of the XUV plasma emission.

Differentiating health statuses using positive health indicators in an occupational context.

OBJECTIVES: To identify different health statuses beyond the dichotomy of 'health' vs 'illness'. To achieve this, positive indicators based on current scientific definitions and concepts were constructed. STUDY DESIGN: Data were collected between 2008 and 2010 in a nationwide representative cross-sectional survey. METHODS: Data for approximately 11,800 wage or salary earners were analysed. Health statuses were modelled using hierarchical cluster analysis, and the relationships between clusters and working conditions were tested using discriminant analyses. RESULTS: According to response patterns in the various health dimensions, four health statuses were found: individuals who were holistically healthy; individuals with multiple health impairments; individuals with stagnating potential for development; and individuals with higher tendency to wear out. CONCLUSIONS: The use of positive health concepts enables better differentiation of health statuses. Under existing working conditions, it is possible to identify group-specific needs for the working population, and to derive appropriate measures as part of workplace health promotion.

Na(+)/H(+) exchange inhibition prevents endothelial dysfunction after I/R injury.

Whereas inhibition of the Na(+)/H(+) exchanger (NHE) has been demonstrated to reduce myocardial infarct size in response to ischemia-reperfusion injury, the ability of NHE inhibition to preserve endothelial cell function has not been examined. This study examined whether NHE inhibition could preserve endothelial cell function after 90 min of regional ischemia and 180 min of reperfusion and compared this inhibition with ischemic preconditioning (IPC). In a canine model either IPC, produced by one 5-min coronary artery occlusion (1 x 5'), or the specific NHE-1 inhibitor eniporide (EMD-96785, 3.0 mg/kg) was administered 15 min before a 90-min coronary artery occlusion followed by 3 h of reperfusion. Infarct size (IS) was determined by 2,3,5-triphenyl tetrazolium chloride staining and expressed as a percentage of the area-at-risk (IS/AAR). Endothelial cell function was assessed by measurement of coronary blood flow in response to intracoronary acetylcholine infusion at the end of reperfusion. Whereas neither control nor IPC-treated animals exhibited a significant reduction in IS/AAR or preservation of endothelial cell function, animals treated with the NHE inhibitor eniporide showed a marked reduction in IS/AAR and a significantly preserved endothelial cell function (P < 0.05). Thus NHE-1 inhibition is more efficacious than IPC at reducing IS/AAR and at preserving endothelial cell function in dogs.

Three thiadiazinone derivatives, EMD 60417, EMD 66430, and EMD 66398, with class III antiarrhythmic activity but different electrophysiologic profiles.

The thiadiazinone derivatives EMD 60417, EMD 66430, and EMD 66398 were developed as class III antiarrhythmic agents. Their chemical structure is closely related to that of their calcium-sensitizing congener [+]-EMD 60263, and EMD 66398 possesses the methylsulfonylaminobenzoyl moiety present in the prototypical IKr blocker E-4031. We compared the electrophysiologic effects of these compounds with standard drugs (almokalant, E-4031, quinidine) in cardiac myocytes from guinea-pig ventricle and human atrium by whole-cell patch-clamp technique. The test compounds' class III action, which is related to impairment of K+ channel function, was confirmed by action potential measurements. EMD 60417, EMD 66430, EMD 66398, and almokalant (1 microM each) reversibly prolonged the action potential duration in guinea-pig myocytes. In the same cells, the rapidly activating component IKr of the delayed rectifier K+ current, which has been defined by its sensitivity to E-4031, was reduced by EMD 60417, EMD 66430, EMD 66398, and almokalant. Inhibition of IKr was concentration-dependent as determined by attenuation of tail currents. The slowly activating component IKs of the delayed rectifier K+ current was not affected. The inward rectifier K+ current IK1 was not influenced at potentials close to the reversal potential. Transient and sustained outward K+ currents (Ito, Iso) measured in human atrial myocytes were not altered by any EMD compound. L-type Ca2+ current was hardly affected at concentrations of 1-10 microM, but sodium current was decreased. Action potential prolongation by EMD 60417, EMD 66430, and EMD 66398 is due to block of IKr. INa is inhibited at higher concentrations by EMD 66430 and EMD 60417. EMD 66398 is more potent and selective for IKr than EMD 60417 and EMD 66430, and thus resembles E-4031 in structure and function.

Structure of the C-domain of human cardiac troponin C in complex with the Ca2+ sensitizing drug EMD 57033.

Ca(2+) binding to cardiac troponin C (cTnC) triggers contraction in heart muscle. In heart failure, myofilaments response to Ca(2+) are often altered and compounds that sensitize the myofilaments to Ca(2+) possess therapeutic value in this syndrome. One of the most potent and selective Ca(2+) sensitizers is the thiadiazinone derivative EMD 57033, which increases myocardial contractile function both in vivo and in vitro and interacts with cTnC in vitro. We have determined the NMR structure of the 1:1 complex between Ca(2+)-saturated C-domain of human cTnC (cCTnC) and EMD 57033. Favorable hydrophobic interactions between the drug and the protein position EMD 57033 in the hydrophobic cleft of the protein. The drug molecule is orientated such that the chiral group of EMD 57033 fits deep in the hydrophobic pocket and makes several key contacts with the protein. This stereospecific interaction explains why the (-)-enantiomer of EMD 57033 is inactive. Titrations of the cCTnC.EMD 57033 complex with two regions of cardiac troponin I (cTnI(34-71) and cTnI(128-147)) reveal that the drug does not share a common binding epitope with cTnI(128-147) but is completely displaced by cTnI(34-71). These results have important implications for elucidating the mechanism of the Ca(2+) sensitizing effect of EMD 57033 in cardiac muscle contraction.

Pharmacokinetic/pharmacodynamic evaluation of the NHE inhibitor eniporide.

The aim of this study was to investigate the pharmacokinetics and pharmacodynamics of the new cardioprotective sodium/proton exchange (NHE-1) inhibitor eniporide in humans. Eniporide was administered intravenously to healthy volunteers in doses between 2.5 and 100 mg. Concentrations of parent drug and its metabolite were measured by HPLC, and the data were analyzed by noncompartmental and compartmental pharmacokinetic methods. Platelet-swelling time was determined in each subject as a biomarker to assess pharmacodynamic activity. Eniporide showed linear pharmacokinetics with an average half-life of approximately 2 hours. The mean total body clearance was 34.4 L/h. The mean volume of distribution (Vdss) was 77.5 L, and the mean residence time was 2.3 hours. An average of 43% of the dose was recovered unchanged from urine. A pharmacokinetic two-compartment model was found suitable to provide excellent curve fits of the measured plasma concentration profiles. Plasma concentrations of the major metabolite were lower than that of the parent drug. An average of 27% of the dose was found in urine as that metabolite. The effect on platelet swelling could be well characterized by a direct Emax model. The average concentration for half-maximum effect (IC50) was 12 ng/mL. Eniporide was found to have predictable linear pharmacokinetics in the investigated dose range. Platelet-swelling time was shown to be a reproducible individual biomarker for pharmacodynamic activity, with great potential for a surrogate that predicts clinical outcome, since this effect is mediated through the same mechanism of action (NHE-1 inhibition) as the desired cardioprotective activity. Pharmacokinetic/pharmacodynamic modeling allowed a first estimate of the degree of NHE inhibition in the investigated dose range.

Interaction of cardiac troponin C with Ca(2+) sensitizer EMD 57033 and cardiac troponin I inhibitory peptide.

The binding of Ca(2+) to cardiac troponin C (cTnC) triggers contraction in cardiac muscle. In diseased heart, the myocardium is often desensitized to Ca(2+), leading to weak cardiac contractility. Compounds that can sensitize cardiac muscle to Ca(2+) would have potential therapeutic value in treating heart failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca(2+) sensitizer', and cTnC is a potential target of the drug. In this work, we used 2D ¿(1)H, (15)N¿-HSQC NMR spectroscopy to monitor the binding of EMD 57033 to cTnC in the Ca(2+)-saturated state. By mapping the chemical shift changes to the structure of cTnC, EMD 57033 is found to bind to the C-domain of cTnC. To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTnC with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence and presence of EMD 57033, respectively. cTnC was also titrated with EMD 57033 in the presence of cIp. The results show that although both the drug and cIp interact with the C-domain of cTnC, they do not displace each other, suggesting noncompetitive binding sites for the two targets. Detailed chemical shift mapping of the binding sites reveals that the regions encompassing helix G-loop IV-helix H are more affected by EMD 57033, while residues located on helix E-loop III-helix F and the linker between sites III and IV are more affected by cIp. In both cases, the binding stoichiometry is 1:1. The binding affinities for the drug are 8.0 +/- 1.8 and 7.4 +/- 4.8 microM in the absence and presence of cIp, respectively, while those for the peptide are 78.2 +/- 10.3 and 99.2 +/- 30.0 microM in the absence and presence of EMD 57033, respectively. These findings suggest that EMD 57033 may exert its positive inotropic effect by not directly enhancing Ca(2+) binding to the Ca(2+) regulatory site of cTnC, but by binding to the structural domain of cTnC, modulating the interaction between cTnC and other thin filament proteins, and increasing the apparent Ca(2+) sensitivity of the contractile system.

Inhibitors of ischemic preconditioning do not attenuate Na+/H+ exchange inhibitor mediated cardioprotection.

Pharmacologic inhibition of the K(ATP) channel with sulfonylureas or the adenosine receptor with methylxanthines has been shown to attenuate ischemic preconditioning (IPC). Both classes of compounds are widely used clinically, and several reports have demonstrated adverse outcomes in patients taking sulfonylureas. Recently inhibition of the sodium/hydrogen exchanger isozyme-1 (NHE-1) has been shown to be equal to IPC at providing myocardial protection in dogs and may be an alternative to IPC in patients taking sulfonylureas or methylxanthines. However, no experiments have examined the pharmacologic overlap between IPC and NHE-1 inhibitor-mediated cardioprotection in dogs. With an in vivo canine infarct model in which the left anterior descending coronary artery was occluded for 60 min and reperfused for 3 h, neither the K(ATP) channel antagonist glibenclamide nor the adenosine-receptor antagonist PD 115199 attenuated NHE-1 inhibitor-mediated reduction in infarct size expressed as a percentage of the area at risk produced by EMD 85131 (Control, 24.2 +/- 3.6%; EMD 85131, 6.4 +/- 2.3%; PD 115199 + EMD 85131, 6.6 +/- 2.4%; glibenclamide + EMD 85131, 3.5 +/- 1.2%). NHE-1 inhibition and IPC do not overlap pharmacologically, and NHE-1 inhibition may be an alternative for cardioprotection in patients taking sulfonylureas or methylxanthines.

New drugs for the Na+/H+ exchanger. Influence of Na+ concentration and determination of inhibition constants with a microphysiometer.

The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensortrade mark microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na+/H+ antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10-138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a KM = 30 +/- 4 mm for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 values of the four compounds could be determined as 23 +/- 7 nm for EMD 84021, 5 +/- 1 nm for EMD 94309, 9 +/- 2 nm for EMD 96785 and 8 +/- 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na+/H+ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.

Bicyclic acylguanidine Na+/H+ antiporter inhibitors.

Blockade of the Na+/H+ exchange has been shown to diminish the serious consequences of myocardial ischemia. The aim of this investigation was to alter the structure of the common benzoylguanidine NHE inhibitors in such a way that the 3-methylsulfonyl and 4-alkyl group form a ring. The new benz-fused five-, six-, and seven-membered ring sulfones were prepared by internal Heck reaction. Benz-fused five-membered ring sulfones could also be prepared by internal aldol-type condensation using ketones or nitriles as acceptor groups. In the final step, the carboxyl groups were converted to acylguanidines preferentially by guanidine treatment of the esters or acid chlorides. The compounds were tested as their methanesulfonate salts. The inhibition of the Na+/H+ antiport activity was determined by observing the uptake of 22Na+ into acidified rabbit erythrocytes. Additionally, the inhibition of the antiport activity was assessed also by the platelet swelling assay (PSA), in which the swelling of human platelets was induced by the incubation in the presence of a weak organic acid. On average, the IC50 values in the PSA turned out to be about 10-fold higher than in the erythrocyte assay primarily due to a higher Na+ concentration in the PSA; however, the order of the compounds' potency was not substantially altered. The new compounds were found to be highly active with peak values ranging within the cariporide and EMD 96785 standards.

A new sodium/hydrogen exchange inhibitor, EMD 85131, limits infarct size in dogs when administered before or after coronary artery occlusion.

Administration of inhibitors of the Na+/H+ exchanger (NHE) have been shown to produce cardioprotective effects in a number of animal models of ischemia-reperfusion injury; however, controversy still exists as to the efficacy of these agents when administered just before reperfusion. To address this question, the efficacy of several doses of a new selective NHE-1 isoform inhibitor (IC50 for inhibition of 22Na uptake in NHE-1 expressing mouse fibroblast cells = 10.4 +/- 1.0 nM), EMD 85131 (2-methyl-5-methylsulfonyl-1-(1-pyrrollyl)-benzoyl-guanidine), was tested in a canine infarct model in which the left anterior descending coronary artery was occluded for 60 min followed by 3 hr of reperfusion. EMD 85131 (0.75 or 3.0 mg/kg) was infused for 15 min before left anterior descending occlusion or 15 min before reperfusion. Infarct size was determined by use of the triphenyltetrazolium chloride histochemical stain and was expressed as a percent of the area at risk. EMD 85131 (0.75 or 3.0 mg/kg) administered before left anterior descending occlusion produced a marked (*P < .05) and dose-related reduction in IS/AAR (24.3 +/- 3.6, control; 9.3 +/- 3.4%, EMD 0.75; 6.4 +/- 2.3%, EMD 3.0). These two doses of EMD also produced significant (*P < .05) reductions in infarct size/area at risk (12.2 +/- 2.1%, EMD 0.75; 13.0 +/- 2.9%, EMD 3.0) when administered 15 min before reperfusion. These results suggest that selective NHE-1 inhibitors are able to markedly reduce infarct size when given before or during ischemia and also suggest that these compounds may have clinical utility when administered after the initiation of an ischemic insult.

(2-Methyl-5-(methylsulfonyl)benzoyl)guanidine Na+/H+ antiporter inhibitors.

The inhibition of the Na+/H+ exchanger during cardiac ischemia and reperfusion has been shown to be beneficial for the preservation of the cellular integrity and functional performance. The aim of the present investigation was to come up with potent and selective benzoylguanidines as NHE inhibitors for their use as an adjunctive therapy in the treatment of acute myocardial infarction. During the course of our investigations it became clear that the substitution ortho to the acylguanidine was of crucial importance for the potency of the compounds. 4-Chloro- and 4-fluoro-2-methylbenzoic acids 6 and 7 were prepared using the directed ortho metalation technique with the carboxylic acid as the directing group. With the LDA/methyl iodide system the 2-methyl group could be extended to an ethyl group. 4-Alkyl groups were inserted by the palladium-catalyzed cross-coupling reaction into the 4-bromo-2-methylbenzoic acid methyl ester (20). Starting with benzoic acids 6-19, the methylsulfonyl group was introduced by a sequence of standard reactions (sulfochlorination, reduction, and methylation). 4-Aryl derivatives 68-75 were synthesized by the palladium-catalyzed Suzuki reaction. A large number of nucleophilic displacement reactions in the 4-position were carried out with S-, O-, and N-nucleophiles as well as with the cyano and trifluoromethyl group. Using the ester method, acid chlorides, or Mukaiyama's procedure, the 5-(methylsulfonyl)benzoic acid derivatives were finally converted to the (5-(methylsulfonyl)benzoyl)guanidines 165-267 with excessive guanidine. In some cases nucleophilic substitutions with pyridinols and piperidine derivatives were carried out at the end of the reaction sequence with the 4-halo-N-(diaminomethylene)-5-(methylsulfonyl)-benzamides. Variations in the 4-position were most reasonable, but the volume of the substituents was of crucial importance. Substitution in the 3- and particularly in the 6-position led to considerable worsening of the inhibitory effects of the Na+/H+ exchanger. The 2-methyl compounds, however, showed without exception higher in vitro activities than their respective demethyl counterparts as they are exemplified by the reference compounds 266 and 267, obviously caused by a conformational restriction of the acylguanidine chain. The development compound (2-methyl-5-(methylsulfonyl)-4-pyrrolobenzoyl)guanidine, methanesulfonate (246) is a NHE-1 subtype specific NHE inhibitor, being 27-fold more potent toward the NHE-1 than the NHE-2 isoform. 246 was found to act cardioprotectively not only when given before an experimentally induced ischemia, but also curatively after the onset of symptoms of acute myocardial infarction when given prior to the induction of reperfusion.

Non-peptide angiotensin II receptor antagonists: synthesis and biological activity of a series of novel 4,5-dihydro-4-oxo-3H-imidazo[4,5-c]pyridine derivatives.

A series of novel non-peptide angiotensin II receptor antagonists containing a 2,3,5-trisubstituted 4,5-dihydro-4-oxo-3H-imidazo[4,5-c]pyridine was prepared via several synthetic routes. Their affinity for angiotensin II receptors was established in a binding assay experiment and in an isolated-organ test. Molecules with small alkyl groups at C-2 and the (methylbiphenylyl)tetrazole moiety at N-3 were the preferred compounds with affinities and potencies in the nanomolar range. Variations at the N-5 position modulate the activity. Substitution at N-5 with various benzyl groups led to derivatives with in vitro potencies in the nanomolar range, which were equivalent to those of losartan in these assays. Replacement of the N-5 hydrogen with acetic acid esters or, in particular, acetamides gave molecules with increased activity. The most potent was 2-butyl-4,5-dihydro-4-oxo-3-[[2'-(1H-tetrazol-5-yl)-4- biphenylyl]methyl]-3H-imidazo[4,5-c]pyridine-5- (N,N-diethylacetamide) (14u), which is superior to L-158,809 in vitro. Two prototypes were selected as their potassium salts for in vivo testing as antihypertensives. Compounds 14a (EMD 61,650) and 14q (EMD 66,684) reduced blood pressure dose dependently in spontaneously hypertensive rats when administered iv. In this assay, acetamide 14q is superior to losartan.

Stereoselective actions of thiadiazinones on canine cardiac myocytes and myofilaments.

Thiadiazinones are cardiotonic agents that have potent, direct, and stereoselective actions on the myofilament response to Ca2+ in intact myocardium. Their mechanism of action is unknown. We studied the effects of racemic thiadiazinone, EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-meth yl-3,6- dihydro-2H-1,3,4-thiadiazin-2-one), and its enantiomers on Ca2+ signaling in myocytes, myofilaments, and myofilament proteins. Intact canine ventricular myocytes responded to the positive enantiomer, EMD 57033, with an increase in the extent of shortening during twitch contractions without increasing the peak amplitude of the Ca2+ transient. The negative enantiomer, EMD 57439, also increased the extent of shortening, but in this case there was a concentration-dependent increase in the peak amplitude of the Ca2+ transient. This is predicted from in vitro data showing that this enantiomer is a relatively potent inhibitor of phosphodiesterase activity. There was no effect of EMD 57439 on the relation between pCa and actomyosin Mg-ATPase activity of canine heart myofibrils. In contrast, EMD 57033 shifted the pCa-Mg-ATPase activity relation to the left. There was no effect of either enantiomer on Ca2+ binding to myofilament troponin C. Moreover EMD 57033, but not EMD 57439, stimulated actomyosin ATPase activity of myofilament preparations in which either troponin or troponin-tropomyosin had been extracted. EMD 57033 had no effect on Mg-ATPase activity of pure ventricular myosin. EMD 57033 also stimulated the velocity of actin filament sliding on myosin heads adhered to nitrocellulose-coated glass coverslips. We propose that the action of EMD 57033 is at the actin-myosin interface on a "receptor" that may be on actin or the crossbridge. Drug binding to this domain appears to reverse the inhibition of actin-myosin interactions by troponin-tropomyosin and also to promote transition of crossbridges from weak to strong force-generating states.

The two mechanisms of action of racemic cardiotonic EMD 53998, calcium sensitization and phosphodiesterase inhibition, reside in different enantiomers.

The novel cardiotonic EMD 53,998 increases contractile force in vitro through both inhibition of phosphodiesterase III (PDE III) activity and increase in the responsiveness of the contractile proteins to calcium ("calcium sensitization"). Because EMD 53,998 is a racemate, the possibility arose that the two modes of action do not reside equally in the enantiomers. Therefore, the effects of the racemate and its two enantiomers [(+)EMD 57,033 and (-)EMD 57,439] were analyzed in guinea pig and rat cardiac tissue with respect to Ca2+ sensitization (Ca(2+)-induced force development in skinned cardiac myofibers and myofibrillar ATPase activity) and PDE III inhibition (isolated PDE isoenzymes and cyclic AMP level in isolated cardiac myocytes). In addition, the positive inotropic effects were compared in isometrically contracting papillary muscles. Enhancement of force of contraction (Fc) in submaximally activated skinned fibers showed a selectivity for the (+)enantiomer with EC50 = 1.7, 4.8, and > 100 microM for EMD 57,033, EMD 53,998, and EMD 57,439, respectively. Ca2+ concentration for half-maximal activation was decreased by 0.5 log units, and Cmax was increased by 15% at 10 microM EMD 57,033. Similarly, myofibrillar ATPase activity was most potently enhanced by the (+)enantiomer, with EC50 values of 1.8, 2.5, and > 30 microM for EMD 57,033, EMD 53,998, and EMD 57,439, respectively. PDE III activity was inhibited with greater potency by the (-)enantiomer, with IC50 values of 0.05, 0.06, and 1.94 microM for EMD 57,439, EMD 53,998, and EMD 57,033, respectively. The cyclic AMP content of isoprenaline-stimulated rat cardiac myocytes was increased by 50% at 13.6 and 0.71 microM for EMD 57,033 and EMD 57,439, respectively. In intact guinea pig papillary muscle, the positive inotropic effect of the (+)enantiomer was insensitive to isoprenaline pretreatment; in contrast, the (-)enantiomer showed only a weak positive inotropic action which was strongly enhanced in the presence of isoprenaline. We conclude that one of the two different mechanisms underlying the overall positive inotropic activity of EMD 53,998 can be assigned, almost exclusively, to one of the two enantiomers. Thus, the (-)enantiomer EMD 57,439 is a "pure" PDE III inhibitor with almost no Ca2+ sensitizing activity; the (+)enantiomer EMD 57,033 is a potent Ca2+ sensitizer with only a weak PDE III inhibitory activity as compared with the racemate. In contrast to other compounds with mixed activity, EMD 57,033 is unique in possessing both a high absolute potency at the level of the contractile elements and a favorable relation of Ca2+ sensitization to PDE inhibition.

Novel diazinone derivatives separate myofilament Ca2+ sensitization and phosphodiesterase III inhibitory effects in guinea pig myocardium.

The inotropic state of the myocardium can be enhanced via an increase in cell Ca2+ loading or in myofilament responsiveness to Ca2+. Although different pharmacological agents combine these properties, no presently available drug acts predominantly as a myofilament sensitizer in situ. We have investigated the effects and the mechanism of action of novel diazinone derivatives, EMD 54622, EMD 53998, and EMD 54650 (developed by E. Merck, Darmstadt), on guinea pig myocardial preparations. Force- and ATPase-pCa relations in skinned fibers show differing potencies of these agents on myofilament sensitization: EMD 54622 greater than EMD 53998 much greater than EMD 54650. This is in contrast to their relative potencies to inhibit isolated myocardial phosphodiesterase III: EMD 54650 greater than EMD 53998 greater than EMD 54622. In isolated hearts studied at constant coronary flow, each of the three diazinone derivatives had a positive inotropic effect. In enzymatically dissociated left ventricular myocytes loaded with the Ca2+ probe indo-1, the positive inotropic effect of EMD 54622 occurred with no change in the amplitude of the cytosolic [Ca2+] (Cai) transient. In contrast, both EMD 53998 and EMD 54650 enhanced Cai transient and twitch contraction amplitudes. Length-indo-1 fluorescence relations were analyzed to determine the effects of the three substances on myofilament responsiveness to Ca2+. EMD 54622 enhanced and EMD 54650 had no effect on myofilament responsiveness to Ca2+. Less uniform results were obtained with EMD 53998 (in two of five cells the myofilament responsiveness to Ca2+ was increased, whereas in three other cells it was unaltered). Our results indicate that structural changes in the diazinone molecule shift the mechanism of action for the positive inotropic effect of the diazinone derivatives in the intact cell from a predominant myofilament sensitization (EMD 54622) to an enhancement in cell Ca2+ loading and an augmentation in the Cai transient (EMD 54650).