ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

Trophoblast antigenicity demonstrated by altered challenge graft survival.

Adult C57BL/6J male mice received either a primary ectopic transplant or a primary and a challenge ectopic transplant of trophoblast tissue obtained from the ectoplacental cones of 7(1/2)-day-old C3H/HeJ x C3H/HeJ embryos. Gross and histologic examinations of these grafts at 5, 7, and 12 days of growth indicated that the challenge grafts were inhibited in growth; there were a smaller percentage of grossly successful grafts, smaller hemorrhagic reactions, fewer viable cells at all stages of growth, and better host containment of the colony. The evidence indicates that exposure to pure trophoblast alters an animal's subsequent reaction to grafts syngeneic with the original trophoblast and can best be explained as evidence of immunologic sensitization of the host.

Quantification of fluorescent dyes in organ tissue samples via HPLC analysis.

The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.

Superagonism at G protein-coupled receptors and beyond.

Ligands targeting GPCRs can be categorized according to their intrinsic efficacy to trigger a specific, receptor-mediated response. A ligand endowed with the same level of efficacy as the endogenous agonist can be classified as a full agonist, whereas a compound that displays greater efficacy, that is, higher receptor signalling output than the endogenous agonist, can be called a superagonist. Subsequent to GPCR activation, an intracellular signalling cascade is set in motion, which may generate substantial amplification of the signal. This may obscure superagonism in pharmacological assays and, therefore, the definition of superagonism necessitates a combination of operational approaches, reduction of spare receptors or estimation of receptor activation close to the receptor level to quantify relative agonist efficacies in a particular system. The first part of this review will compare GPCR superagonism with superagonism in the field of immunology, where this term is well established. In the second part, known GPCR superagonists will be reviewed. Then, the experimental and analytical challenges in the deconvolution of GPCR superagonism will be addressed. Finally, the potential benefit of superagonism is discussed. The molecular mechanisms behind GPCR superagonism are not completely understood. However, crystallography shows that agonist binding alone is not sufficient for a fully active receptor state and that binding of the G protein is at least equally important. Accordingly, the emerging number of reported superagonists implies that ligand-induced receptor conformations more active than the ones stabilized by the endogenous agonist are indeed feasible. Superagonists may have therapeutic potential when receptor function is impaired or to induce negative feedback mechanisms. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation.

Allosteric modulation of a receptor may be proved experimentally by demonstrating an altered radioligand dissociation in the presence of the allosteric modulator. Two-point kinetic experiments provide a screening-type approach to determine the delay of radioligand dissociation caused by allosteric modulation. In this article, Evi Kostenis and Klaus Mohr describe a pitfall in the data analysis that may lead to a suboptimum determination of the allosteric potency in the case of monophasic dissociations, and suggest how this problem may be resolved.

Lack of inotropic effect of cyclosporin A in isolated guinea pig left atria.

OBJECTIVE: Cyclosporin A is being widely used to prevent graft rejection in organ transplantation and to treat autoimmune diseases. Since various toxic side effects have been observed, the aim of this study was to look for even a subtle deleterious effect of cyclosporin A on cardiac inotropy in electrically stimulated guinea pig left atria. METHODS: The left atrial muscles of guinea pigs, in Tyrode's solution containing 2.7 or 5.4 mM potassium, were electrically stimulated by one of two methods: (1) continuously at 3 Hz, during which cyclosporin A was applied cumulatively (from 10(-9) to 10(-5) M); or (2) stimulated intermittently at 2.5 Hz in 5 mM cyclosporin A, with rest periods of 4 s duration interposed every 4 min. The effects of cyclosporin A on contractile force were observed for 150 min in the first stimulation method, and the effects on the steady state contractile force and amplitude of post-rest contraction were observed for 240 min in the second method. RESULTS: The steady state contractile force of the atria declined within the 4 h period at 2.7 mM potassium in Tyrode's solution both in the cyclosporin A group (n = 10) and in the control group (n = 5) to 68(SD 11)% and to 63(4)%, respectively. After 4 h the amplitudes of the post-rest contraction were 101(16)% and 101(4)% in cyclosporin A and control groups, respectively. At 5.4 mM potassium, the following values were obtained (cyclosporin A v control): steady state force 70(8)% (n = 11) v 69(8)% (n = 5); post-rest force 105(9)% v 102(7)%. CONCLUSIONS: Cyclosporin A does not influence the steady state contractile force or the amplitude of the post-rest contraction, suggesting the absence of inotropic effects on isolated guinea pig left atria.

Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

Search for the pharmacophore of bispyridinium-type allosteric modulators of muscarinic receptors.

The bis(dichlorobenzyl) ether of the bispyridinium oxime TMB 4 stabilizes antagonist binding to M2-cholinoceptors which is indicative of an allosteric action. More than 10 derivatives of the lead compound were synthesized to investigate structure-activity relationships. The allosteric potency of the compounds was indicated by the concentrations which retarded the rate of dissociation of [3H]N-methylscopolamine from porcine cardiac cholinoceptors by a factor of 2 (EC50). Compared with TMB 4, the bis(dichlorobenzyl) derivative 4a displayed a more than 200-fold higher potency (EC50 = 4.7 microM). One of the dichlorobenzyl groups could be replaced by a methyl group without loss of activity (EC50 = 4.5 microM). Further shortening of this end of the molecule was accompanied by a moderate decline in potency to a minimum of EC50 = 26 microM. The second quaternary nitrogen was not a prerequisite for an allosteric activity. It is concluded that one half of the lead compound is pivotal for an interaction with the allosteric site of the M2-cholinoceptor, whereas the opposite end of the molecule modulates the allosteric activity.

Synthesis, pharmacological and biophysical characterization, and membrane-interaction QSAR analysis of cationic amphiphilic model compounds.

Cationic amphiphilic drugs have a propensity to interact with biological interphases. This study was designed to gain more insight into the molecular properties of catamphiphilic drugs which govern this type of interaction. A series of phenylpropylamine model compounds were synthesized in which modifications were incorporated at the aromatic part of the molecule. The replacement of (45)Ca(2+) from phosphatidylserine monolayers served to monitor drug binding to the phospholipid. The influence on the phase-transition temperature of liposomes of dipalmitoylphosphatidic acid was measured to assess the perturbing action of the drugs on the structural organization of phospholipid assemblies. The antiarrhythmic activity of the compounds was determined in Langendorff preparations of guinea pig hearts to assess the membrane-stabilizing action. Quantitative structure-activity relationship (QSAR) models for these endpoints were developed using both intra- and intermolecular QSAR descriptors. Intermolecular membrane-interaction descriptors were derived from molecular dynamics simulations of the compounds in a model phospholipid monolayer. QSAR models were derived for all endpoints using partial least-squares regression (PLS) and a genetic algorithm tool, the genetic function approximation (GFA). Membrane-interaction descriptors appear to be of a particular importance in explaining the influence of the compounds on the phase-transition temperature of DPPA liposomes, while the other endpoints can be adequately modeled by intramolecular descriptors. The calcium-displacing activity at phosphatidylserine monolayers is governed by the electrostatic properties of the compounds. Measures of lipophilicity and molecular size are of particular importance for antiarrhythmic activity. Possible improvements to both the molecular modeling and the applied computational protocol of membrane-solute systems are identified and discussed.

Bisquaternary ligands of the common allosteric site of M2 acetylcholine receptors: search for the minimum essential distances between the pharmacophoric elements.

Structurally diverse molecules, such as alcuronium, gallamine, and tubocurarine as well as W84 and WDUO, are known to interact allosterically with ligand binding to muscarinic M2 acetylcholine receptors. Preliminary molecular modeling studies revealed two positive charges in the middle and two lateral aromatic areas to be essential elements of a high allosteric potency. To find out the optimum distances between these pharmacophoric elements, a systematic variation of the spacer in the series of W84, WDUO, and IWDUO compounds was performed. The allosteric reduction of the rate of dissociation of the antagonist [3H]-N-methylscopolamine from porcine heart M2 receptors served as a test system. The minimal essential distance between the positive charges was found to be 10 A. The length of the peripheral spacers connecting the positive charge and the lateral aromatic moiety appears to depend on the chemical functionality; the peripheral spacers have to be long and flexible enough to position the aromatic skeletons in the spatial neighborhood of the alkane middle chain: in the case of an oxime ether containing peripheral spacer, six atoms are required, and in the case of an alkane chain, four carbon atoms are necessary to adopt the pharmacophoric S-shape conformation.

Structure-activity relationships in a series of bisquaternary bisphthalimidine derivatives modulating the muscarinic M(2)-receptor allosterically.

Hexane-bisammonium-type compounds containing lateral phthalimide moieties are well-established ligands of the common allosteric binding site of muscarinic M(2) receptors. Previous structure-activity relationships (SAR) revealed two positively charged centers and two lateral phthalimide moieties in a defined arrangement to be essential of a high allosteric potency. The purpose of this study was to replace one carbonyl group of the phthalimides with hydrogens, hydroxy, alkoxy, phenyl, benzyl, and benzylidene groups in order to check the influence of these substituents on the allosteric activity in antagonist-linked receptors. The analysis of the quantitative SAR indicated that a high allosteric potency is related to a certain amount of rigidity as well as polarizibility and the ability to form hydrophobic interactions.

Action of ouabain on rat heart: comparison with its effect on guinea-pig heart.

The inotropic dose-response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 X 10(-7) M to 3 X 10(-5) M) displaying an intermediate plateau phase. In guinea-pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 X 10(-7) M-1 X 10(-6) M). Ouabain-intoxication in guinea-pig ventricular strips occurring at 3 X 10(-6) M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 X 10(-4) M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain-induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10(-4) M. Ouabain reduced the activity of Na/K-ATPase prepared from rat and guinea-pig cardiac ventricles to half of its maximum at 6.5 X 10(-5) M in rat and 1.0 X 10(-6) M in guinea-pig, rat heart Na/K-ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]-ouabain binding to membrane suspensions prepared from rat and guinea-pig ventricles was characterized by a similar affinity in rat (KD = 4 X 10(-8) M) and guinea-pig (KD = 13 X 10(-8) M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea-pig membranes. In rat the presence of additional ouabain-binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea-pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K-ATPase complex and mediate a positive inotropic effect after ouabain-binding in an identical manner.

High and concentration-proportional accumulation of [3H]-nitrendipine by intact cardiac tissue.

The binding of [3H]-nitrendipine to intact, electrically driven isolated left atria of guinea-pigs was investigated over the concentration range 10(-10) M to 3 X 10(-5) M. A high affinity binding site saturable in the nM range as found in ventricular homogenates could not be detected. Instead the accumulation of nitrendipine in intact atria was found to be proportional to the concentration from 10(-10) M to 10(-6) M; beyond 10(-6) M the binding started to become saturated. Nitrendipine was highly accumulated in atrial tissue. The cell:medium ratios amounted to about 120 in the range from 10(-10) M to 10(-6) M. The concentration-response curve for the negative inotropic action of nitrendipine yielded an ED50 of 3 X 10(-7) M, thus lying within the range of concentration-proportional accumulation. The reduction of the contractile force proceeded faster (t1/2 less than 10 min) than the uptake process t1/2 approximately 40 min) suggesting that it is the binding of nitrendipine into a superficial compartment, which interferes with the excitation-contraction coupling. The results suggest that the high concentration of nitrendipine present in hydrophobic cellular compartments such as the plasmalemma might be involved in its pharmacological action.

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.

On the binding of a 3-alpha-methylated digitoxigenin-glucoside to ouabain receptors in heart muscle homogenate.

3-alpha-Methyl substituted digitoxigenin-3-beta-glucoside (methyl-dtg-gluc) displays unusual features, e.g. a high dissociation rate constant from its binding site leading to rapid reversibility of the inotropic and toxic effects, and a flat dose-response curve attaining higher inotropic maxima thus indicating an increased therapeutic index in animal experiments. In order to check whether or not methyl-dtg-gluc is specifically bound to the same receptors as classic cardiac glycosides we compared binding of ouabain and of methyl-dtg-gluc to guinea-pig heart muscle homogenate. For both compounds specific binding required the addition of ATP (2.5 mM). The binding curve for ouabain yielded half maximum binding at 1.3 x 10(-7) M and maximum number of binding sites of 6 pmole/mg protein; the corresponding values for methyl-dtg-gluc amounted to 1.4 x 10(-6) M and 6 pmole/mg protein, respectively. A mutual competition could be demonstrated between the two compounds. Since the provided data are equilibrium values they do not exclude higher turnover rates of methyl-dtg-gluc in comparison with ouabain at a given glycoside-ATPase complex concentration which can be expected from the fast dissociation rate constant of the methyl-dtg-gluc-ATPase complex. The results are briefly discussed with respect to the molecular mole of action of cardiac glycosides.

Differential influence of anionic and cationic charge on the ability of amphiphilic drugs to interact with DPPC-liposomes.

The compounds clofibric acid and chlorphentermine have identical aromatic ring systems, but when charged their side chains are anionic or cationic, respectively. The drugs were applied as tools to investigate whether the interaction of amphiphilic drugs with the zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) depends on the charge of the polar side chain. In suspensions of DPPC-liposomes, the drug-effect on the phase-transition temperature (Tt) was evaluated by means of differential scanning calorimetry. The drug-binding was determined spectrophotometrically. The clofibric acid anion had a much weaker depressing effect on Tt than the chlorphentermine-cation and a considerably lower ability to bind to the DPPC-liposomes. Furthermore, a plot of the effect versus the binding suggested that the clofibric acid-anion had a lower intrinsic activity to reduce Tt compared with the chlorphentermine-cation. In contrast, when the dissociation-equilibrium was shifted towards the uncharged state both drugs were indistinguishable with respect to effect and binding, suggesting that the differences observed with the charged forms could indeed be attributed to the opposite charges. The findings are tentatively explained to result from a different ability of the anionic and the cationic form to reach the hydrophobic interior of the DPPC-bilayer.

Influence of cationic amphiphilic drugs on the characteristics of ouabain-binding to cardiac Na+/K+-ATPase.

The influence of 12 cationic amphiphilic compounds on the equilibrium and kinetic characteristics of the binding of tritium-labelled ouabain to the lipoprotein Na+/K+-ATPase present in a crude membrane suspension of guinea pig myocardium was investigated. The drugs, e.g. local anaesthetic, antiarrhythmic and psychotropic agents, inhibited specific binding of ouabain in a concentration-dependent manner by reducing its affinity without affecting the number of binding sites. In the presence of chlorpromazine, propranolol and dibucaine, the decreased affinity of ouabain was due to both a diminished association rate and an increased dissociation rate, while in the presence of the weakly potent procaine only the association rate of ouabain was found to be reduced. The different potency of the catamphiphilic drugs was well correlated to the degree of their hydrophobicity. Evidence is presented that the protonized form of the drugs is the effective one. Concerning the mode of action, the catamphiphilic drugs are proposed to interact with the phospholipid part of the lipoprotein Na+/K+-ATPase, thereby indirectly altering the conformation of the embedded protein moiety and thus reducing the proper fit between ouabain and its receptor.

Comparison between the activities of cationic amphiphilic drugs to affect phospholipid-membranes and to depress cardiac function.

The activity of cationic amphiphilic compounds to affect artificial phospholipid-membranes was compared with the membrane-stabilizing cardiodepressant potency of the drugs. The twenty-one investigated catamphiphilic compounds belonged to various pharmacological groups including antiarrhythmic, local anaesthetic, beta-blocking, antimalarial, and psychoactive drugs. The perturbing action of the drugs on phospholipid-membranes was evaluated by determining the drug-effect on the temperature of the phase-transition from the gel to the liquid crystalline state in liposomes of dipalmitoylphosphatidic acid by means of differential scanning calorimetry. The ability to interact with the polar headgroups of phospholipid-membranes was measured by recording the effects of the cationic compounds on the binding of 45Ca2+ to monomolecular layers of phosphatidylserine. The cardiodepressant action was observed in Langendorff-preparations of guinea-pig hearts. The drug-effect on excitability was indicated by the elevation of the threshold-intensity of 50 Hz alternating current to induce ventricular arrhythmia. For the sake of comparison, the negative chronotropic and inotropic effects were evaluated. There was only a moderate correlation found between the activities of the drugs to reduce the transition temperature and to inhibit 45Ca2+ -binding (r = 0.69). This result probably reflects that both methods look upon different consequences of the drug-phospholipid interaction. The membrane-stabilizing, anti-excitatory potency corresponded favourably with the ability of the drugs to affect the phospholipids. Almost 80% of the variance between the anti-excitatory potencies could be accounted for by the drug-phospholipid interactions. The negative chronotropic and inotropic effects accompanying the anti-excitatory actions were similar for most of the drugs. The results of the study are compatible with the hypothesis that the interaction with phospholipid-membranes is a major determinant of the membrane-stabilizing cardiodepressant potency of cationic amphiphilic drugs.

Influence of various cationic amphiphilic drugs on the phase-transition temperature of phosphatidylcholine liposomes.

The influence of 16 cationic amphiphilic compounds from various pharmacological groups on the phase-transition temperature (Tt) of dipalmitoyl-phosphatidylcholine (DPPC) liposomes was investigated using the method of differential scanning calorimetry. All drugs, the hydrophobicity of which varied in a wide range, depressed Tt. Biphasic dose-effect curves were obtained when the reduction of Tt (delta Tt) was plotted vs the molar ratio of drug/DPPC; beyond a plateau, Tt could again be reduced markedly by increasing the molar ratio. Concomitantly with the depression of Tt, the width of the transition peak changed in a characteristic way: it broadened during the (first) steep part of the dose-effect curves and became narrow like a control transition when the plateau of the dose-effect curves was reached. At still higher ratios the peak broadened again and eventually vanished, probably due to a detergent-like effect of the drug. Increasing hydrophobicity of the compounds shifted the dose-effect curves to lower molar ratios and enhanced the delta Tt attained at the plateau phase. It is proposed that the different potencies of the drugs to depress Tt result from different binding equilibria between the compounds and DPPC membranes, the individual equilibrium being determined by hydrophobic attraction and electrostatic repulsion.

The ability of cationic amphiphilic compounds to depress the transition temperature of dipalmitoylphosphatidic acid liposomes depends on the spatial arrangement of the lipophilic moiety.

The hypothesis was tested with the help of model compounds that the ability of cationic amphiphilic drugs to depress the phase-transition temperature Tt of dipalmitoylphosphatidic acid (DPPA) liposomes depends on the spatial arrangement of the lipophilic moiety. The main structure of the compounds with identical cationic side chain was 1-dimethylamino-3-phenylpropane (compound I). A further phenyl ring was introduced either at C3 of the propane chain (compound II) to broaden the lipophilic moiety, or in para-position of the phenyl ring (compound III) to elongate it. As shown by differential scanning calorimetry, the reduction of Tt (control 64 degrees) amounted for compound I to 29 degrees, for compound II to 28 degrees and for compound III to 53 degrees. In order to assess the binding affinity of the compounds to DPPA, their inhibitory effect on 45Ca(2+)-binding to DPPA films was measured. The IC50 values were 2100 microM for compound I, 40 microM for compound II, and 9 microM for compound III. Thus, binding affinity corresponded with the hydrophobicity of the compounds. In contrast, the depressing effect on the transition temperature was only augmented by the additional phenyl ring when substituted in the elongating position.