Andreas Wüthrich

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions.

Abstract Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca ++ -ATPase as well as Ca ++ transport into inside-out red blood cell vesicles with an IC 50 -value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca ++ -transport ATPase as compared to the basal Ca ++ -transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca ++ -transport ATPase. Mg ++ -ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC 50 = 20 μM) whereas (Na + + K + )-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).

Compound 4880 is a selective and powerful inhibitor of calmodulin-regulated functions

Compound 48/80, a condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde, is composed of a family of cationic amphiphiles differing in the degree of polymerization. Compound 48/80 was found to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and red blood cell Ca2+-transport ATPase, with IC50 values of 0.3 and 0.85 micrograms/ml, respectively. However, the basal activity of both enzymes is not at all suppressed by the drug at concentrations up to 300 micrograms/ml. Inhibition of Ca2+ transport into inside-out red blood cell vesicles by compound 48/80 follows a similar pattern in that basal, calmodulin-independent, transport is also not affected by the drug. Kinetic analysis revealed that the stimulation of Ca2+-transport ATPase induced by calmodulin is inhibited by compound 48/80 according to a competitive mechanism. It was demonstrated that the inhibitory constituents of compound 48/80 bind to calmodulin in a Ca2+-dependent fashion. Comparison of the specificity of several anti-calmodulin drugs showed that compound 48/80 is the most specific inhibitor of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase that has been described hitherto. In addition, compound 48/80 was found to be a rather specific inhibitor of the calmodulin-induced activation of Ca2+-transport ATPase when compared with the stimulation induced by an anionic amphiphile or by limited proteolysis. Half-maximal inhibition of the activity stimulated by oleic acid or mild tryptic digestion required 8- and 32-times higher concentrations of compound 48/80, respectively, compared with the calmodulin-dependent fraction of the ATPase activity. Moreover, calmodulin-independent systems as rabbit skeletal muscle sarcoplasmic reticulum Ca2+-transport ATPase or calf cardiac sarcolemma (Na+ + K+)-transport ATPase are far less influenced by compound 48/80 as compared with trifluoperazine and calmidazolium. Because of its high specificity compound 48/80 is proposed to be a promising tool for studying calmodulin-dependent processes.

Inhibition of the red cell calcium pump by quercetin

Abstract 1. 1. Quercetin inhibits active Ca2+ transport into inside-out vesicles (IOVs) of human red cell membranes with an apparent KI of 6 μM. In the same preparation KI for inhibition of the (Ca2++Mg2+)-ATPase was found to be 4 μM. 2. 2. The apparent affinity of disrupted red cell membranes for quercetin is 5 times less than that of IOVs. 3. 3. Quercetin crosses the red cell membrane rapidly at pH 7.4. 4. 4. Quercetin is not competitive with ATP or Ca2+. 5. 5. The action of quercetin is not specific for the Ca2+ transport system. The compound is equally active in inhibiting the (Na++K+)-ATPase of human red cell membranes. The Mg+-ATPase requires higher quercetin concentrations for inhibition (KI = 240 μM) and is stimulated by 10–20% at quercetin concentrations below 50 μM.

Topical application of synthetic pyrethroids to cattle as a source of persistent environmental contamination

Following the application of permethrin or cyhalothrin to cattle for the control of ectoparasites, the occurrence and persistence of these chemicals was assessed on the animals and in their environment. The release of permethrin from ear tags containing 1 g of the drug on cattle was followed for 65 days and lead to concentrations of 5 to 35 micrograms of permethrin per gram of hair on the shoulders. On the flanks of the animals, the corresponding values were 10 times lower. Across the 1.5 acre pasture, high concentrations of permethrin were measured at various locations and long after treatment: 6 micrograms/g on bark of a birch after one week, 5 micrograms/g on a pole of the fence after two weeks, 1 microgram/g in grass from a resting site of the animal after six weeks, and 0.5 microgram/g in bark of a pine tree after three month and two weeks after the animals had left the pasture. In similar assays, cyhalothrin applied to milk cows as a pour-on preparation was monitored. One week following treatment with 0.2 g/animal, hair cut from the shoulders contained 5 micrograms/g of the insecticide, which disappeared with a half-life of 12 days. Dust collected two weeks after the pour-on treatment from the milk barn where the cows were milked twice daily contained 47 micrograms/g of cyhalothrin, which disappeared with a half-life of 44 days. These results show that synthetic pyrethroids used on farm animals can be the source of widespread and persistent contamination.

Bovine hereditary cardiomyopathy: an animal model of human dilated cardiomyopathy

UNLABELLED Bovine hereditary cardiomyopathy (bCMP) displays clinical characteristics of human idiopathic dilated cardiomyopathy (DCM). We studied isometric force of contraction in right ventricular trabeculae, plasma and tissue catecholamines, beta- and alpha 1-adrenoceptor density, Gi proteins and adenylyl cyclase activity in eight hearts with bCMP and eight control hearts (right and left atria and ventricles each). RESULTS Compared to control, the potency of isoprenaline in bCMP was eight-fold decreased, whereas the maximal positive inotropic effect of isoprenaline as well as the efficacy and potency of calcium were unchanged. Plasma noradrenaline was increased by 240%. Tissue noradrenaline and adrenaline were decreased by 36-63% and 58-69%, whereas dopamine was increased by 105-218%. beta-adrenoceptor density was drastically reduced by 90%, but binding affinity was unchanged. alpha-Adrenoceptor density and binding affinity were unchanged. Total PTX-substrates were increased in bCMP by 28-99%. Basal adenylyl cyclase activity was decreased by 36-47%. Similarly, stimulation by GTP, GMPPNP, isoprenaline, sodium fluoride, manganese or forskolin was attenuated by 26-62% (atria) and 45-66% (ventricles). In conclusion, we found marked activation of the sympatho-adrenergic system, downregulation of beta-adrenoceptors, upregulation of Gi proteins, global desensitization of adenylyl cyclase and selective subsensitivity to beta-adrenergic inotropic stimulation. These results closely resemble the characteristic alterations in the beta-adrenoceptor-G protein-adenylyl cyclase pathway in human heart failure, indicating that they are general features of heart failure. The similarity to human DCM, the inheritance and the availability of large tissue samples make bCMP a suitable model for human DCM.

Dependence of the red blood cell calcium pump on the membrane potential

(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.

Isolation from haemolysate of a proteinaceous inhibitor of the red cell Ca2+-pump ATPase. Its action on the kinetics of the enzyme

The purification to apparent homogeneity of a small protein from the cytosol of human red cells is described. The procedure consists of a combination of anion-exchange-chromatography, ultrafiltration, (NH4)2SO4- and heat-precipitation. The resulting protein is a potent inhibitor of (Ca2+ + Mg2+)-ATPase of erythrocyte membranes and of Ca2+-uptake into inside-out vesicles. Membrane (Na+ + K+)-ATPase is not affected by the inhibitor. The peptide migrates as a single band in SDS gels. Its apparent molecular weight is 19,000. It causes inhibition of the Ca2+-pump by decreasing Ca2+-affinity at all calmodulin concentrations.

Effects of Vinca Alkaloids on Calmodulin-Dependent Ca2+-Transport ATPase

SummaryThe Vinca alkaloids vinblastine, vindesine and vincristine were found to be inhibitors of the calmodulin-dependent fraction of red blood cell Ca2+-transport ATPase with IC50-values of 35, 100 and 220 μM, respectively. However, in the concentration range of 1–10 uM all three substances inhibit 10–15 % of the calmodulin-dependent ATPase activity. The concentration of vinblastine for 50 % inhibition of the calmodulin-dependent fraction of the Ca2+transport into inside-out vesicles is very similar to that found for the Ca2+-ATPase. It was shown that the inhibitory potency of vinblastine can be reduced by increasing calmodulin concentrations, suggesting a competitive mechanism. With respect to other ATPases the action of vinblastine on Ca2+-transport ATPase seems to be specific since Mg2+-ATPase and (Na+ + K+)-ATPase are hardly affected up to 300 μM.

Cell culture in nephrotoxicity testing

The need to develop alternative methods has become obvious thanks to organizations concerned with animal welfare who ask for more humane and ethically acceptable methods, but also thanks to scientists who call for finer and more differentiated methods. In industrial toxicology where the numbers of animals used are particularly large there is increasing concern as to reducing the number of animals, replacing animal experiments by other methods or at least refining the tests performed on animals in order to diminish pain and discomfort. Being responsible for the elimination of many xenobiotics, the kidneys are exposed to much higher quantities of toxicants than other organs. The high blood flow compared to the organ weight and the ability of the tubular cells to concentrate toxins within them are two further factors which account for the vulnerability of the kidney to a large number of toxic substances. Considering that damage to the kidney can be a threat to the whole organism, it is clear that nephrotoxicity testing is an essential step in the development of new drugs. Cell culture is one of many in vitro methods in renal toxicology. Its advantages in comparison to in vivo tests are: (1) the opportunity to investigate the mechanisms of toxicity at the cellular level, (2) the possibility of long-term storage and repeated experimentation under similar conditions (especially when using cell lines), and (3) saving of time and test substance. However, since the cells are taken out of their normal environment and since some important factors of nephrotoxicity are omitted such as the metabolism of the drugs in the liver or the dynamics of blood flow in the kidney, cells in culture may not be considered as a replacement for animal experiments.

THE CALCIUM-PUMP OF THE HUMAN RED BLOOD CELL

Publisher Summary Red cells are able to extrude Ca2+-ions across the plasma membrane against a large gradient by way of an ATP-fueled pumping mechanism, located in the plasma membrane. In isolated, disrupted red cell membranes, the existence of the system is reflected by an ATPase activity requiring the simultaneous presence of Ca2+ and Mg2+, which shows the same characteristics as the Ca-pump in a large number of details. This system is also present in the plasma membranes of many of the more highly organized animal cells, like kidney tubule cells, intestinal mucosa cells, liver cells, squid giant axons, nerve terminals, and heart muscle. The low concentration of ionised calcium inside the free cellular space is a prerequisite for minute Ca2+ influx variations to exert regulatory influences on cell functions in response to electrical, hormonal, or other stimulations. Such cell functions are contraction in muscle and nonmuscle cells, exocytosis in exocrine and endocrine glands, disaggregation of microtubuli and cell division, egg development, and protein synthesis. An intriguing problem is the action of cellular Ca2+ on the synthesis and degradation of cyclic AMP. The importance of low intracellular Ca2+ concentration is emphasized by the fact that many deadly poisons act by inducing an uncontrolled rise in cellular Ca2+.