Klaus Gietzen

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 human erythrocyte Ca++-transport ATPase by phenothiazines and butyrophenones

Abstract Antipsychotic agents such as the phenothiazines-trifluoperazine, fluphenazine and chlorpromazine- and the butyrophenones-penfluridol, fluspirilene and pimozide — which were shown to interact with calmodulin in a Ca++-dependent fashion, competitively inhibit Ca++-transport ATPase of human erythrocyte membranes. Activation of the ATPase by calmodulin is half-maximally antagonized by these agents in the concentration range of 2.6–22 μM whereas inhibition of the basal ATPase (in the absence of calmodulin) is achieved at about 10-fold higher concentrations.

Calmodulin antagonism: A pharmacological approach for the inhibition of mediator release from mast cells

Several Ca2+ antagonists with either Ca2+-entry blocking or calmodulin (CaM) antagonistic properties and antiallergic drugs were investigated for their effects on mediator release from mast cells induced by different secretagogues (compound 48/80, concanavalin A, antigen-IgE and Ca2+ ionophore A23187) and for their ability to inhibit the function of CaM or phospholipid/Ca2+-dependent protein kinase (C-kinase). The effects of the different agents--with the only exception of cromolyn sodium--on histamine release elicited by compound 48/80 correlated well with their actions on two CaM-dependent enzymes whereas the activity of C-kinase was far less altered, or not altered at all. CaM antagonism of cloxacepride, picumast, oxatomide, fendiline and bepridil correlated not only with the inhibition of exocytosis evoked by compound 48/80 but also with that induced by A23187, concanavalin A and antigen-IgE. This indicates an action of these substances distal to the generation of the Ca2+ signal since the various secretagogues elevate the intracellular Ca2+ concentration by different mechanisms. However, prenylamine and thioridazine inhibited concanavalin A- and antigen-IgE-induced mediator release more potently and more effectively than that elicited by compound 48/80 or A23187. Therefore inhibition of allergic histamine release by these drugs may in part be dependent on an impairment of the Ca2+ signal. Since for each of two agents inhibition of histamine release (evoked by different releasers) parallels that of serotonin release it may be concluded that these mediators are secreted via the same mechanism. The results obtained with agents exhibiting different pharmacological properties but which share one common property, namely antagonism of CaM, strengthen the view that CaM is involved in exocytosis of mediators from mast cells.

Imidazole fungicides provoke histamine release from mast cells and induce airway contraction.

The effects of imidazole fungicides on rat mast cells and on guinea-pig airway smooth muscle contraction were studied. The dose-effect studies on mast cells were performed to prove our hypothesis that imidazole fungicides are potential histamine releasing agents and thus may induce bronchoconstriction in vivo. Indeed, all imidazole fungicides tested (i. e. ketoconazole, miconazole, prochloraz) and an agricultural formulation of prochloraz (i. e. Sportak) were able to elicit histamine release from mast cells in the concentration range of 30-300 microM, although there were marked differences in potency and efficacy. The in vivo experiments clearly showed that inhaled Sportak aerosols induce a significant bronchoconstriction in guinea-pigs. Moreover, after a single 5 min exposure to Sportak aerosols the animals developed airway hyperreactivity against histamine. From the results of our study it may be concluded that certain imidazole fungicides provoke histamine release by a non-immunological mechanism, induce airway constriction in guinea-pigs and hence may be harmful to spray operators who might inhale fungicide aerosols used for plant protection.

Induction of histamine release and calmodulin antagonism are two distinct properties of compound 4880

Compound 48/80, a mixture of oligomers, was fractionated by passing it in the presence of Ca2+ over a calmodulin-Sepharose column. The fraction not retained by the gel was shown by mass spectrometry to consist mainly of trimers, tetramers and pentamers. A second fraction consisting of hexamers and heptamers was eluted from the column at high ionic strength in the presence of Ca2+. Finally, in the presence of EGTA at high ionic strength, a third fraction eluted mainly consisting of higher oligomers (hexamers to dodecamers). The different fractions were characterized by testing their influence on calmodulin-sensitive Ca2+-transporting ATPase and their ability to elicit histamine release from mast cells. The third fraction showed the highest potency as calmodulin antagonist, however, the second fraction was the most potent in inducing histamine secretion. This would imply that the ability of compound 48/80 to evoke histamine release and to inhibit the function of calmodulin are distinct properties of the agent which are unrelated.

Target sizes of human erythrocyte membrane Ca2+-ATPase and Mg2+-ATPase activities in the presence and absence of calmodulin

We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase.

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.

Effects of the novel dihydropyridine derivative niguldipine on the cytoplasmic free calcium concentration of mouse thymocytes.

Niguldipine, a novel dihydropyridine derivative, was tested for its effects on the cytoplasmic free Ca2+ concentration of mouse thymocytes. In quin-2-loaded cells, a concentration-dependent rise of cytoplasmic Ca2+ can be detected, which requires extracellular Ca2+. The effect of niguldipine reaches a maximum after about 5 min; a similar time course has been observed, when using concanavalin A as a stimulus. Niguldipine provokes influx of Ca2+ into thymocytes, but not of Mn2+. Moreover, the effect of niguldipine exhibits some degree of stereospecificity, since (-)-niguldipine was more effective than its (+)-enantiomer. The action of niguldipine could be reversed by addition of bovine serum albumin, but not by addition of nitrendipine. None of several agents tested (e.g. felodipine, nitrendipine, trifluoperazine, cloxacepride, phenylephrine and ouabain) could mimic the effect of niguldipine at a concentration of 1 microM.