R 24 571: A potent inhibitor of calmodulin-activated enzymes

104

A liver plasma-membrane fraction capable of CaZ+ uptake was isolated. The fraction exhibited high Na', K + -ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent, and has an acid pH optimum.The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 x lop8 M and a Vmax of transport of 30 nmol x mg protein-' x min-l. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na', and incubation of the vesicles in a high Na+ medium (as compared to high K + medium) resulted in lower (about 25 %) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H'. The releasing effect of H', taken together with the absence of CaZ . The distribution of Ca2+ among the various cell phases is regulated at different sites. Mitochondria and the endoplasmic reticulum are of special importance [4,5], but it is also likely that calmodulin, which is present in the cytosol in micromolar concentration, influences the cytosolic level of free Ca2+. It is obvious, however, that the plasma membrane plays the ultimate role in the balancing of extracellular and intracellular Ca2+. This is so because most plasma membranes are somewhat permeable to Caz+. Since the Ca2+ gradient between the extracellular and intracellular compartements is in favour of influx, the CaZ+ that has entered the cell will eventually have to be ejected by energy-linked mechanisms. Indeed, perfusion of liver with 45Ca2 + In ' the presence of inhibitors of glycolysis and oxidative phosphorylation results in increased cellular Ca2+ [6]. Energy-dependent Caz + extrusion has now been demonstrated in many plasma membranes (for a review, see [7]), and a high-affinity Caz+-stimulated ATPase, possibly involved in CaZ + extrusion, has been recently isolated from those of liver [8]. The isolated liver enzyme is apparently regulated by an activator different from calmodulin [8].The problem of the extrusion of Ca2+ from liver has been approached in this work by studying the properties of the Ca2+ transport process in isolated plasma membrane vesicles. Extending previous work in this Laboratory [9] the results obtained have shown that an ATP-linked Ca2+ transport mechanism indeed exists. A Na+/Ca2+ exchange system, Abbreviations. Hepes = 4-(2-hydroxyethyl)-l-piperazineethanesul-

Section: Calmodulin Sensitivity Of the Transportmentioning

confidence: 99%

“…The possibility of calmodulin involvement in the regulation of Ca2+ transport in hepatic plasma membrane vesicles was examined by adding purified calmodulin to the vesicles, and by using two anticalmodulin drugs, trifluoperazine and R24571 [24]. Fig.…”

Section: Calmodulin Sensitivity Of the Transportmentioning

confidence: 99%

Calcium Uptake in Isolated Hepatic Plasma‐Membrane Vesicles

Kraus‐Friedmann

Biber

Murer

et al. 1982

104

“…As shown in Table 3, the putative phosphorylation process requires Ca2+, Mg2 + and ATP. Exogenously added calmodulin has no effect, but the calmodulin dependence of the process can be inferred from the strong inhibitory effect of low concentrations of trifluoperazine [34] or of the other calmodulin antagonist R24571 [35] (about 90%, inhibition of the activation reaction at a trifluoperazine/phospholipid ratio of 20 nmol/mg). Vanadate, an inhibitor of the ATPase activities of the sarcolemmal vesicles 15,361 and digitoxigenin, a membrane permeant inhibitor of the Na,K-ATPase activity have no effect on the activation reaction (Table 3).…”

Section: Inhibition Of the Na+-ca'+-exchange Activity By Phosphorylusmentioning

confidence: 99%

The Regulation of the Na⁺ ‐Ca²⁺ Exchanger of Heart Sarcolemma

Caroni¹,

Carafoli²

1983

215

The Na+/Ca2+ ‐exchange of calf‐heart sarcolemma is activated by a treatment with ATP, Mg2+, and Ca2+, and deactivated by a treatment with phosphorylase phosphatase. The effect of th e latter can be substituted by a treatment with Mg2+, Ca2+, and calmodulin. the activating treatment does not require added calmodulin, but is inhibited by calmodulin antagonists. Evidently, engdogenous calmodulin is required and sufficient. Activation is half‐maximal at about 2μM Ca2+. Added clamodulin, however, decreases the Km (Ca2+) of the activating process to about 0.8 μM. Deactivation is half‐maximal, at optimal calmodulin concentrations, at about 1.5 μM Ca2+. Experiments with adenosine 5′‐[γ‐thio]triphosphate have shown that the activating treatment is mediated by a kinase and the deactivating treatment by a phosphatase. The concerted operation of the two enzymes is made possible by their different Ca2+ affinity. At saturating Ca2+ concentrations, the level of ATP may also influence the balance of the two enzymes.

“…None of the calcium channel blockers (at a concentration of 100 pM) had a significant effect on the release of PGE2 by rat Kupffer cells after addition of zymosan, phorbol ester, A23187 or arachidonic acid (data not shown). The calmodulin antagonist R 24571 [23] inhibited only partially the PGE, synthesis elicited by zymosan and A 23187, whereas the generation of PGE2 after exposure of the cells to phorbol ester or added arachidonic acid was not significantly affected by the calmodulin antagonist (Table 2). R 24571 inhibited to about the same extent the zymosan-induced release of TxB,, PGE2 and PGD, from cells prelabeled with [3H]arachidonic acid (data not shown).…”

Section: Resultsmentioning

confidence: 97%

Ca²⁺ requirement of prostanoid but not of superoxide production by rat Kupffer cells

Dieter

Schulze‐Specking

Decker

1988

The release of the prostanoids prostaglandin D2 (PGDJ, prostaglandin E2 (PGE,) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE, release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 pM free extracellular Ca2+ and 2 100 pM free Ca2+ was required to stimulate PGE, formation maximally.The calmodulin antagonist R24571 partially inhibited the release of PGEz elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE, irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE, induced by zymosan and phorbol ester.The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.