The inhibition of receptor-mediated and voltage-dependent calcium entry by the antiproliferative L-651,582

D.J Hupe,R Boltz,C.J Cohen,J Felix,E Ham,D Miller,D Soderman,D Van Skiver

doi:10.1016/s0021-9258(18)99200-8

Abstract

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1,2,3-triazole-4- carboxamide, an antiproliferative and antiparasitic agent previously shown to affect 45Ca2+ uptake into mammalian cells, inhibits both receptor-mediated and voltage-dependent calcium entry in well characterized in vitro systems. Indo 1 fluorescence measurements of cytosolic calcium levels indicate that the drug has no effect on the initial transient release of internal stores of calcium stimulated by fMet-Leu-Phe in rat polymorphonuclear leukocytes. It does decrease the levels maintained subsequently, however, indicating blockage of calcium influx through receptor-operated channels. L-651,582 also blocks the stimulation of leukotriene B4 (LTB4) production by fMet-Leu-Phe with an IC50 = 0.5 micrograms/ml equal to that for calcium entry inhibition. The LTB4 inhibition is likely due to calcium entry inhibition since L-651,582 does not inhibit calmodulin or enzymes producing arachidonate metabolites. L-651,582 also inhibits potassium-stimulated 45Ca2+ influx into GH3 cells with an IC50 of 0.5 microgram/ml, indicating a block of voltage-gated L-type calcium channels. Patch voltage clamp measurements of current through L- and T-type calcium in guinea pig atrial cells also indicate that L-651,582 is a calcium antagonist. Block of L-type calcium channels is voltage-dependent, and the apparent dissociation constant for the high affinity state is 0.2 micrograms/ml. The IC50 for block of T-type calcium channels is 1.4 micrograms/ml. The inhibition of cellular proliferation and the production of arachidonate metabolites by L-651,582 may be the result of the nearly equipotent block of receptor-operated and voltage-gated calcium channels.

Highlights

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dich-nucleotide pools [1].The inhibition of purine base incorpolorobenzyl]-1,2,3-triazole-4-carboxamidea,n antipro- ration into soluble nucleotide pools in Madin-Darby bovine liferative and antiparasitic agent previously shown tkoidney (MDBK)’ cells was shown to be the result of a previaffect 4sCa2+uptakeintomammalian cells, inhibits ously unrecognized Ca2+-dependentregulation of thefirst bothreceptor-mediated and voltage-dependentcal- enzyme in nucleotide biosynthesis, PRPP synthetase [5]
1fluorescence measurements of cytosolic calcium levels indicate that the drug has no effect on the initial transient release of internal stores of calcium stimulated by met-Leu-Phe in rat polymorphonuclear leukocytes
L-651,582 decreased the rate of production of PRPP and PRPP-dependent nucleotide biosynthesis

Summary

Introduction

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dich-nucleotide pools [1].The inhibition of purine base incorpolorobenzyl]-1,2,3-triazole-4-carboxamidea,n antipro- ration into soluble nucleotide pools in Madin-Darby bovine liferative and antiparasitic agent previously shown tkoidney (MDBK)’ cells was shown to be the result of a previaffect 4sCa2+uptakeintomammalian cells, inhibits ously unrecognized Ca2+-dependentregulation of thefirst bothreceptor-mediated and voltage-dependentcal- enzyme in nucleotide biosynthesis, PRPP synthetase [5]. 1fluorescence measurements of cytosolic calcium levels indicate that the drug has no effect on the initial transient release of internal stores of calcium stimulated by met-Leu-Phe in rat polymorphonuclear leukocytes. It does decrease the levels maintainedsubsequently, indicating blockage of calcium influx through receptor-operated channels. 651,582to inhibit both intracellular parasgitreowth in MDBK blocks the stimulation of leukotriene B4 (LTB4) cells and to inhibit the rapid proliferation rate of the production by met-Leu-Phe with anICso = 0.5 rg/ml MDBK cells themselves inculture,both mayhavebeen equal to that for calcium entry inhibition.

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