Elsevier

Biochemical Pharmacology

Volume 65, Issue 3, 1 February 2003, Pages 329-338
Biochemical Pharmacology

Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells

https://doi.org/10.1016/S0006-2952(02)01488-0Get rights and content

Abstract

A series of 1,4-dihydropyridines (DHPs) were investigated as inhibitors of capacitative calcium influx through store-operated calcium (SOC) channels. Such channels activate after ATP-elicited release of inositol trisphosphate (IP3)-sensitive calcium stores in leukemia HL-60 cells. The most potent DHPs were those containing a 4-phenyl group with an electron-withdrawing substituent, such as m- or p-nitro- or m-trifluoromethyl (ic50 values: 3–6 μM). Benzyl esters, corresponding to the usual ethyl/methyl esters of the DHPs developed as L-type calcium channel blockers, retained potency at SOC channels, as did N-substituted DHPs. N-Methylation reduced by orders of magnitude the potency at L-type channels resulting in DHPs nearly equipotent at SOC and L-type channels. DHPs with N-ethyl, N-allyl, and N-propargyl groups also had similar potencies at SOC and L-type channels. Replacement of the usual 6-methyl group of DHPs with larger groups, such as cyclobutyl or phenyl, eliminated activity at the SOC channels; such DHPs instead elicited formation of inositol phosphates and release of IP3-sensitive calcium stores. Other DHPs also caused a release of calcium stores, but usually at significantly higher concentrations than those required for the inhibition of capacitative calcium influx. Certain DHPs appeared to cause an incomplete blockade of SOC channel-dependent elevations of calcium, suggesting the presence of more than one class of such channels in HL-60 cells. N-Methylnitrendipine (ic50 2.6 μM, MRS 1844) and N-propargylnifrendipine (ic50 1.7 μM, MRS 1845) represent possible lead compounds for the development of selective SOC channel inhibitors.

Introduction

Cell function is regulated in a wide variety of cells by changes in cytosolic free calcium. In non-excitable cells, changes in intracellular calcium are often associated with receptor-mediated release of calcium from intracellular calcium stores and subsequent capacitative calcium entry due to the activation of SOC channels in the plasma membrane. The activation of SOC channels appears to be linked to the filling state of the calcium stores, but the pathways of activation and inactivation remain unclear [1], [2], [3]. A major problem in studying SOC channels has been the lack of selective probes for detection, activation, blockade, or modulation of such channels. A variety of structurally different compounds have been reported to cause some degree of SOC channel blockade [4], [5], [6], [7], [8], [9], but there is no clear pattern regarding the structural requirements for the blockade of SOC channels, nor insights into the mechanisms involved. Many of the putative blockers of SOC channels also cause the release of intracellular calcium, rendering them unsatisfactory as specific probes for SOC channels [5], [7], [9], [10].

DHPs are known as a class of potent and selective inhibitors of voltage-dependent L-type calcium channels [11]. Other classes of voltage-sensitive calcium channels, however, can be inhibited by certain DHPs [12]. In addition, certain structurally novel DHPs have proven to be highly selective as antagonists of A3-adenosine receptors [13], [14], [15]. DHPs, such as nicardipine, nitrendipine (6), and nifedipine (8), have been reported to cause inhibition of capacitative calcium influx in HL-60 and U937 cells, which lack L-type calcium channels, but high micromolar concentrations are required [10], [16]. Certain p-substituted 4-phenyl DHPs inhibit a capacitative calcium current in skeletal muscle cells [17], while nifedipine (8), which is an o-nitro-substituted 4-phenyl DHP, enhances the current in muscle cells [18].

A series of DHPs have now been assayed with HL-60 cells, which lack L-type calcium channels, to provide insight into the structural requirements for inhibition of the SOC channels by DHPs in this cell line. The IP3-sensitive calcium stores were depleted in the HL-60 cells using the receptor agonist ATP to initiate the formation of IP3, the release of intracellular calcium, and the subsequent activation of the SOC channels [10]. The inhibition of SOC channels by the DHPs was determined by monitoring the effects after ATP on intracellular calcium levels using a fluorescent calcium probe. Several DHPs also were tested in GH4C1 cells, labeled with a fluorescent calcium probe, for effects on calcium influx through voltage-sensitive L-type calcium channels to ascertain if there was any correlation between inhibition of L-type calcium channels and SOC channels by the DHPs.

Section snippets

Materials

Nitrendipine (6), nifedipine (8), diltiazem, and methoxyverapamil (D-600) were obtained from Research Biochemicals International (RBI). Dibutyryl cyclic AMP was obtained from the Sigma Chemical Co., and ATP from Fluka. Roswell Park Memorial Institute (RPMI) 1640 medium, F10 nutrient mixture (Ham), fetal bovine serum, horse serum, l-glutamine, trypsin-EDTA, and penicillin/streptomycin (10,000 units/mL of penicillin G sodium and 10,000 μg/mL of streptomycin sulfate) were purchased from Gibco BRL,

Results and discussion

Although a variety of SOC channel inhibitors are now known, there has been little investigation into the structural requirements necessary for SOC channel inhibition, and no potent (sub-micromolar) or selective inhibitors are available. Imidazoles, such as SKF 96365 and miconazole [5], [6], [7], tricyclics, such as trifluoperazine [7], and DHPs, such as nitrendipine [10], represent distinct structural classes of SOC channel inhibitors. Most imidazoles and tricyclics, in addition to blocking SOC

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