Curves fitted with GraphPad Prism (GraphPad Software)

Curves fitted with GraphPad Prism (GraphPad Software). DHP 1 having a phenyl group in the 4-position of the DHP ring showed much higher potency than the 4-phenethynyl DHP 3 or the 4-thienyl DHP 4, both of which were inactive at 100 M (Table 1). calcium stores. Additional DHPs also caused a launch 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. em N /em -Methylnitrendipine (IC50 2.6 M, MRS 1844) and em N /em -propargylnifrendipine (IC50 1.7 M, MRS 1845) symbolize possible lead compounds for the development of GLPG0634 selective SOC channel inhibitors. strong class=”kwd-title” Keywords: Calcium channels, Dihydropyridines, Capacitative calcium influx, Inositol trisphosphate, Store-operated calcium channels 1. Intro Cell function is definitely 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 launch 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 [1C3]. 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 [4C9], but GLPG0634 there is no clear pattern concerning 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 GLPG0634 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]. Additional classes of voltage-sensitive calcium channels, however, can be inhibited by particular DHPs [12]. In addition, particular structurally novel DHPs have proven to be highly selective as antagonists of A3-adenosine receptors [13C15]. 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 em p /em -substituted 4-phenyl DHPs inhibit a capacitative calcium current in skeletal muscle mass cells [17], while nifedipine (8), which is an em o /em -nitro-substituted 4-phenyl DHP, enhances the current in muscle mass 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 with this cell collection. 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 LEG8 antibody channels from 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 having 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 from the DHPs. 2. Materials and methods 2.1. Materials Nitrendipine (6), nifedipine (8), diltiazem, and methoxyverapamil (D-600) were obtained from Study Bio-chemicals International (RBI). Dibutyryl cyclic AMP was from the Sigma Chemical Co., and ATP from Fluka. Roswell Park Memorial Institute (RPMI) 1640 medium, F10 nutrient combination (Ham), fetal bovine serum, horse serum, L-glutamine, trypsin-EDTA, and penicillin/streptomycin (10,000 models/mL of penicillin G sodium and 10,000 g/mL of streptomycin sulfate) were purchased from Gibco BRL, Existence Systems. Fluo3-AM, fluo4-AM, and fura2-AM were from Molecular Probes, Inc. [3H] em myo /em -Inositol was from NEN Existence Science Products. 3-Nitrobenzaldehyde, 3-trifluoromethylbenzaldehyde, ethyl 3-aminocrotonate, ethyl acetoacetate, 3-aminocrotonitrile, 2-methoxyethyl acetoacetate, benzyl acetoacetate, em p /em -nitrobenzyl acetoacetate, ethyl 4,4,4-trifluoroacetoacetate, methyl acetoacetate, ethyl propionylacetate, iodomethane, zinc, ammonium acetate, and sodium hydride GLPG0634 GLPG0634 were purchased from your.