ABBREVIATIONS: NSC, nonselective cation; Me2SO, dimethylsulfoxide; Bt2cAMP, dibutyryl-cAMP; EGTA, ethylene glycol bis(f3-aminoethyl ether)- N,N,N’,N’-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; GTP-YS, guanosine-5’-O-(3-thio)thphosphate; G protein, guanine nucleotide-binding protein; TEA, tetraethanolamine. 655 0026-895X/93/050655-05$03.OO/O Copyright @ by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 43:655-659 ACCELERA TED COMMUNICATION Novel Potent Inhibitor of Receptor-Activated Nonselective Cation Currents in HL-60 Cells D. KRAUTWURST, J. HESCHELER, D. ARNDTS, W. LOSEL, R. HAMMER, and G. SCHULTZ Institut f#{252}r Pharmakologie, Freie Universitt Berlin, D-1000 Berlin 33, Germany (D.K., J.H., G.S.), and Boehringer Ingelheim KG, D-6507 Ingelheim am Rhein, Germany (D.A., W.L., RH.) Received January 13, 1993; Accepted February 9, 1993 SUMMARY A pharmacological classification of receptor-activated nonselec- tive cation channels has not been possible because of the lack of specific and potent pharmacological blockers. In dibutyryl- cAMP-differentiated HL-60 cells, we recently identified ATP- and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulated cation currents that were blocked by an organic inhibitor of receptor-mediated Ca2’ entry, 1 -fl-[3-(4-methoxyphenyl)- propoxy]-4-methoxyphenethyl-l H-imidazole hydrochloride (SK&F 96365), with an IC50 of about 3 M. Here, we describe a new compound, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1- yl )-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]acetamide (LOE 908), thatfully blocked these currents at 3 Half-maximal inhibition of agonist-activated nonselective inward currents was seen at 40 nM LOE 908, whereas voltage-dependent K currents in undifferentiated HL-60 cells were blocked with an IC of 620 ni. fMLP-induced single-channel currents of 4-5-pS conduct- ance were abolished when the excised inside-out patch was exposed to 3 M LOE 908. The rank order of potency of cations blocking ATP- and fMLP-induced inward currents was Gd3 > Ni2> Cd2. The activation of neutrophil leukocytes during inflammatory events results in chemotaxis, exocytosis, and superoxide anion production. These reactions depend strongly on extracellular Ca2 but are not influenced by activators or blockers of voltage- dependent Ca2 channels (1, 2). These observations point to the existence of another class of ion channels that are charac- terized by a lack of voltage dependence and cation selectivity, because Ca2 influx is parallelled by a substantial Na influx. There are a variety of nonexcitable cells, including platelets, lymphocytes, and neutrophils, in which transmembranous Ca2 and Na influx involves NSC channels (1-il). These channels lack a voltage-dependent gating mechanism but are opened by receptor agonists (for reviews, see Refs. 6 and 7). It has been suggested that these NSC channels play a prominent role in prolonged cell activation and in the refilling of internal Ca2 stores (12, 13). Under pathophysiological conditions, NSC channels may be involved in cell degradation and necrosis (14). In contrast to voltage-operated Ca2 channels, which are clas- sified on the basis oftheir different sensitivities to Ca2 channel This work was supported by grants from the Deutsche Forschungsgemein- schaft and Fonds der Chemischen Industrie. D.K. was supported by a predoctoral fellowship from the Freie Universit#{228}t Berlin. This work contains data from the doctoral thesis of D.K. blockers (15), a pharmacological classification of NSC channels has not yet been possible because of the lack of specific and potent blockers. Me2SO- or Bt2cAMP-differentiated HL-60 cells are widely used as a model system for neutrophils (16-21). Like neutro- phils (4, 22), HL-60 cells possess NSC channels that can be activated by the chemotactic peptide fMLP or ATP (3, 23-25). In the present study, we characterized agonist-induced NSC channels in undifferentiated and Bt2cAMP-differentiated HL- 60 cells, using the whole-cell and single-channel patch-clamp techniques (26). We compared the efficacy of inorganic and organic blockers, and we introduce a new potent blocker (LOE 908) of receptor-activated NSC channels. Experimental Procedures Materials. SK&F 96365 was kindly provided by Dr. J. E. Merritt, Smith Kline Beecham (Welwyn, UK). LOE 908 [(RS)-(3,4-dihydro- 6,7-dimethoxyisoquinoline-1--yl)-2-phenyl-N,N-di-[2-(2,3,4-trime- thoxyphenyl)ethyl]acetamide] was prepared by Bischler-Napieralsky cyclization (CH3CN/POC13) from (RS)-phenyl-N-[2-(3,4-dimethoxy- phenyl)ethyl}-N’ -[2-(2,3,4-trimethoxyphenyl)ethyl]malonyldiamide and was transformed to its monooxalate. Stock solutions of LOE 908 (30 mM) and SK&F 96365 (30 mM) were prepared in Me2SO and were at Universitaetsbibliothek der TU Munchen on June 30, 2014 molpharm.aspetjournals.org Downloaded from